CN1183118A - 具有降低的吸附作用和增加的水解作用的热酶变异体 - Google Patents
具有降低的吸附作用和增加的水解作用的热酶变异体 Download PDFInfo
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- CN1183118A CN1183118A CN96193608A CN96193608A CN1183118A CN 1183118 A CN1183118 A CN 1183118A CN 96193608 A CN96193608 A CN 96193608A CN 96193608 A CN96193608 A CN 96193608A CN 1183118 A CN1183118 A CN 1183118A
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Abstract
本发明涉及具有野生型热酶氨基酸序列的修饰氨基酸序列的热酶(thermitase)变异体,野生型氨基酸序列包括第一环区域、第二环区域、第三环区域、第四环区域、第五环区域和第六环区域;其中,修饰的氨基酸序列在一个或多个环区域中的特定标记的位点上含有与野生型热酶不同的氨基酸(即取代),从而与野生型热酶相比,热酶变异体具有对不溶解底物降低的吸附作用和增加的水解作用。本发明还涉及编码这种热酶变异体的DNA序列。本发明还涉及含有这种热酶变异体的用于洗涤各种表面的组合物。
Description
技术领域
本发明涉及新的用于各种洗涤组合物的酶变异体和编码这种酶变异体的DNA序列。
发明背景
酶构成了最大种类的天然产生的蛋白质。每一类酶通常催化(加速反应而不被消耗)不同种类的化学反应。称为蛋白酶的一种酶已知能够水解(分解化合物成两个或更多个更简单的化合物，在断裂的化学键的两侧接受水分子的H和OH部分)其它蛋白质。将天然产生的和蛋白质工程蛋白酶作为添加剂加入洗衣洗涤剂制剂中即利用了水解蛋白质的这种能力。在衣服上的许多污垢是蛋白质的，广谱的蛋白酶可显著改善这种污垢的除去。
不幸的是，这些蛋白质在其天然的，细菌的环境中的功效水平通常不能转移至相对非天然的洗涤环境。尤其是蛋白酶特性，例如热稳定性、pH稳定性、氧化稳定性和底物特性对于酶的天然环境之外的应用未必达到最佳。
蛋白酶的氨基酸序列决定了蛋白酶的特征。根据氨基酸序列中改变的位点、性质和/或幅度，改变蛋白酶的氨基酸序列可改变酶的特性至各不相同的程度，甚至可失活酶。人们已做了许多尝试以改变蛋白酶的野生型氨基酸序列，以改善它们的性质，从而增加蛋白酶在洗涤环境中的效率。这些尝试包括改变氨基酸序列以便在相当不同的条件下提高热稳定性和改善氧化稳定性。
尽管在现有技术中描述了各种各样的尝试，但人们仍不断需要用于洗涤各种表面的蛋白酶的新有效变异体。
发明目的
本发明的目的在于提供与野生型酶相比较具有改善的水解作用的热酶(Thermitase)变异体。
本发明的另一个目的在于提供含有这些枯草杆菌蛋白酶变异体的洗涤组合物。
发明概述
本发明涉及具有野生型热酶氨基酸序列的修饰氨基酸序列的热酶变异体，野生型氨基酸序列包括第一环区域、第二环区域、第三环区域、第四环区域和第五环区域；其中，修饰的氨基酸序列在一个或多个环区域中的特定标记的位点上含有与野生型热酶不同的氨基酸(即取代)，与野生型热酶相比，热酶变异体具有对不溶解底物降低的吸附作用和增加的水解作用。本发明还涉及编码这种热酶变异体的DNA序列。本发明还涉及含有这种热酶变异体的用于洗涤各种表面的组合物。
发明详述I.热酶变异体
本发明涉及枯草杆菌蛋白酶类，尤其是热酶，它们通过编码酶的各种核苷酸顺序的突变，从而改变酶的氨基酸序列而被修饰。本发明的修饰枯草杆菌蛋白酶(下文称为“热酶变异体”)与野生型热酶相比，具有对不溶解底物降低的吸附作用和增加的水解作用。本发明还涉及编码这种热酶变异体的DNA序列。
本发明的枯草杆菌蛋白酶属于称为蛋白酶类。蛋白酶是断裂肽键的催化剂。一种类型的蛋白酶是丝氨酸蛋白酶。丝氨酸的特征在于在活性位点上存在必需的丝氨酸残基。
随着酶浓度的增加酶对可溶解底物的水解速率增加的现象已经作了充分报导。因而，对于表面结合的底物，例如在许多洗涤应用中遇到的情况，水解速率随着表面浓度的增加而增加似乎是可能的，这已由事实说明。(Brode，P.F.III和D.S.Rauch，LANGMUIR，“枯草杆菌蛋白酶BPN’“固定化底物上的活性”，卷8，1325-1329页(1992))。事实上，当酶的表面浓度变化时，对于不溶解底物已经发现了表面浓度与速率的直线关系。(Rubingh，D.N.和M.D.Bauer.“在蛋白质溶液界面上的蛋白酶的催化水解”多聚物溶液：混和与界面(，POLYMER SOLUTIONS，BLENDSAND INTERFACES)，I.Noda和D.N.Rubingh，Elsevier，编著第464页(1992))。出乎意料的是，当试图将这种原理用于研究得到较好洗涤性能的变异体蛋白酶时，我们不能发现吸附更多的酶得到较好的性能。事实上，我们惊奇地发现与之相反的事实：酶与底物的下降的吸附导致增加的底物水解(即，较好的洗涤性能)。
尽管不希望受任何理论束缚，但我们相信，当将变异体相互比较时，改善的性能是如下事实的结果，即吸附较少的酶也不太紧密地结合，因而在欲除去不溶解蛋白质底物的表面更易移动。在可比的酶溶液浓度下，增加的移动能力足以胜过通过向表面提供较高浓度酶而给予的任何优点。
本发明描述的突变用于改变(即降低)酶与表面结合的污垢的吸附。在热酶中，某些氨基酸在酶分子上形成外环。为了讨论，这些环可称为第一、第二、第三、第四和第五环区域。具体地说，位点66-73形成第一环区域；位点103-115形成第二环区域；位点134-141形成第三环区域；位点162-171形成第四环区域；位点191-195形成第五环区域；和位点204-224形成第六环区域(位点编号类似于野生型枯草杆菌蛋白酶热酶(SEQ ID NO：1)的氨基酸序列)。
我们相信，这些环区域在酶分子与表面结合的肽的吸附中起着重要的作用，在一个或多个环区域中具体的突变对吸附具有明显的效果。尽管不希望受任何理论束缚，但我们相信出于至少两个原因，这些环区域对热酶分子的吸附是重要的。首先，含有环区域的氨基酸能够密切地与分子暴露的任何表面接触。其次，热酶分子的环区域与活性位点的接近和结合囊使得它们在酶与表面结合的底物(肽/蛋白质污垢)的催化产生的吸附中起作用。
本文使用的术语“变异体”是指含有不同于野生型氨基酸序列的酶。
本文使用的术语“突变热酶DNA”指的是编码热酶变异体的DNA序列。
本文使用的术语“野生型热酶”指的是由SEQ ID NO：1表示的酶。该热酶的氨基酸序列在Meloun，B.，Baudys，M，Kostka，V.，Hausdorf，G，Frommel，C.，和Hohne，W.E.，FEBA LETT.，卷183，第195-200页(列为参考文献)中进一步描述。
本文使用的术语“热酶野生型氨基酸序列”包括SEQ ID NO：1以及除位点66、67、68、69、70、72、73、103、104、105、106、107、108、109、110、111、112、113、114、115、134、135、136、137、138、139、140、141、162、163、164、165、166、167、168、169、170、171、191、192、193、194、195、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223或224之外对氨基酸序列修饰的SEQ ID NO：1。
本文使用的术语“更亲水的氨基酸”指的是参考下表中的亲水性值比目标氨基酸具有更大亲水性值的任何其它氨基酸。如下亲水性值表(表1)以增加的亲水性值的下降顺序列出氨基酸(参见Hopp，T.P.，和Woods.K.R.，“由氨基酸序列判断蛋白质抗原决定簇”，美国国家科学院院刊，卷78，3824-3828页，1981，列为参考文献)。
表1氨基酸 亲水性值Trp -3.4Phe -2.5Tyr -2.3Leu，Ile -1.8Val -1.5Met -1.3Cys -1.0Ala，His -0.5Thr -0.4Pro，Gly -0.0Gln，Asn 0.2Ser 0.3Arg+，Lys+，Glu-，Asp- 3.0
表l还说明了带有电荷(该特征基于约8-9的pH)。正电荷的氨基酸是Arg和Lys，负电荷氨基酸是Glu和Asp，其余的氨基酸是中性的。在本发明的优选实施方案中，取代氨基酸是中性的或负电荷的，更优选负电荷(即Glu或Asp)。
因而，例如说明“用相等或更亲水的中性或带有负电荷的氨基酸取代的Gln”指的是用Asn(它与Gln的亲水性相等)或Ser、Glu或Asp(它们比Gln更亲水)取代的Gln。它们每个均是中性或具有负电荷，与Gln相比较，它们具有较大的亲水性值。同样，说明“用更亲水的中性或带有负电荷的氨基酸取代的Pro”指的是用Gln、Asn、Ser、Glu或Asp取代的Pro。
在本发明的一个实施方案中，热酶变异体具有热酶野生型氨基酸序列的修饰氨基酸序列，其中野生型氨基酸序列含有在第一环区域、第二环区域、第三环区域、第四环区域、第五环区域或第六环区域的一个或多个中的一个或多个位点上的取代基；从而热酶变异体与野生型热酶相比较，对不溶解底物具有降低的吸附作用和增加的水解作用。
在本发明的优选实施方案中，在一个或多个环区域中一个或多个位点取代氨基酸，参考表1，是中性的或负电荷的，与在野生型氨基酸序列中目标位点的氨基酸相比较，是相等或更亲水性的，优选是更亲水性的。A.第一环区域中的取代
当取代发生在第一环区域中时，取代发生在位点66、67、68、69、70、72或73的一个或多个上。
当取代发生在位点66时，取代氨基酸是Asn、Asp、Glu或Ser。
当取代发生在位点67时，取代氨基酸是Asn、Asp、Glu或Ser。
当取代发生在位点68时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在位点69时，取代氨基酸是Asp、Gln、Glu或Ser。
当取代发生在位点70时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在位点72时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在位点73时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。B.第二环区域的取代
当取代发生在第二环区域中时，取代发生在位点103、104、105、106、107、108、109、110、111、112、113、114或115的一个或多个上。
当取代发生在位点103时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Met、Pro、Ser或Thr。
当取代发生在位点104时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Met、Pro、Ser、Thr或Val。
当取代发生在位点105时，取代氨基酸是Glu。
当取代发生在位点106时，取代氨基酸是Asp、Gln、Glu或Ser。
当取代发生在位点107时，取代氨基酸是Asp或Glu。
当取代发生在位点108时，取代氧基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在位点109时，取代氨基酸是Asp或Glu。
当取代发生在位点110时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在位点111时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
当取代发生在位点112时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Phe、Pro、Ser、Thr、Tyr或Val。
当取代发生在位点113时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
当取代发生在位点114时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、His、Pro、Ser或Thr。
当取代发生在位点115时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Met、Pro、Ser或Thr。C.第三环区域中的取代
当取代发生在第三环区域中时，取代发生位点134、135、136、137、138、139、140或141的一个或多个上。其中
当取代发生在位点134时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Met、Pro、Ser、Thr或Val。
当取代发生在位点135时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在位点136时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在位点137时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
当取代发生在位点138时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Met、Pro、Ser或Thr。
当取代发生在位点139时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在位点140时，取代氨基酸是Asp、Gln、Glu或Ser。和
当取代发生在位点141时，取代氨基酸是Asp或Glu。D.第四环区域中的取代
当取代发生在第四环区域中时，取代发生在位点162、163、164、165、166、167、168、169、170或171的一个或多个上。
当取代发生在位点162时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在位点163时，取代氨基酸是Asp、Gln、Glu或Ser。
当取代发生在位点164时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、His、Pro、Ser或Thr。
当取代发生在位点165时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在位点166时，取代氨基酸是Asp、Gln、Glu或Ser。
当取代发生在位点167时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
当取代发生在位点168时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、His、Pro、Ser或Thr。
当取代发生在位点169时，取代氨基酸是Asn、Asp、Gln、Glu、Gly或Ser。
当取代发生在位点170时，取代氨基酸是Asp、Gln、Glu或Ser；和
当取代发生在位点171时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Pro、Ser、Thr或Val。E.第五环区域中的取代
当取代发生在第五环区域中时，取代发生在位点191、192、193、194或195一个或多个上。
当取代发生在位点191时，取代氨基酸是Asp或Glu。
当取代发生在位点192时，取代氨基酸是Asp或Glu。
当取代发生在位点193时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Pro、Ser、Thr、Tyr或Val。
当取代发生在位点194时，取代氨基酸是Asp或Glu。
当取代发生在位点195时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。F第六环区域中的取代
当取代发生在第六环区域中时，取代发生在位点204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223或224的一个或多个上。
当取代发生在位点204时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、His、Pro、Ser或Thr。
当取代发生在位点205时，取代氨基酸是Asn、Asp、Gln、Glu、Gly或Ser。
当取代发生在位点206时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在位点207时，取代氨基酸是Asp或Glu。
当取代发生在位点208时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Phe、Pro、Ser、Thr、Tyr或Val。
当取代发生在位点209时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Leu、Met、Pro、Ser、Thr或Val。
当取代发生在位点210时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Pro、Ser、Thr或Val。
当取代发生在位点211时，取代氨基酸是Asp或Glu。
当取代发生在位点212时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
当取代发生在位点213时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Phe、Pro、Ser、Thr或Val。
当取代发生在位点214时，取代氨基酸是Asn、Asp、Gln、Glu、Gly或Ser。
当取代发生在位点215时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
当取代发生在位点216时，取代氨基酸是Asp或Glu。
当取代发生在位点217时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
当取代发生在位点218时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Pro、Ser、Thr或Val。
当取代发生在位点219时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、His、Pro、Ser或Thr。
当取代发生在位点220时，取代氨基酸是Asp或Glu。
当取代发生在位点221时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、 Gly、His、Ile、Met、Pro、Ser、Thr或Val。
当取代发生在位点222时，取代氨基酸是Asp或Glu。
当取代发生在位点223时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在位点224时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。G.酶变异体的制备
实施例1
突变体热酶DNA
构建了含有野生型热酶基因的噬菌粒(“TP”)。将质粒pUC119的2.8Kbp Pvu II限制酶片段(Vieira，J.和Messing，J，“单链的质粒DNA的制备”，153酶学方法3-11(1989))克隆至质粒pUB110(Bacillus GeneticStock Center，Columbus，OH1E9)的PvuII位点。pUC 119-pUB110杂交质粒被称为pJMA601。在pJMA601的Bam H1限制位点克隆入具有TP的聚合酶链反应扩增的热酶基因。噬菌粒TP被转化入大肠杆菌Ung-菌株CJ236，用VCSM13辅助噬菌体(Kunkel，T.A，J.D.Roberts和R.A.Zakour，“无需表型选择的快速和有效的定点突变”，酶学方法，卷154，367-382页(1987)；由Yuckenberg，P.D.，F.Witney，J.Geisselsoder和J.McClary改进，“使用含尿嘧啶的DNA和噬菌粒载体的体外定点突变”，定点突变 操作指南M.J.McPherson编，第27-48页(1991)；均列为本文参考文献)制备单链的含尿嘧啶DNA模板。使用Zoller和Smith方法的单引物定点诱变修饰方法(Zoller，M.J.和M Smith，“使用M13衍生的载体的寡核苷酸定向诱变：一种在DNA任何片段中制备点突变的有效和常用方法”，核酯研究，卷10，6487-6500页(1982)，列为本文参考文献)制备所有突变体(基本上如上述Yuckenberg等人，1991中说明)。寡核苷酸使用Applied Biosystem Inc.380B DNA合成仪制备。突变反应产物转移至大肠杆菌菌株MM294(美国典型培养物保藏中心大肠杆菌33625)中。所有突变体由DNA测序证实，分离的DNA转化入至枯草芽孢杆菌表达菌株BG2036(Yang，M.Y.，E.Ferrari和D.J.Henner，(1984)，“枯草芽孢杆菌的中性蛋白酶基因的克隆和克隆的基因用于产生体外得到的缺失突变”，细菌学杂志，卷160，15-21页)。对于某些突变体，用在相应环中移码终止的密码子突变的修饰TP制备尿嘧啶模板。寡核苷酸用来恢复合适的阅读框和编码在位点66、67、68、69、70、72、73、103、104、105、106、107、108、109、110、111、112、113、114，115、134、135、136、137、138、139、140、141、162、163、164、165、166、167、168、169、170、171、191、192、193、194、195、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223或224的随机取代(在这些密码子中对于所有三种碱基所有四种寡核苷酸的等摩尔和/或可变混合物)。校正移码终止和制备功能酶的突变由它们消化酪蛋白的能力说明。随机取代用DNA测序确定。
实施例2
发酵
将含有感兴趣的枯草芽孢杆菌突变体的枯草芽孢杆菌细胞(BG2036)在1升LB-葡萄糖肉汤培养基中的培养至对照中期，在总体积10升的Biostat ED发酵罐(B.Braun Biotech，Inc.，Allentown，Pennsylvania)中培养。发酵培养基含有酵母提取物、淀粉、消泡剂、缓冲剂和痕量矿物质(参见发酵，操作指南，B.McNeil和L.M.Harvey编，1990)。在发酵试验过程中，肉汤保持恒定pH7.0。加入氯霉素以进行诱变质粒的抗生素选择。细胞在37℃生长过夜至A600约60，并收获。
实施例3
提纯
将发酵肉汤进行如下步骤以得到纯酶。通过离心除去肉汤中的枯草芽孢杆菌细胞，通过用100K截断膜除去细的粒子而澄清。然后用10K截断膜上浓缩，进行流体透析以降低离子强度，用0.025M MES缓冲剂(2-(N-吗啉代)乙磺酸)调节pH至5.5。通过将酶加入阳离子交换层析柱或亲合吸附层析柱，由柱中用氯化钠或丙二醇梯度洗脱而进一步纯化(参见，Scopes，R.K.，蛋白质纯化理论和实践，Springer-Verlag，NewYork(1984)，列为本文参考文献)。
使用pNA试验(DelMar，E.G.，C.Largman，J.W.Brodrick和M.C.Geokas，生物化学年鉴，卷99，316-320页(1979)，列为本文参考文献)测定在梯度洗脱过程中收集的级分的活性酶浓度。该试验测量当酶水解溶解的合成底物，琥珀酰-丙氨酰-丙氨酰-脯氨酰-苯基丙氨酰-对-硝基苯胺(sAAPF-pNA)时释放对-硝基苯胺的速率。用分光光度计在410nm测量水解反应产生黄色的速率，与活性酶浓度成比例。此外，在280nm的吸附测量用来测定总蛋白质浓度。活性酶/总蛋白质比率得到酶的纯度，并用于鉴定用作存贮溶液的收集的级分。
为避免在贮存过程中酶的自溶，在由层析柱得到的收集的级分中加入相等重量的丙二醇。在提纯过程完成后，存贮酶溶液用SDS-PAGE(十二烷基磺酸钠聚丙烯酰胺凝胶电泳)校验，用胰蛋白酶抑制剂型II-T：由Sigma Chemical Company(St.Louis，Missouri)购买的火鸡卵白经活性位滴定法测定绝对酶浓度。测量的转化系数将说明在酶分子中不同位点发生的改变使得酶变异体与野生型相比较对溶解底物pNA具有增加的活性。
在使用的制剂中，酶存贮溶液通过Sephadex-G25(Pharmacia，Piscataway，New Jersey)大小排除柱以除去丙二醇和交换缓冲剂。用含有0.01M氯化钙的0.1M Tris缓冲剂(三(羟基甲基-氨基)甲烷)代替在酶存贮溶液中的MES缓冲剂，用盐酸调节pH至8.6。在恒温25℃下在Tris缓冲剂中在pH8.6下进行所有试验。H.酶变异体的特性
实施例4
模型表面制备
由CPG Inc.(Fairfield，New Jersey)购买的氨基丙基控制的孔玻璃(CPG)用作共价连接由Bachem，Inc.(Torence，California)购买的sAAPD-pNA底物的载体。反应在二甲基亚砜中进行，用(1-乙基-3-[3-(二甲基氨基)丙基]碳化二亚胺盐酸盐)(EDC)作偶联剂。在反应完成后(用pNA试验监测)，除去过量的溶剂，用二甲基亚砜(DMSO)和双蒸馏水漂洗CPG：sAAPF-pNA。然后在烘箱中氮气吹扫在约70℃下干燥。反应方案和固定的底物的制备方法如Brode，P.F.III，和D.S.Rauch，“枯草杆菌蛋白酶BPN’：在固定底物上的活性”，LANGMUIR，卷8，1325-1329页(1992)(列为本文参考文献)中描述进行。
CPG表面含有62000±7000pNA分子/μm2。从CPG Inc.报导的CPG的50.0m2/g的值来看，表面积保持未变。这说明用于在CPG中加入sAAPf-pNA的方法未破坏孔结构(平均直径486A)。
实施例5
表面水解试验
使用CPG：sAAPf-pNA，酶变异体的吸附和CPG-结合的肽的水解可在同一试验中测量。在含有Tris缓冲剂和已脱气的CPG：sAAPF-pNA的烧瓶中加入少量的酶变异体存贮溶液。烧瓶在活塞作用的振荡器中振荡90分钟，在此期间，以不同的时间间隔停止振荡器(例如，在吸附水解作用的最初阶段，例如最初20分钟为每2分钟-在实验结束时为每10分钟)。使CPG：sAAPF-pNA静置，取样溶液。如上述Brode等人，1991中所述进行吸附和水解的实验过程和计算。
测定所有的酶的自溶稳定性，在实验的时间内，应未显示明显的自溶损失。因此，酶吸附可通过测量溶液消耗测定。最初酶变异体浓度与在每个间隔的时间点测量的浓度之间的差值得到吸附的酶变异体的量。由表面水解的pNA的量由在410nm时试样的吸附读数测定。水解的pNA的总量由取样的量加上残余在烧瓶中的量计算。该值通过减去当不存在酶时在pH8.6下由Tris缓冲剂水解的pNA的量而校准。根据酶的效率，基准水解范围为总水解的7-29％。
实施例6
可溶解底物的动力学分析
可溶解底物sAAPF-pNA的水解速率通过用DU-70分光光度计在410nm下测量作为时间函数的吸附增量而监测。酶浓度保持恒定，并制备成6-10纳摩尔的范围，而对于每次动力学测定，底物浓度在90-700μMsAAPF-pNA之间变化。在900秒的时间内，每秒收集吸附数据点，数据传递至LOTUSTM记录纸(Lotus Development Corporation，Cambridge，Massachu setts)。用标准Linewweaver Burk分析法进行动力学参数的分析，其中在试验最初(通常为最初几分钟)的数据符合于线性回归曲线以得到vO。以标准倒数方式标绘的vO和sO数据得到KM和Kcat。I.热酶变异体的实例
降低表面结合的底物的吸附作用和增加的水解作用的本发明热酶变异体在如下表2-36中说明。在描述的具体突变中，首先给出野生型中出现的原始氨基酸，其次是位点编号，第三是取代氨基酸。
表2
环1-单突变变异体
Gln66Asn
Gln66Asp
Gln66Glu
Gln66Ser
Asn67Asp
Asn67Gln
Asn67Glu
Asn67Ser
Gly68Asn
Gly68Asp
Gly68Gln
Gly68Glu
Gly68Pro
Gly68Ser
Asn69Asp
Asn69Gln
Asn69Glu
Asn69Ser
Gly70Asn
Gly70Asp
Gly70Gln
Gly70Glu
Gly70Pro
Gly70Ser
Gly72Asn
Gly72Asp
Gly72Gln
Gly72Glu
Gly72Pro
Gly72Ser
Thr73Asn
Thr73Asp
Thr73Gln
Thr73Glu
Thr73Gly
Thr73Pro
Thr73Ser
表3
环1-双突变变异体Asn68Asp + Gly72ProGly69Asn + Thr73ProAsn66Glu + Gly69SerAsn66Glu + Gly67SerGly69Gln + Thr73ProGly67Ser + Gly72SerGly67Asp + Thr73GlyGly69Pro + Ser70AspGly67Pro + Gly69ProAsn66Glu + Thr73GlyGly69Gln + Gly72AspAsn66Gln + Thr73GlyAsn68Asp + Thr73SerAsn66Gln + Gly69AsnGly67Glu + Thr73SerGly72Glu + Thr73ProAsn66Glu + Thr73ProGly72Asp + Thr73GlnAsn68Ser + Gly69GluGly67Pro + Gly69SerGly67Pro + Gly72AspGly69Ser + Gly72GluSer70Asp + Thr73GlnGly67Ser + Asn68GluGly69Ser + Thr73SerAsn68Asp + Gly69SerAsn66Ser + Gly72AspGly69Asp + Gly72SerAsn68Ser + Thr73ProSer70Asp + Thr73SerSer70Glu + Gly72AsnGly69Asp + Thr73ProGly67Glu + Thr73GlyGly72Asp + Thr73SerSer70Asp + Thr73AsnAsn66Ser + Gly69GluGly72Ser + Thr73GlnSer70Glu + Thr73SerAsn66Gln + Gly69Ser
表4
环1-三突变变异体Ser70Asp + Gly72Gln + Thr73GlyGly67Ser + Asn68Gln + Ser70AspAsn66Ser + Gly67Asn + Thr73GlnAsn66Gln + Ser70Asp + Gly72AsnSer70Glu + Gly72Gln + Thr73SerGly67Pro + Asn68Glu + Thr73SerAsn66Ser + Gly69Gln + Gly72GlnAsn66Ser + Ser70Asp + Thr73GlnGly67Gln + Gly69Asp + Thr73GlnGly69Pro + Gly72Glu + Thr73SerGly67Gln + Gly69Asn + Thr73AsnAsn66Ser + Gly69Asp + Thr73AsnGly69Asp + Gly72Ser + Thr73SerAsn68Asp + Gly72Gln + Thr73SerAsn66Gln + Asn68Ser + Thr73AsnAsn68Ser + Gly69Ser + Ser70AspAsn68Gln + Gly69Ser + Thr73GlnGly67Ser + Gly69Pro + Gly72GluGly67Asn + Gly69Ser + Thr73AsnGly67Gln + Gly72Asn + Thr73GlnGly67Pro + Asn68Gln + Gly69ProGly69Asn + Ser70Glu + Thr73SerAsn66Ser + Ser70Glu + Gly72AsnGly69Gln + Ser70Asp + Gly72AsnGly67Ser + Ser70Glu + Thr73GlnAsn66Glu + Gly67Gln + Thr73ProGly67Ser + Gly72Glu + Thr73SerGly67Pro + Gly69Gln + Gly72AspAsn66Ser + Gly67Glu + Thr73ProAsn66Ser + Gly67Glu + Asn68SerGly67Asn + Asn68Glu + Gly69AspAsn68Glu + Gly69Glu + Gly72ProAsn68Glu + Gly69Glu + Thr73AsnGly67Glu + Asn68Asp + Gly69GlnGly67Glu + Asn68Asp + Thr73GlnAsn66Asp + Gly67Asp + Gly72SerAsn66Glu + Gly67Glu + Thr73Pro
表5
环1-四突变变异体Asn66Gln + Gly67Ser + Gly69Asp + Gly72ProAsn68Ser + Ser70Glu + Gly72Gln + Thr73ProAsn66Gln + Gly67Gln + Ser70Asp + Thr73AsnGly67Ser + Gly69Asp + Gly72Gln + Thr73GlyAsn66Glu + Gly67Ser + Asn68Ser + Thr73AsnGly67Ser + Asn68Asp + Gly72Asn + Thr73GlyAsn66Ser + Gly67Glu + Gly69Gln + Thr73GlyAsn68Glu + Gly69Asp + Gly72Asn + Thr73AsnGly67Glu + Asn68Glu + Gly72Asn + Thr73AsnAsn66Glu + Gly67Glu + Asn68Gln + Gly72ProAsn66Glu + Gly67Glu + Asn68Ser + Gly72AsnAsn66Glu + Gly67Glu + Gly72Pro + Thr73AsnGly67Ser + Gly69Asp + Ser70Glu + Thr73GlnAsn66Ser + Gly69Glu + Ser70Glu + Thr73GlyGly67Pro + Asn68Glu + Gly69Glu + Ser70GluGly67Ser + Asn68Glu + Gly69Asp + Ser70AspGly67Asn + Asn68Asp + Gly69Asp + Ser70GluGly67Asp + Asn68Gln + Gly69Glu + Ser70AspAsn66Gln + Gly67Asp + Gly69Asp + Ser70AspGly67Asp + Asn68Asp + Ser70Asp + Thr73AsnGly67Asp + Asn68Asp + Gly69Pro + Ser70GluGly67Asp + Asn68Gln + Ser70Glu + Gly72ProAsn66Gln + Gly67Glu + Ser70Asp + Gly72GlnGly67Glu + Ser70Glu + Gly72Gln + Thr73SerGly67Asp + Asn68Gln + Ser70Asp + Gly72SerGly67Glu + Ser70Asp + Gly72Ser + Thr73Ser
表6
环2-单突变变异体
Val103Ala
Val103Asn
Val103Asp
Vsl103Gln
Val103Gln
Val103Glu
Val103Gly
Val103His
Val103Met
Val103Pro
Val103Ser
Val103Thr
Leu104Ala
Leu104Asn
Leu104Asp
Leu104Cys
Leu104Gln
Leu104Glu
Leu104Gly
Leu104His
Leu104Ile
Leu104Met
Leu104Pro
Leu104Ser
Leu104Thr
Leu104Val
Asp105Glu
Asn106Asp
Asn106Gln
Asn106Glu
Asn106Ser
Ser107Asp
Ser107Glu
Gly108Asn
Gly108Asp
Gly108Gln
Gly108Glu
Gly108Pro
Gly108Ser
Ser109Asp
Ser109Glu
Gly110Asn
Gly110Asp
Gly110Gln
Gly110Glu
Gly110Pro
Gly110Ser
Thr111Asn
Thr111Asp
Thr111Gln
Thr111Glu
Thr111Gly
Thr111Pro
Thr111Ser
Trp112Ala
Trp112Asn
Trp112Asp
Trp112Cys
Trp112Gln
Trp112Glu
Trp112Gly
Trp112His
Trp112Ile
Trp112Leu
Trp112Met
Trp112Phe
Trp112Pro
Trp112Ser
Trp112Thr
Trp112Tyr
Trp112Val
Thr113Asn
Thr113Asp
Thr113Gln
Thr113Glu
Thr113Gly
Thr113Pro
Thr113Ser
Ala114Asn
Ala114Asp
Ala114Gln
Ala114Glu
Ala114Gly
Ala114His
Ala114Pro
Ala114Ser
Ala114Thr
Val115Ala
Val115Asn
Val115Asp
Val115Cys
Val115Gln
Val115Glu
Val115Gly
Val115His
Val115Met
Val115Pro
Val115Ser
Val115Thr
表7
环2-双突变变异体Val103Gln + Ser109GluAsp105Glu + Gly108GlnAsp105Glu + Gly110AsnAsp105Glu + Gly110ProVal103Asp + Thr111GlnLeu104Gln + Gly108AspLeu104Gly + Ser107GluThr111Pro + Ala114SerAsn106Gln + Ser109AspAsp105Glu + Gly108ProThr113Asp + Val115GlyVal103Met + Leu104GlnGly108Gln + Thr113AsnGly110Glu + Ala114GlyThr111Gln + Val115GlnGly110Glu + Thr111GlnGly108Pro + Ala114GluGly108Ser + Val115GlyAsp105Glu + Thr113GlnLeu104His + Ser107AspAsp105Glu + Ala114GlnGly110Gln + Thr113SerVal103His + Ala114GlnAsp105Glu + Trp112IleThr111Ser + Thr113AspLeu104His + Thr111GluAsn106Gln + Trp112TyrLeu104Gly + Thr113GlnVal103Thr + Val115AsnVal103Thr + Asn106SerVal103Pro + Thr111ProSer109Glu + Val115MetVal103Ala + Asn106GlnVal103Cys + Thr111GlnTrp112Gly + Thr113GlyVal103Gly + Asp105GluGly110Asn + Thr113GlyThr111Asn + Thr113GluVal103Glu + Trp112MetLeu104Pro + Ser109AspGly108Gln + Ala114GlyLeu104Met + Val115GluLeu104Gly + Ala114SerThr113Ser + Val115AlaAsp105Glu + Asn106GlnVal103Thr + Asp105GluGly110Glu + Val115MetVal103Gly + Gly110GluSer109Asp + Gly110GlnThr111Asp + Ala114SerAsn106Glu + Ala114ProSer109Glu + Thr113GlyGly108Asn + Trp112GluVal103Pro + Asn106GluAsp105Glu + Trp112LeuLeu104Cys + Asp105GluLeu104Cys + Thr111SerLeu104Thr + Ser109AspVal103Asn + Val115GluVal103Ser + Gly108Glu
表8
环2-三突变变异体Val103Gln + Leu104Thr + Ser109GluLeu104Ser + Asp105Glu + Trp112PheAsn106Gln + Gly108Ser + Gly110GlnSer109Asp + Thr111Asn + Trp112ThrAsn106Ser + Gly108Gln + Ser109AspAsp105Glu + Gly108Pro + Val115GlyAsn106Ser + Ser107Asp + Val115GlyAsn106Ser + Trp112Gln + Val115GluLeu104Pro + Ser109Asp + Thr113GlyLeu104Asp + Gly110Gln + Val115ProGly108Asn + Thr111Ser + Ala114ProLeu104Ser + Asn106Ser + Thr111GluVal103His + Leu104Gln + Asn106AspAsn106Gln + Trp112Asn + Ala114AspVal103Met + Gly110Pro + Thr111ProLeu104Thr + Gly108Glu + Gly110AsnLeu104Ala + Ser109Asp + Trp112CysGly108Glu + Gly110Pro + Val115GlyLeu104Pro + Thr113Asn + Ala114AspVal103Asn + Leu104Met + Asn106GluVal103Cys + Ser109Glu + Val115CysVal103Gly + Leu104Val + Ser107AspGly108Pro + Ser109Glu + Trp112GlnLeu104Met + Trp112Cys + Thr113AsnAsn106Asp + Trp112Ile + Ala114SerVal103Gly + Ser109Glu + Ala114ProLeu104Ile + Trp112Leu + Thr113GlyAsn106Gln + Gly108Asn + Ser109GluVal103Asn + Trp112Pro + Ala114AspGly108Asn + Gly110Ser + Trp112GlyGly110Asp + Trp112Leu + Val115HisVal103Gly + Leu104Gly + Gly108AspLeu104Ala + Thr111Ser + Val115AlaThr111Ser + Thr113Asn + Val115HisAsp105Glu + Gly108Gln + Val115GlyLeu104Gly + Ala114Ser + Val115AlaVal103His + Leu104Gly + Asn106GluVal103Ser + Gly110Glu + Thr113ProVal103Pro + Gly110Asn + Ala114GluLeu104Met + Gly110Asp + Val115ProLeu104Asp + Thr111Gln + Trp112AlaSer109Asp + Gly110Pro + Trp112AsnLeu104Asp + Asp105Glu + Trp112CysGly108Glu + Ser109Glu + Ala114ThrGly108Asp + Ser109Asp + Thr111ProGly108Glu + Ser109Glu + Ala114AsnAsn106Ser + Ser109Glu + Gly110AspAsn106Gln + Ser109Asp + Gly110AspVal103Asp + Leu104Asp + Gly108ProLeu104Thr + Asp105Glu + Asn106GluAsp105Glu + Asn106Asp + Thr113AsnGly108Gln + Thr113Glu + Ala114GluLeu104Ser + Thr111Glu + Trp112GluAsn106Asp + Ser107Asp + Gly108AspAsp105Glu + Ser109Asp + Gly110GluTrp112Ala + Thr113Asp + Val115AspVal103Gln + Asp105Glu + Gly108AspLeu104Gln + Asp105Glu + Ser107GluSer107Asp + Ser109Asp + Thr111GlyVal103Cys + Ser107Asp + Ser109Glu
表9
环2-四突变变异体Asp105Glu + Gly110Ser + Trp112Phe + Thr113SerVal103Ala + Ser107Asp + Gly110Pro + Ala114AsnVal103Gln + Gly108Pro + Thr111Ser + Ala114GluVal103Thr + Ser107Asp + Thr113Gln + Val115CysLeu104Asn + Gly110Ser + Thr113Glu + Val115GlnVal103Pro + Gly108Glu + Trp112His + Val115MetVal103Ala + Asn106Ser + Gly108Asp + Thr111AsnVal103Ala + Leu104Asp + Gly110Ser + Val115AlaSer109Glu + Trp112Pro + Ala114Asn + Val115MetVal103Thr + Asn106Asp + Gly108Ser + Gly110GlnVal103Pro + Leu104Pro + Asn106Ser + Gly110AsnSer109Glu + Gly110Pro + Thr111Ser + Thr113SerAsn106Gln + Gly108Ser + Trp112Tyr + Ala114ThrLeu104Cys + Gly110Gln + Thr111Asp + Thr113GlyLeu104Ser + Asp105Glu + Gly108Gln + Val115AsnVal103Asp + Gly108Asn + Thr113Pro + Ala114ProVal103Asp + Leu104Asn + Thr113Asn + Val115AlaVal103Asn + Gly110Asp + Thr111Asn + Val115CysVal103Ser + Asn106Glu + Gly110Gln + Val115MetLeu104Val + Thr111Gln + Trp112His + Ala114SerLeu104Ser + Asp105Glu + Gly110Gln + Thr111AsnVal103Ala + Asp105Glu + Asn106Ser + Gly110AsnVal103Glu + Gly110Ser + Trp112Phe + Val115GlyAsn106Glu + Thr113Asn + Ala114Pro + Val115AsnLeu104Glu + Asp105Glu + Gly108Ser + Gly110AsnGly108Glu + Ser109Glu + Thr111Asn + Ala114ProGly108Glu + Ser109Glu + Thr111Gln + Ala114ThrLeu104Ile + Gly108Pro + Ser109Glu + Gly110AspVal103Glu + Leu104Asp + Trp112Cys + Thr113SerAsp105Glu + Asn106Glu + Gly110Pro + Thr113GlyGly110Gln + Thr111Glu + Trp112Glu + Thr113GlySer107Asp + Gly108Asp + Ser109Asp + Trp112ValAsn106Asp + Ser107Asp + Gly108Glu + Val115ProAsp105Glu + Ser107Glu + Gly108Asp + Gly110SerAsp105Glu + Gly108Asp + Ser109Glu + Thr111GlnAsp105Glu + Gly108Glu + Ser109Glu + Thr111ProLeu104Asp + Ser109Glu + Trp112Asn + Ala114GlyVal103Ser + Leu104Glu + Gly108Asn + Ser109GluAsp105Glu + Ser109Glu + Gly110Glu + Trp112GlnVal103Ser + Asp105Glu + Gly108Glu + Val115ProAsp105Glu + Ser107Asp + Trp112Leu + Thr113GlyLeu104His + Asp105Glu + Asn106Ser + Ser107AspAsp105Glu + Ser107Glu + Ser109Asp + Val115GlnAsp105Glu + Ser107Glu + Ser109Glu + Thr113GlySer107Glu + Ser109Glu + Thr111Gln + Thr113GlnSer107Glu + Ser109Glu + Trp112Val + Val115ProSer107Glu + Ser109Glu + Trp112Ile + Val115GlnAsp105Glu + Ser109Glu + Gly110Gln + Val115ProLeu104Glu + Asp105Glu + Ser107Glu + Ala114GlyAsp105Glu + Asn106Glu + Gly108Pro + Ser109AspLeu104Asn + Asp105Glu + Gly110Asp + Ala114GlySer107Glu + Gly108Gln + Ser109Asp + Gly110AspGly108Gln + Gly110Glu + Trp112Asp + Val115GluVal103Asp + Leu104Ser + Asp105Glu + Val115GlnLeu104Glu + Asn106Glu + Gly110Glu + Val115AsnLeu104Asp + Gly110Glu + Thr111Ser + Trp112AspSer109Asp + Gly110Glu + Thr113Asn + Val115GluVal103Ser + Gly108Asp + Gly110Glu + Thr111GluLeu104Gly + Asn106Glu + Gly108Asn + Ser109GluVal103Asp + Leu104Gly + Thr111Glu + Val115Met
表10
环3-单突变变异体
Leu134Ala
Leu134Asn
Leu134Asp
Leu134Cys
Leu134Gln
Leu134Glu
Leu134Gly
Leu134His
Leu134Ile
Leu134Met
Leu134Pro
Leu134Ser
Leu134Thr
Leu134Val
Gly135Asn
Gly135Asp
Gly135Gln
Gly135Glu
Gly135Pro
Gly135Ser
Gly136Asn
Gly136Asp
Gly136Gln
Gly136Glu
Gly136Pro
Gly136Ser
Thr137Asn
Thr137Asp
Thr137Gln
Thr137Glu
Thr137Gly
Thr137Pro
Thr137Ser
Val138Ala
Val138Asn
Val138Asp
Val138Cys
Val138Gln
Val138Glu
Val138Gly
Val138His
Val138Met
Val138Pro
Val138Ser
Val138Thr
Gly139Asn
Gly139Asp
Gly139Gln
Gly139Glu
Gly139Pro
Gly139Ser
Asn140Asp
Asn140Gln
Asn140Glu
Asn140Ser
Ser141Asp
Ser141Glu
表11
环3-双突变变异体Leu134Val + Ser141AspLeu134Val + Thr137GluGly135Asn + Ser141AspGly135Ser + Thr137SerLeu134Met + Gly139AspLeu134Thr + Gly139GlnGly139Gln + Asn140GluGly139Pro + Ser141AspLeu134Glu + Val138AsnLeu134Asn + Val138AlaLeu134Ser + Asn140GluVal138Asn + Ser141AspGly135Glu + Val138SerLeu134Ile + Gly139GluLeu134Ile + Gly135AsnLeu134Met + Asn140AspGly136Ser + Gly139AspGly136Pro + Val138AsnLeu134Gly + Gly135ProLeu134Gly + Ser141GluGly135Pro + Gly139ProLeu134Thr + Gly139ProGly135Glu + Asn140SerThr137Gln + Gly139AspGly135Asp + Asn140SerLeu134Ser + Asn140AspGly136Gln + Gly139AspLeu134Pro + Ser141GluLeu134His + Asn140GlnLeu134Pro + Gly135GluGly135Pro + Gly136AspVal138Thr + Ser141AspVal138Pro + Ser141AspLeu134Gln + Thr137GluVal138Ser + Ser141AspVal138Thr + Asn140GlnLeu134Glu + Val138GlnGly139Asn + Ser141GluGly139Pro + Ser141GluGly135Gln + Gly139GluThr137Pro + Val138GlyGly135Asp + Gly136GlnGly135Asn + Val138ThrThr137Asn + Ser141GluGly135Pro + Val138GluGly135Glu + Thr137SerGly135Asp + Gly139ProLeu134Ala + Gly135GlnVal138Gln + Ser141AspGly135Ser + Gly139AspThr137Ser + Val138MetLeu134Ala + Gly136GlnLeu134Glu + Asn140GlnGly136Asn + Asn140GlnGly136Gln + Ser141AspVal138Ser + Ser141GluLeu134Thr + Thr137GluGly135Ser + Gly139ProVal138Cys + Gly139AspThr137Gly + Gly139Asp
表12
环3-三突变变异体Gly135Ser + Val138Cys + Ser141AspGly135Gln + Thr137Glu + Val138CysLeu134Ser + Gly135Pro + Gly136GluLeu134Pro + Val138Cys + Asn140SerLeu134Val + Gly135Asp + Val138MetLeu134Pro + Gly135Pro + Gly139GluGly135Gln + Val138Asp + Gly139SerGly136Pro + Val138His + Ser141AspLeu134Val + Gly136Ser + Asn140AspThr137Asp + Val138Met + Gly139GlnLeu134Val + Gly136Pro + Thr137GlyGly135Gln + Val138Gln + Asn140AspLeu134His + Gly136Glu + Asn140GlnGly135Pro + Gly139Gln + Ser141AspGly136Asn + Val138Asn + Ser141AspGly135Asp + Val138Thr + Gly139GlnGly136Pro + Thr137Asp + Gly139SerLeu134Gly + Gly136Gln + Gly139SerLeu134Val + Gly135Glu + Thr137GlyLeu134Thr + Val138Gln + Gly139SerGly136Asp + Thr137Asn + Val138GlyGly135Asp + Val138Ser + Asn140GlnGly136Asn + Val138Glu + Asn140GlnLeu134Cys + Thr137Glu + Val138GlyLeu134Asn + Gly135Glu + Gly139SerGly135Ser + Val138His + Asn140GluLeu134Ala + Gly139Ser + Asn140GlnGly135Asp + Thr137Pro + Val138CysLeu134Cys + Val138Gly + Asn140AspLeu134Thr + Val138Ser + Gly139AspLeu134Cys + Thr137Gln + Ser141GluLeu134Val + Gly136Pro + Gly139GluLeu134Ala + Gly135Asn + Ser141GluGly136Gln + Thr137Gln + Val138ProLeu134Asp + Gly135Pro + Gly139SerGly135Ser + Val138Gly + Ser141AspLeu134Val + Thr137Asp + Gly139GlnLeu134His + Gly135Pro + Gly139GlnThr137Pro + Val138Gln + Asn140AspThr137Gln + Val138Cys + Gly139GluThr137Ser + Gly139Glu + Asn140GlnGly135Gln + Thr137Gln + Asn140AspGly135Gln + Thr137Gln + Val138AspLeu134His + Gly139Gln + Asn140AspLeu134Ala + Thr137Asn + Gly139GluVal138Pro + Gly139Pro + Asn140GlnGly135Asn + Asn140Ser + Ser141GluLeu134Ile + Gly135Asp + Val138CysThr137Gln + Gly139Ser + Asn140SerLeu134Ala + Gly136Pro + Asn140AspGly135Ser + Gly139Asp + Asn140SerLeu134Thr + Gly135Asn + Ser141GluLeu134Gly + Thr137Ser + Val138AlaLeu134Thr + Gly135Gln + Val138ProGly135Gln + Gly136Asp + Thr137SerGly135Ser + Val138Thr + Asn140AspLeu134Cys + Thr137Ser + Val138GluGly136Ser + Thr137Asn + Ser141AspLeu134Thr + Gly135Glu + Gly139GlnLeu134Thr + Gly135Asp + Thr137Ser
表13
环3-四突变变异体Leu134Met + Thr137Pro + Asn140Ser + Ser141GluLeu134Asn + Gly136Pro + Thr137Ser + Val138GlnLeu134Gln + Gly136Gln + Val138Ser + Gly139SerGly135Glu + Gly136Asn + Thr137Pro + Asn140GlnLeu134Ser + Gly136Asn + Gly139Gln + Ser141GluLeu134Gln + Val138Pro + Gly139Ser + Ser141AspLeu134Pro + Gly135Asn + Thr137Glu + Asn140GlnGly135Asp + Thr137Gly + Val138Gln + Gly139SerLeu134Val + Gly135Asp + Thr137Asn + Asn140SerLeu134Thr + Gly135Glu + Gly136Ser + Thr137SerGly136Pro + Thr137Gln + Gly139Gln + Asn140GluLeu134His + Gly135Glu + Val138Asn + Asn140GlnLeu134Gln + Gly135Ser + Thr137Asn + Ser141GluGly135Gln + Gly139Asn + Asn140Gln + Ser141AspThr137Gln + Val138His + Gly139Asn + Ser141GluLeu134Thr + Gly135Gln + Val138Thr + Asn140AspLeu134Met + Gly135Glu + Thr137Ser + Val138SerVal138Thr + Gly139Ser + Asn140Ser + Ser141GluLeu134Met + Gly136Pro + Val138Cys + Gly139ProLeu134His + Gly136Pro + Thr137Gln + Gly139GluGly135Asn + Gly136Gln + Val138Cys + Ser141GluLeu134Asn + Thr137Ser + Val138Gln + Ser141AspGly135Gln + Gly136Pro + Thr137Asn + Asn140SerGly135Asn + Gly139Ser + Asn140Gln + Ser141AspLeu134Met + Gly136Pro + Val138Met + Ser141GluLeu134Cys + Thr137Gln + Val138Pro + Ser141AspGly136Asn + Thr137Gln + Gly139Asn + Asn140SerLeu134Asn + Thr137Ser + Gly139Ser + Asn140AspGly135Asp + Gly136Asp + Val138Met + Asn140SerGly135Glu + Gly136Asp + Val138His + Gly139GlnLeu134Ser + Gly136Glu + Thr137Glu + Val138ThrLeu134Thr + Val138Gln + Asn140Glu + Ser141GluGly135Gln + Thr137Asn + Asn140Glu + Ser141GluGly135Gln + Thr137Gly + Val138Asp + Gly139AspLeu134Thr + Gly135Glu + Gly136Asp + Thr137AspVal138Asn + Gly139Asp + Asn140Glu + Ser141AspLeu134Ile + Gly139Glu + Asn140Asp + Ser141AspGly136Pro + Gly139Glu + Asn140Glu + Ser141AspLeu134Gln + Gly139Asp + Asn140Asp + Ser141GluLeu134Val + Gly136Glu + Val138Asp + Asn140GlnGly136Asp + Thr137Gln + Val138Asp + Gly139AsnGly136Glu + Thr137Glu + Val138His + Gly139AspLeu134His + Thr137Gln + Val138Glu + Asn140GluLeu134Gly + Gly135Ser + Thr137Glu + Gly139GluLeu134Gln + Gly135Pro + Thr137Asp + Gly139AspLeu134His + Gly135Asn + Thr137Asp + Gly139GluLeu134Val + Gly135Glu + Thr137Asp + Gly139GlnGly135Asn + Thr137Ser + Gly139Asp + Ser141AspLeu134Cys + Thr137Asn + Gly139Glu + Ser141AspLeu134Ile + Thr137Pro + Gly139Asp + Ser141AspLeu134Thr + Val138Asn + Gly139Glu + Ser141AspGly135Pro + Thr137Gly + Gly139Glu + Ser141AspGly136Ser + Val138Ala + Gly139Glu + Ser141GluLeu134Thr + Gly139Asp + Asn140Ser + Ser141AspGly135Ser + Thr137Gly + Gly139Glu + Ser141AspLeu134Met + Gly135Asn + Gly139Asp + Ser141GluThr137Pro + Val138Gly + Gly139Asp + Ser141AspGly136Glu + Thr137Pro + Val138Gln + Gly139AspThr137Asn + Val138Glu + Gly139Asp + Ser141AspLeu134Thr + Val138Glu + Gly139Glu + Ser141Glu
表14
环4-单突变变异体
Gly162Asn
Gly162Asp
Gly162Gln
Gly162Glu
Gly162Pro
Gly162Ser
Asn163Asp
Asn163Gln
Asn163Glu
Asn163Ser
Ala164Asn
Ala164Asp
Ala164Gln
Ala164Glu
Ala164Gly
Ala164His
Ala164Pro
Ala164Ser
Ala164Thr
Gly165Asn
Gly165Asp
Gly165Gln
Gly165Glu
Gly165Pro
Gly165Ser
Asn166Asp
Asn166Gln
Asn166Glu
Asn166Ser
Thr167Asn
Thr167Asp
Thr167Gln
Thr167Glu
Thr167Gly
Thr167Pro
Thr167Ser
Ala168Asn
Ala168Asp
Ala168Gln
Ala168Glu
Ala168Gly
Ala168His
Ala168Pro
Ala168Ser
Ala168Thr
Pro169Asn
Pro169Asp
Pro169Gln
Pro169Glu
Pro169Gly
Pro169Ser
Asn170Asp
Asn170Gln
Asn170Glu
Asn170Ser
Tyr171Ala
Tyr171Asn
Tyr171Asp
Tyr171Cys
Tyr171Gln
Tyr171Glu
Tyr171Gly
Tyr171His
Tyr171Ile
Tyr171Leu
Tyr171Met
Tyr171Pro
Tyr171Ser
Tyr171Thr
Tyr171Val
表15
环4-双突变变异体Ala164Gln + Pro169GluAsn163Asp + Thr167SerAla164Pro + Ala168AspAla164Gln + Asn166SerGly165Asp + Thr167ProAla164Gln + Ala168SerGly165Gln + Asn166GlnGly162Gln + Asn163GluGly165Gln + Thr167GluGly165Ser + Ala168GlnAla164Gln + Ala168GluAla164His + Tyr171MetGly165Pro + Pro169GlnGly165Gln + Ala168ProGly162Pro + Ala164GlyThr167Pro + Asn170SerAsn163Gln + Tyr171HisAsn163Ser + Ala164GlnAla164Asp + Asn166GlnGly165Asn + Tyr171IleAla168Asp + Asn170GlnAsn166Asp + Ala168GlyAsn163Glu + Thr167SerAsn166Glu + Ala168SerGly162Gln + Ala168GluGly162Asn + Asn170SerAsn163Gln + Ala168HisThr167Pro + Ala168ThrGly162Asn + Gly165GluAsn166Glu + Tyr171ProAla168Asn + Asn170GlnGly162Glu + Thr167SerThr167Asp + Tyr171ValGly162Asp + Ala168HisGly165Ser + Ala168GluThr167Asp + Tyr171MetAla168Thr + Pro169GlyGly165Ser + Pro169GluGly162Glu + Ala164ThrAla168Ser + Tyr171AspAsn163Ser + Gly165ProAla168Asn + Tyr171SerAla164Gln + Gly165GlnThr167Asp + Asn170SerGly165Ser + Asn170GlnThr167Gly + Tyr171GluGly162Asn + Tyr171GlnGly165Asn + Asn166GluAla168Thr + Tyr171GluGly165Ser + Asn166GluThr167Gly + Ala168ThrAsn166Asp + Ala168AsnPro169Glu + Tyr171ValGly162Glu + Asn170SerGly162Ser + Pro169GlyAla164Asn + Asn166GluThr167Gly + Asn170AspThr167Glu + Asn170SerAsn163Asp + Ala164GlnGly165Gln + Pro169Glu
表16
环4-三突变变异体Ala164Gly + Gly165Glu + Thr167SerGly162Asn + Ala164Thr + Asn166AspAla164Asn + Gly165Asp + Pro169GlnGly165Pro + Thr167Asn + Pro169GluAla168Gly + Pro169Gly + Asn170AspGly162Asp + Gly165Pro + Ala168SerGly165Asp + Thr167Asn + Tyr171GlyThr167Ser + Pro169Asn + Tyr171ThrAsn166Ser + Ala168Gly + Tyr171IleGly165Gln + Thr167Pro + Ala168GluGly162Ser + Asn163Asp + Gly165GlnGly165Glu + Thr167Gly + Ala168SerThr167Gly + Ala168Asp + Tyr171ValAsn163Ser + Gly165Gln + Ala168AspAsn163Asp + Ala164Gly + Gly165AsnAla164Gln + Pro169Ser + Asn170GlnAla164His + Thr167Gln + Pro169GlyThr167Ser + Ala168Asn + Tyr171AsnGly162Asp + Gly165Ser + Ala168AsnGly162Pro + Asn163Gln + Tyr171ValAsn166Glu + Ala168Ser + Asn170GlnThr167Pro + Pro169Ser + Tyr171HisGly162Gln + Asn163Gln + Gly165AspAla164Ser + Thr167Ser + Ala168GluThr167Gln + Pro169Glu + Asn170GlnAsn163Gln + Pro169Asn + Asn170GlnGly162Glu + Ala168Gln + Asn170GlnAla164Gly + Gly165Ser + Asn166AspAla164Ser + Ala168Gly + Asn170GlnAsn163Glu + Ala164Asn + Ala168SerThr167Ser + Pro169Glu + Tyr171SerAla168Glu + Pro169Gln + Tyr171IleGly162Pro + Ala164Pro + Tyr171ProGly162Pro + Asn166Ser + Pro169GluAla164Gly + Ala168Asn + Tyr171SerAla164Thr + Asn170Asp + Tyr171LeuAsn163Gln + Thr167Glu + Pro169SerAsn166Asp + Ala168Gln + Tyr171ProGly162Asn + Ala164Glu + Tyr171AlaAsn166Glu + Thr167Gln + Ala168ProAla164Pro + Ala168Gln + Asn170AspAla164Thr + Gly165Gln + Ala168AspAla164Ser + Ala168Gly + Asn170GluAla164His + Gly165Glu + Tyr171MetAla164Gln + Gly165Asp + Asn166GlnGly162Gln + Thr167Asn + Ala168AsnAla168His + Pro169Gln + Tyr171GluAla164Gln + Asn166Gln + Tyr171ValGly162Pro + Ala164Ser + Asn170GlnThr167Glu + Ala168Gly + Asn170SerThr167Ser + Pro169Gly + Asn170SerAsn163Glu + Asn166Ser + Ala168SerAsn163Gln + Ala168Glu + Tyr171AlaThr167Ser + Ala168Ser + Asn170AspGly165Asp + Asn166Glu + Tyr171ProAsn163Asp + Ala164Asp + Thr167SerAla164Asp + Gly165Glu + Asn166GlnGly162Ser + Ala164Glu + Gly165AspGly165Asn + Asn170Glu + Tyr171GluGly165Pro + Asn170Glu + Tyr171Glu
表17
环4-四突变变异体Ala164Asn + Asn166Asp + Thr167Asn + Ala168GlnGly162Asn + Asn163Gln + Ala164Asn + Asn170GluAla164Thr + Asn166Ser + Ala168Asn + Pro169GluAla164Gln + Pro169Asn + Asn170Glu + Tyr171ThrAsn163Ser + Ala164Thr + Thr167Asp + Tyr171ThrThr167Ser + Pro169Ser + Asn170Asp + Tyr171SerGly162Asn + Thr167Gly + Pro169Gln + Tyr171SerGly162Pro + Asn163Ser + Ala168Glu + Tyr171ValAsn163Gln + Ala168Asp + Pro169Gln + Asn170GlnAla164Asn + Gly165Ser + Pro169Gln + Tyr171IleGly162Glu + Thr167Gln + Ala168Ser + Tyr171SerGly165Ser + Ala168Gly + Asn170Asp + Tyr171LeuAla164His + Gly165Asp + Asn166Gln + Tyr171ThrAsn163Gln + Ala164Ser + Asn166Ser + Ala168GluAsn166Asp + Thr167Asn + Asn170Ser + Tyr171ThrGly162Asp + Gly165Gln + Asn170Ser + Tyr171GlnGly162Pro + Ala168Asn + Pro169Glu + Tyr171GlyAsn166Gln + Thr167Gln + Ala168Thr + Asn170AspAsn163Gln + Thr167Asp + Ala168Ser + Tyr171ProAla164Pro + Thr167Glu + Pro169Ser + Tyr171ValGly165Asn + Asn166Gln + Pro169Gly + Asn170AspAsn166Ser + Thr167Pro + Ala168Asn + Asn170GluAla164Gly + Ala168Asn + Pro169Asp + Tyr171AlaAla164Gln + Gly165Gln + Thr167Ser + Pro169GluAsn163Glu + Gly165Ser + Thr167Gln + Ala168ThrGly162Pro + Ala164Thr + Thr167Ser + Pro169GlnGly162Gln + Asn163Glu + Gly165Gln + Thr167ProAsn163Ser + Pro169Gly + Asn170Glu + Tyr171SerAsn163Gln + Gly165Ser + Asn170Asp + Tyr171LeuAla164His + Gly165Pro + Pro169Ser + Asn170GlnGly162Pro + Asn166Gln + Ala168Asn + Asn170GluAsn163Gln + Gly165Pro + Thr167Ser + Tyr171GlyAsn163Gln + Gly165Asn + Asn166Ser + Tyr171CysAsn163Asp + Gly165Pro + Asn166Ser + Tyr171GlyAla164Pro + Thr167Pro + Pro169Glu + Asn170SerAsn163Asp + Ala164Glu + Pro169Gln + Tyr171HisAsn163Glu + Ala164Glu + Ala168Gly + Tyr171HisThr167Asp + Ala168Glu + Pro169Gln + Asn170SerAla164Asp + Gly165Asp + Ala168Asn + Pro169GlnGly162Ser + Ala164Gly + Asn170Asp + Tyr171GluAsn163Gln + Thr167Asn + Ala168Asp + Pro169GluAsn163Ser + Gly165Pro + Ala168Asp + Pro169GluGly165Ser + Ala168Asp + Pro169Glu + Asn170SerAsn166Gln + Ala168Gln + Pro169Asp + Asn170AspAla164Gly + Thr167Gly + Pro169Glu + Asn170AspGly162Pro + Ala164Gly + Pro169Asp + Asn170GluAla164Pro + Thr167Glu + Ala168Asp + Pro169AspGly162Asp + Asn163Gln + Ala168His + Asn170AspGly162Glu + Ala164Ser + Asn166Ser + Asn170GluGly162Glu + Ala164Ser + Asn166Gln + Asn170GluThr167Asp + Ala168His + Pro169Glu + Tyr171ProAsn163Glu + Ala164His + Gly165Asp + Asn166GlnAsn163Asp + Ala164Gln + Gly165Asp + Asn166SerAsn163Glu + Ala164Asp + Gly165Gln + Ala168AspGly162Glu + Ala164Glu + Asn166Ser + Pro169AspAla164Glu + Asn166Asp + Asn170Ser + Tyr171GlyAla164Glu + Gly165Gln + Asn166Glu + Tyr171CysAla164Asp + Asn166Glu + Thr167Asn + Pro169AsnThr167Asp + Pro169Glu + Asn170Glu + Tyr171SerAsn166Asp + Thr167Ser + Ala168Asp + Tyr171His
表18
环5-单突变变异体
Ser191Asp
Ser191Glu
Ser192Asp
Ser192Glu
Phe193Ala
Phe193Asn
Phe193Asp
Phe193Cys
Phe193Gln
Phe193Glu
Phe193Gly
Phe193His
Phe193Ile
Phe193Leu
Phe193Met
Phe193Pro
Phe193Ser
Phe193Thr
Phe193Tyr
Phe193Val
Ser194Asp
Ser194Glu
Thr195Asn
Thr195Asp
Thr195Gln
Thr195Glu
Thr195Gly
Thr195Pro
Thr195Ser
表19
环5-双突变变异体Ser192Asp + Phe193SerSer192Asp + Phe193MetSer191Glu + Phe193AsnSer191Asp + Phe193IleSer191Glu + Phe193GlnSer191Asp + Thr195SerPhe193Gly + Thr195AspSer192Asp + Phe193CysSer191Glu + Thr195ProSer191Glu + Phe193MetPhe193Thr + Thr195GluSer192Asp + Thr195GlnPhe193Asp + Thr195AsnSer191Asp + Phe193GlnPhe193Gly + Thr195ProSer192Asp + Thr195SerSer191Glu + Phe193IlePhe193Gln + Thr195AsnSer191Glu + Thr195GlySer191Glu + Phe193HisSer191Asp + Phe193LeuSer192Glu + Phe193ProPhe193His + Thr195GluSer192Asp + Thr195GlySer192Glu + Phe193AlaSer192Glu + Phe193ValSer192Asp + Phe193LeuSer192Glu + Phe193AsnPhe193Tyr + Thr195SerPhe193His + Thr195AsnSer192Glu + Thr195SerPhe193Ile + Thr195GluPhe193Val + Thr195AsnPhe193Met + Thr195GlnSer191Asp + Thr195ProSer192Glu + Phe193TyrPhe193Ser + Thr195AsnSer192Glu + Phe193GlnSer191Asp + Phe193SerSer192Asp + Phe193IleSer192Asp + Phe193HisSer192Asp + Phe193GlySer191Glu + Phe193SerSer192Glu + Thr195GlnSer191Glu + Thr195SerSer191Asp + Thr195AsnSer191Glu + Phe193ValPhe193Tyr + Thr195AspPhe193His + Thr195SerSer192Glu + Phe193IlePhe193Met + Thr195GlySer191Glu + Phe193TyrSer191Asp + Thr195GlnSer192Asp + Phe193AsnSer192Asp + Phe193ThrSer192Glu + Phe193CysPhe193Asp + Thr195GlnPhe193Val + Thr195GlnSer192Glu + Phe193ThrPhe193Leu + Thr195Glu
表20
环5-三突变变异体Ser192Glu + Phe193Thr + Thr195GlySer191Glu + Phe193His + Thr195SerSer192Asp + Phe193Leu + Thr195AsnSer191Asp + Phe193Ala + Thr195SerSer192Asp + Phe193Val + Thr195GlnSer191Asp + Phe193Ser + Thr195ProSer191Asp + Phe193Ile + Thr195GlySer192Glu + Phe193His + Thr195GlySer192Glu + Phe193Leu + Thr195AsnSer192Asp + Phe193Gly + Thr195GlnSer192Asp + Phe193Ile + Thr195ProSer191Glu + Phe193Asn + Thr195SerSer192Asp + Phe193Met + Thr195AsnSer191Glu + Phe193Gly + Thr195AsnSer192Glu + Phe193His + Thr195AsnSer192Asp + Phe193Met + Thr195ProSer191Glu + Phe193Leu + Thr195AsnSer191Glu + Phe193Met + Thr195AsnSer192Glu + Phe193Ala + Thr195GlnSer191Glu + Phe193Tyr + Thr195GlySer192Asp + Phe193Ala + Thr195GlySer191Glu + Phe193Met + Thr195GlySer192Glu + Phe193Thr + Thr195GlnSer191Asp + Phe193Asn + Thr195GlySer192Glu + Phe193Asn + Thr195SerSer192Asp + Phe193Ser + Thr195GlySer192Glu + Phe193Leu + Thr195ProSer191Asp + Phe193Cys + Thr195GlySer191Asp + Ser192Asp + Phe193TyrSer191Asp + Ser192Asp + Phe193GlySer191Asp + Ser192Glu + Phe193MetSer191Asp + Ser192Glu + Thr195GlnSer191Glu + Ser192Asp + Phe193ValSer191Glu + Ser192Glu + Phe193ThrSer191Glu + Ser192Asp + Phe193ThrSer191Asp + Ser192Asp + Thr195GlySer191Glu + Ser192Glu + Phe193AsnSer191Asp + Ser192Glu + Phe193AlaSer191Asp + Ser192Asp + Phe193AsnSer191Asp + Ser192Asp + Phe193ProSer191Asp + Ser192Glu + Phe193ThrSer191Asp + Ser192Asp + Thr195AsnSer191Asp + Ser192Asp + Thr195GlnSer191Glu + Ser192Asp + Phe193AsnSer191Glu + Ser192Glu + Phe193GlnSer191Glu + Ser192Asp + Phe193LeuSer191Asp + Ser192Asp + Thr195SerSer191Asp + Ser192Glu + Phe193SerSer191Glu + Ser192Asp + Phe193TyrSer191Asp + Ser192Asp + Phe193ThrSer191Asp + Ser192Glu + Phe193ProSer191Glu + Ser192Asp + Phe193IleSer191Asp + Ser192Glu + Phe193TyrSer191Glu + Ser192Glu + Phe193TyrSer191Asp + Ser192Asp + Phe193SerSer191Glu + Ser192Glu + Thr195ProSer191Asp + Ser192Glu + Thr195SerSer191Glu + Ser192Asp + Phe193MetSer191Asp + Ser192Glu + Thr195ProSer191Glu + Ser192Asp + Phe193His
表21
环5-四突变变异体Ser191Asp + Ser192Glu + Phe193Gly + Thr195GlnSer191Glu + Ser192Asp + Phe193His + Thr195GlySer191Glu + Ser192Glu + Phe193Cys + Thr195ProSer191Asp + Ser192Glu + Phe193Cys + Thr195GlnSer191Asp + Ser192Glu + Phe193Thr + Thr195ProSer191Glu + Ser192Glu + Phe193Val + Thr195AsnSer191Asp + Ser192Glu + Phe193Val + Thr195AsnSer191Glu + Ser192Glu + Phe193Leu + Thr195SerSer191Glu + Ser192Asp + Phe193Cys + Thr195GlnSer191Glu + Ser192Asp + Phe193Pro + Thr195ProSer191Glu + Ser192Asp + Phe193Ser + Thr195GlySer191Glu + Ser192Asp + Phe193Pro + Thr195AsnSer191Glu + Ser192Glu + Phe193Ser + Thr195ProSer191Glu + Ser192Asp + Phe193Ala + Thr195GlySer191Asp + Ser192Glu + Phe193His + Thr195AsnSer191Asp + Ser192Glu + Phe193Asn + Thr195AsnSer191Glu + Ser192Glu + Phe193Gln + Thr195SerSer191Glu + Ser192Glu + Phe193Thr + Thr195GlnSer191Glu + Ser192Glu + Phe193Asn + Thr195GlnSer191Glu + Ser192Glu + Phe193Tyr + Thr195SerSer191Asp + Ser192Glu + Phe193Ile + Thr195ProSer191Glu + Ser192Asp + Phe193Val + Thr195GlnSer191Glu + Ser192Asp + Phe193Val + Thr195AsnSer191Asp + Ser192Glu + Phe193Tyr + Thr195SerSer191Asp + Ser192Glu + Phe193Ala + Thr195SerSer191Glu + Ser192Glu + Phe193His + Thr195AsnSer191Asp + Ser192Asp + Phe193Pro + Thr195AsnSer191Asp + Ser192Asp + Phe193Gln + Thr195SerSer191Asp + Ser192Asp + Phe193Leu + Thr195AsnSer191Asp + Ser192Glu + Phe193Val + Thr195GlnSer191Glu + Ser192Glu + Phe193Gly + Thr195GlySer191Glu + Ser192Glu + Phe193Thr + Thr195ProSer191Glu + Ser192Glu + Phe193Thr + Thr195GlySer191Glu + Ser192Asp + Phe193Ile + Thr195SerSer191Glu + Ser192Glu + Phe193Ile + Thr195GlySer191Asp + Ser192Asp + Phe193Pro + Thr195ProSer191Asp + Ser192Asp + Phe193Ile + Thr195GlySer191Glu + Ser192Asp + Phe193Thr + Thr195GlySer191Asp + Ser192Glu + Phe193Ser + Thr195GlySer191Glu + Ser192Asp + Phe193Tyr + Thr195SerSer191Asp + Ser192Asp + Phe193Thr + Thr195GlnSer191Asp + Ser192Glu + Phe193Ser + Thr195GlnSer191Glu + Ser192Glu + Phe193His + Thr195SerSer191Glu + Ser192Asp + Phe193Leu + Thr195AsnSer191Asp + Ser192Glu + Phe193Leu + Thr195ProSer191Asp + Ser192Asp + Phe193Ile + Thr195ProSer191Asp + Ser192Asp + Phe193Tyr + Thr195SerSer191Asp + Ser192Glu + Phe193Ile + Thr195GlnSer191Asp + Ser192Glu + Phe193Pro + Thr195SerSer191Asp + Ser192Glu + Phe193Leu + Thr195GlnSer191Glu + Ser192Asp + Phe193Asn + Thr195SerSer191Glu + Ser192Glu + Phe193Ser + Thr195AsnSer191Asp + Ser192Asp + Phe193Glu + Thr195GlnSer191Asp + Ser192Glu + Phe193Glu + Thr195ProSer191Glu + Ser192Asp + Phe193Glu + Thr195GlnSerl91Glu + Ser192Glu + Phe193Glu + Thr195GlySer191Glu + Ser192Glu + Phe193Asp + Thr195GlySer191Asp + Ser192Asp + Phe193Glu + Thr195SerSer191Glu + Ser192Asp + Phe193Asp + Thr195ProSer191Glu + Ser192Glu + Phe193Asp + Thr195Gln
表22
环6-单突变变异体
Ala204Asn
Ala204Asp
Ala204Gln
Ala204Glu
Ala204Gly
Ala204His
Ala204Pro
Ala204Ser
Ala204Thr
Pro205Asn
Pro205Asp
Pro205Gln
Pro205Glu
Pro205Gly
Pro205Ser
Gly206Asn
Gly206Asp
Gly206Gln
Gly206Glu
Gly206Pro
Gly206Ser
Ser207Asp
Ser207Glu
Trp208Ala
Trp208Asn
Trp208Asp
Trp208Cys
Trp208Gln
Trp208Glu
Trp208Gly
Trp208His
Trp208Ile
Trp208Leu
Trp208Met
Trp208Phe
Trp208Pro
Trp208Ser
Trp208Thr
Trp208Tyr
Trp208Val
Ile209Ala
Ile209Asn
Ile209Asp
Ile209Cys
Ile209Gln
Ile209Glu
Ile209Gly
Ile209His
Ile209Leu
Ile209Met
Ile209Pro
Ile209Ser
Ile209Thr
Ile209Val
Tyr210Ala
Tyr210Asn
Tyr210Asp
Tyr210Cys
Tyr210Gln
Tyr210Glu
Tyr210Gly
Tyr210His
Tyr210Ile
Tyr210Leu
Tyr210Met
Tyr210Pro
Tyr210Ser
Tyr210Thr
Tyr210Val
Ser211Asp
Ser211Glu
Thr212Asn
Thr212Asp
Thr212Gln
Thr212Glu
Thr212Gly
Thr212Pro
Thr212Ser
Tyr213Ala
Tyr213Asn
Tyr213Asp
Tyr213Cys
Tyr213Gln
Tyr213Glu
Tyr213Gly
Tyr213His
Tyr213Ile
Tyr213Leu
Tyr213Met
Tyr213Pro
Tyr213Ser
Tyr213Thr
Tyr213Val
Pro214Asn
Pro214Asp
Pro214Gln
Pro214Glu
Pro214Gly
Pro214Ser
Thr215Asn
Thr215Asp
Thr215Gln
Thr215Glu
Thr215Gly
Thr215Pro
Thr215Ser
Ser216Asp
Ser216Glu
Thr217Asn
Thr217Asp
Thr217Gln
Thr217Glu
Thr217Gly
Thr217Pro
Thr217Ser
Tyr218Ala
Tyr218Asn
Tyr218Asp
Tyr218Cys
Tyr218Gln
Tyr218Glu
Tyr218Gly
Tyr218His
Tyr218Ile
Tyr218Leu
Tyr218Met
Tyr218Pro
Tyr218Ser
Tyr218Thr
Tyr218Val
Ala219Asn
Ala219Asp
Ala219Gln
Ala219Glu
Ala219Gly
Ala219His
Ala219Pro
Ala219Ser
Ala219Thr
Ser220Asp
Ser220Glu
Leu221Ala
Leu221Asn
Leu221Asp
Leu221Cys
Leu221Gln
Leu221Glu
Leu221Gly
Leu221His
Leu221Ile
Leu221Met
Leu221Pro
Leu221Ser
Leu221Thr
Leu221Val
Ser222Asp
Ser222Glu
Gly223Asn
Gly223Asp
Gly223Gln
Gly223Glu
Gly223Pro
Gly223Ser
Thr224Asn
Thr224Asp
Thr224Gln
Thr224Glu
Thr224Gly
Thr224Pro
Thr224Ser
表23环6-双突变变异体Ser207Glu + Thr215GlnPro205Gly + Ala219GlyThr217Asn + Thr224GlyGly206Gln + Pro214AspAla204Asn + Thr212SerPro205Gly + Ser220AspTyr213Gln + Tyr218GluPro205Gln + Leu221GluThr212Gln + Ser220AspTyr210Gly + Thr224GlyTyr213Gly + Ser216GluSer207Glu + Gly223AsnTyr210Ser + Tyr218LeuSer216Glu + Thr217SerSer216Glu + Thr224AsnPro205Asn + Ala219AsnThr217Gly + Leu221ThrThr215Glu + Ala219GlnThr217Asn + Tyr218GluAla204Asn + Gly206ProThr212Ser + Thr224GlnThr217Gly + Ser222GluTrp208Glu + Ile209AsnPro205Ser + Ala219GlyTyr213Ser + Ala219SerAla204Ser + Gly206SerAla204Gly + Thr215Glu
42Thr212Gln + Ala219GluAla204Gly + Trp208GlnAla204Thr + Ser216AspThr212Gln + Tyr218GlnSer207Glu + Leu221MetThr215Pro + Gly223GlnAla204Gly + Ser216AspTyr210Asp + Ala219HisIle209Gln + Tyr218AlaIle209Ala + Ala219ProAla204Asn + Ser222GluTyr210Asp + Tyr213AlaTyr218Leu + Thr224GluTyr218Asn + Ser220GluTrp208Met + Ala219ProPro205Gly + Ser222AspLeu221Val + Thr224GlnTrp208Ile + Pro214SerTrp208Asp + Thr212ProAla204Thr + Gly223GlnThr217Gln + Tyr218IleTyr213His + Ser216AspThr212Ser + Thr215AspThr217Asn + Leu221AsnPro214Asp + Thr217ProSer207Glu + Ala219HisThr217Asp + Tyr218HisTrp208Ala + Thr217SerPro205Gln + Ser220AspIle209Gly + Ser216GluTyr218Ser + Ser220GluThr215Asn + Ser216GluThr215Gly + Tyr218GlnThr217Ser + Leu221AlaAla219Pro + Leu221HisPro205Gln + Leu221MetTyr213Met + Leu221IleTrp208Asn + Thr212SerSer207Glu + Thr224GlyPro205Ser + Leu221CysAla204Gly + Tyr210ValTrp208Phe + Ser222AspIle209Thr + Ser222GluLeu221Ile + Ser222GluTyr213Pro + Thr217GluIle209Thr + Ser216GluTyr210Cys + Ser222AspLeu221Glu + Thr224SerTyr213Ala + Thr217ProTrp208Cys + Thr215GluAla204Ser + Thr217SerIle209His + Gly223SerAla204Asn + Trp208Tyr
43Pro205Gln + Ser207GluAla204Asn + Trp208MetTyr210Pro + Leu221AspTyr210Gln + Thr215SerAla204Pro + Ser216GluAla204Gly + Ser222AspSer216Asp + Leu221MetTyr210Ile + Ala219AsnPro214Glu + Thr224SerPro205Gln + Thr215SerAla204Gly + Pro205AsnTyr210Asp + Tyr218ValTrp208Ser + Thr215GlySer222Glu + Thr224SerTrp208Gln + Ser216AspSer207Asp + Gly223SerPro205Gly + Tyr210CysThr212Gly + Thr224AspSer220Glu + Gly223ProGly206Pro + Tyr210MetTyr210Ser + Ser222AspSer207Glu + Pro214GlnPro205Gly + Gly223AsnTyr213Ser + Pro214AspPro214Gln + Ser220AspAla204Thr + Ser222GluAla219Asn + Leu221AspGly206Ser + Thr215ProThr212Asn + Tyr218ValThr217Pro + Gly223AspGly206Pro + Tyr218AsnGly206Gln + Thr217AspTyr210Ala + Ser220AspPro214Asp + Tyr218ValLeu221His + Gly223AspPro214Gln + Gly223AspIle209Pro + Ser216AspIle209Thr + Gly223AspPro214Asn + Ala219AspTyr218Cys + Ser220GluSer207Glu + Tyr218AsnTyr218Gly + Leu221GlnIle209Thr + Tyr218ValPro214Glu + Gly223SerTyr213Met + Ala219ThrSer222Glu + Thr224AsnTrp208Asp + Ile209MetThr212Gln + Pro214GlnSer207Asp + Tyr213LeuThr212Gln + Gly223ProTyr210Leu + Ser216GluSer216Asp + Ala219ThrTyr213Gln + Thr217AspGly206Pro + Ser222GluAla204Thr + Ser220AspAla219Thr + Thr224ProTrp208Ile + Ala219GluIle209Gly + Thr215SerGly206Gln + Tyr210GluPro214Gln + Ser220GluSer207Glu + Thr217AsnAla204Pro + Gly223AspIle209Cys + Ser222AspPro214Gly + Gly223ProIle209Met + Thr224ProPro214Glu + Thr217SerPro205Ser + Ser207AspTyr210Thr + Thr212GlnGly206Gln + Tyr218ProTyr218Val + Ser222GluLeu221Asn + Gly223GluPro205Gln + Tyr218ValPro205Gln + Thr217AsnTrp208His + Ile209AlaPro205Ser + Gly223GlnPro214Ser + Thr217GlyTyr218His + Ser222AspThr212Gln + Gly223SerThr215Glu + Ala219ThrTyr213Gly + Tyr218AspThr212Gln + Tyr213GlnTyr210Ile + Thr215AspAla204Asn + Gly223ProSer207Glu + Ile209AsnThr215Gln + Ala219AspSer220Asp + Gly223ProPro205Ser + Tyr213AlaTrp208Gly + Ser222GluThr215Glu + Tyr218AlaAla204Gln + Ala219GlnPro205Asn + Tyr213ThrTyr218Thr + Thr224GlnSer222Asp + Thr224SerAla204Pro + Tyr213CysGly206Gln + Leu221GlySer216Glu + Thr224SerSer207Asp + Ala219AsnTrp208Leu + Ile209HisPro205Ser + Tyr213ValThr212Gly + Thr215Asp
表24
环6-三突变变异体Gly206Ser+Thr212Ser+Ala219GluAla204Asn + Thr212Ser + Ser216GluPro205Gln + Leu221Glu + Gly223AsnTrp208Thr + Ile209Cys + Thr224AspIle209Met + Thr212Gln + Ser220AspTyr210Gly + Tyr213Gly + Thr224GlySer207Glu + Tyr210Ser + Gly223AsnTyr213Gly + Ser216Asp + Thr217SerPro205Asn + Ser216Glu + Thr224AsnThr215Glu + Ala219Gln + Leu221ThrAla204Asn + Thr217Asn + Tyr218GluThr212Ser + Thr217Gly + Thr224GlnAla204Ser + Tyr218His + Ala219GlnPro205Ser + Tyr213Ser + Leu221AsnAla204Ser + Gly206Ser + Ala219SerAla204Gly + Trp208Gln + Ser216AspSer207Glu + Tyr218Gln + Leu221MetAla204Gly + Thr215Pro + Gly223GlnIle209Gln + Tyr218Ala + Ala219ProAla204Asn + Trp208Met + Ser222GluTrp208Met + Ala219Pro + Ser222AspPro205Gly + Leu221Val + Thr224GlnAla204Thr + Thr212Pro + Gly223GlnGly206Gln + Ile209Gln + Thr212GlnGly206Pro + Ser220Glu + Leu221SerGly206Gln + Tyr210Gly + Ser222AspAla204Gly + Tyr213Thr + Pro214GlnAla204His + Thr212Gln + Thr224GlnGly206Gln + Pro214Asp + Tyr218SerThr212Asn + Ser216Asp + Thr217SerSer207Asp + Tyr213Ile + Thr224ProPro205Gln + Tyr213Met + Leu221HisTrp208Asn + Thr212Ser + Gly223AspSer207Glu + Leu221Gly + Thr224GlyPro205Ser + Tyr210Val + Leu221CysPro205Gly + Ile209His + Tyr213ThrIle209Pro + Tyr210Asn + Ser220AspPro205Gly + Thr212Pro + Thr224GlyTrp208Cys + Thr217Asp + Ala219AsnPro205Gly + Leu221Cys + Gly223ProAla204His + Ser216Glu + Leu221SerThr217Asp + Gly223Ser + Thr224GlyPro205Gln + Trp208Gln + Ile209AlaThr212Gly + Thr217Gly + Leu221HisGly206Asn + Thr212Gly + Tyr213GlyGly206Asn + Ser207Glu + Thr212SerThr212Gln + Leu221Glu + Gly223SerTyr210His + Thr215Asp + Thr224ProPro214Gln + Thr215Pro + Ser216GluPro205Gly + Tyr213Val + Gly223AspTrp208Phe + Thr215Asn + Thr224GlyTyr213Met + Ala219Pro + Ser220GluTrp208Phe + Ile209Ser + Tyr213GlnGly206Ser + Ser216Glu + Tyr218ProAla204Gln + Tyr210His + Thr224GluPro205Ser + Thr212Pro + Ala219SerPro205Gln + Tyr210Met + Thr217ProAla204Gly + Ile209His + Ser216GluThr212Asn + Thr217Ser + Thr224AsnGly206Pro + Tyr210Gly + Gly223GlnPro205Gln + Thr217Asp + Ala219ThrPro205Ser + Ile209Met + Ser216AspThr215Asn + Ser216Glu + Thr224SerPro205Gln + Leu22lThr + Ser222GluTyr210Cys + Ala219Asn + Leu221ThrAla204Ser + Gly206Ser + Ala219GlnIle209Pro + Ser216Asp + Leu221CysTrp208Thr + Tyr210Val + Thr212SerThr212Gly + Pro214Asp + Gly223GlnPro214Gln + Thr215Ser + Thr224GluThr212Ser + Pro214Gln + Leu221HisTrp208Gly + Ser220Glu + Gly223SerPro205Ser + Ser220Asp + Leu221ThrGly206Asn + Thr215Pro+ Leu221AlaTyr213Leu + Leu221His + Gly223AspSer207Asp + Thr212Gly + Tyr218CysGly206Gln + Ile209Val + Ala219ThrTrp208Ile + Tyr218Ile + Gly223AsnAla204His + Ile209Gly + Ser216AspTrp208Leu + Thr212Gly + Ala219SerThr212Gln + Pro214Glu + Ala219ThrPro205Gln + Ser207Asp + Thr212ProPro205Ser + Tyr210Pro + Tyr218IleLeu221Ala + Gly223Asn + Thr224GluAla204Thr + Gly206Gln + Ser207AspSer216Glu + Ala219Gln + Gly223GlnPro205Gly + Trp208Leu + Leu221GlnThr217Gln + Gly223Gln + Thr224GluThr212Gly + Thr217Ser + Tyr218AlaGly206Asn + Gly223Asp + Thr224AsnAla204Gly + Tyr210Cys + Tyr213ThrGly206Ser + Thr212Asn + Thr215GlyAla204Asn + Thr212Asn + Ser216GluGly206Gln + Tyr218Cys + Gly223GluAla204Ser + Pro205Ser + Gly206AsnIle209Gly + Leu221Val + Thr224ProGly206Asn + Trp208His + Thr217GlyGly206Ser + Trp208Leu + Ser222AspPro205Gly + Thr212Gln + Ser222AspAla204Thr + Tyr213Gln + Thr224ProThr212Ser + Thr217Ser + Gly223AspPro205Ser + Tyr213Ile + Ser222GluIle209Pro + Tyr210Asp + Ala219ProGly206Gln + Thr215Pro + Ser220GluTrp208Cys + Tyr210Leu + Thr215GlyTrp208Tyr + Thr212Gln + Ser222GluGly206Asn + Ser222Asp + Gly223AsnPro205Gly + Gly206Asn + Leu221CysPro205Gln + Tyr213Ile + Ser216GluAla204His + Tyr210His + Ser220AspAla204Thr + Thr212Asn + Ala219AspSer207Glu + Tyr210Ala + Thr212GlyTyr210His + Thr217Gly + Leu221MetAla204Thr + Ile209Met + Ser222GluTrp208Pro + Ala219Pro + Ser222AspAla204His + Ile209Ser + Ser220GluPro214Ser + Leu221Glu + Gly223GlnGly206Pro + Leu221Met + Gly223AspAla204His + Tyr213Thr + Ser222AspTyr213Ala + Leu221Met + Ser222AspTrp208Leu + Thr212Ser + Pro214AspTyr213Ile + Ser216Glu + Thr217ProPro205Gln + Pro214Gln + Thr224GlyTyr213Cys + Thr217Gly + Ser220GluGly206Asn + Thr212Gly + Tyr218ProGly206Pro + Ser216Glu + Gly223ProThr212Gln + Thr215Gly + Ser222GluAla204Pro + Thr217Asp + Thr224SerPro205Ser + Thr212Pro + Ala219AspIle209Cys + Pro214Ser + Thr215AspIle209Cys + Ser220Asp + Gly223GlnThr217Gly + Ser220Asp + Leu221GlnAla204His + Pro205Asn + Trp208GluAla204Gln + Thr212Ser + Thr215AspPro214Asn + Ser220Glu + Leu221GlyIle209Gly +Thr215Asn + Ala219ProSer207Glu + Thr217Gln + Thr224SerAla204Gly + Thr215Asn + Gly223ProIle209Ser + Tyr213Ile + Leu221MetTyr210Thr + Pro214Ser + Ser222AspTyr210Leu + Thr212Ser + Leu221AsnGly206Asn + Pro214Asp + Tyr218MetGly206Asn + Pro214Gln + Ala219AsnSer207Glu + Pro214Ser + Ala219HisAla204Asn + Trp208Ala + Ile209GlnThr217Glu + Tyr218Leu + Gly223AsnTrp208Pro + Pro214Gly + Leu221AlaGly206Pro + Thr215Ser + Leu221CysAla204Ser + Ile209Val + Thr224AspTrp208Ile + Tyr210Pro + Thr224AspThr212Gln + Thr215Asn + Thr217AspTyr210Ser + Tyr213Cys + Thr217AsnThr212Gln + Ser222Asp + Gly223GlnTyr210Asn + Thr217Glu + Tyr218MetPro214Asn + Ala219Gln + Ser220GluPro205Ser + Trp208Met + Leu221AspAla204Asn + Tyr210Cys + Tyr213AsnTyr218Leu + Leu221His + Ser222GluAla204His + Tyr218Ser + Ser222AspGly206Pro + Tyr213Asn + Pro214AsnGly206Pro + Thr215Gln + Tyr218SerAla204Pro + Ile209His + Leu221ThrGly206Gln + Thr212Gly + Gly223GluThr215Gly + Tyr218Ala + Thr224ProPro205Gln + Tyr213Gly + Gly223GluAla204Gly + Tyr213Asn + Leu221ThrTrp208Gly + Ile209His + Thr215SerAla204His + Ser207Glu + Tyr213CysTrp208His + Tyr210His + Ser222AspPro205Gly + Tyr210Asp + Leu221GlnPro205Asn + Tyr210Ala + Pro214SerThr212Pro + Thr215Gln + Thr224AspAla204Thr + Tyr213Val + Pro214GluAla204Gly + Tyr210Pro + Pro214GluPro205Gly + Trp208His + Tyr218GlyGly206Ser + Ala219Gln + Gly223SerTyr213Thr + Tyr218Cys + Ser220GluThr215Pro + Ser216Glu + Leu221AsnPro205Gly + Thr212Asn + Thr224AspPro205Gly + Ser207Asp + Thr212Ser
表25
环6-四突变变异体Gly206Asn + Thr215Gln + Tyr218Ile + Leu221IleTyr210Gly + Thr212Gln + Tyr213Gly + Thr224GlyPro205Gly + Gly206Pro + Trp208Asn + Thr224SerIle209Thr + Pro214Asp + Thr217Asn + Tyr218CysAla204Ser + Tyr218His + Ala219Gln + Leu221AsnAla204Ser + Pro205Ser + Tyr213Ser + Ala219SerAla204Gly + Thr212Gln + Ser216Asp + Gly223SerSer207Glu + Thr215Pro + Tyr218Gln + Leu221MetAla204Gly + Ser216Asp + Ala219His + Gly223GlnIle209Gln + Tyr210Asp + Tyr218Ala + Ala219ProTrp208Met + Tyr218Leu + Ala219Pro + Ser222GluPro205Gly + Leu221Val + Ser222Asp + Thr224GlnAla204Thr + Thr212Pro + Ser222Glu + Gly223GlnGly206Gln + Ile209Gln + Thr212Gln + Leu221HisAla204Ser + Gly206Pro + Ser220Asp + Leu221SerAla204Gly + Thr212Gln + Tyr213Thr + Thr224GlnTyr210Ala + Pro214Ser + Tyr218Val + Ser222AspAla204Gln + Pro205Ser + Gly206Ser + Ser222GluTrp208Cys + Pro214Gln + Ala219Thr + Thr224AspAla204Gly + Gly206Ser + Tyr210Asp + Leu221GlyAla204Gln + Gly206Asn + Tyr210Gln + Ala219AspPro205Ser + Gly206Pro + Ile209Val + Tyr213ProGly206Pro + Pro214Glu + Thr215Pro + Leu221MetSer207Asp + Trp208Met + Tyr218Val + Leu221ProTyr210Gly + Thr212Pro + Pro214Gly + Ser222AspGly206Pro + Thr212Gln + Thr217Ser + Ser222AspAla204Pro + Trp208Met + Ile209Val + Ser216GluTrp208Ser + Tyr218His + Ala219His + Gly223Pro
49Ala204Gln + Trp208Ala + Tyr210Asn + Ser222GluGly206Gln + Ser220Asp + Leu221Ala + Thr224SerIle209Gln + Thr212Ser + Ala219Gln + Ser220GluPro205Asn + Thr212Gly + Tyr218Ala + Ser222GluPro205Gln + Trp208Cys + Tyr213Cys + Pro214AspPro205Gly + Thr215Gln + Thr217Pro + Gly223SerAla204His + Gly206Pro + Ser216Glu + Leu221SerSer207Asp + Leu221Gly + Gly223Gln + Thr224AsnTyr210His + Thr212Ser + Leu221Glu + Gly223SerPro205Gly + Thr215Asn + Gly223Asp + Thr224GlyTrp208Phe + Tyr213Met + Thr215Pro + Ser220GluGly206Ser + Trp208Phe + Ile209Ser + Tyr213GlnAla204Gln + Pro205Ser + Thr212Pro + Ala219SerAla204Gly + Tyr210Met + Ser216Glu + Thr217ProGly206Pro + Thr212Asn + Thr217Gln + Thr224AsnPro205Ser + Ile209Met + Thr215Asn + Thr224SerTyr210Thr + Thr212Pro + Tyr218Asn + Ser220GluTrp208Thr + Tyr210Val + Thr212Ser + Leu221CysThr212Gly + Pro214Asp + Thr215Ser + Gly223GlnSer207Glu + Thr212Ser + Pro214Gln + Leu221ProPro205Asn + Gly206Gln + Ser207Asp + Leu221ThrPro205Ser + Thr215Pro + Ser220Asp + Leu221CysGly206Asn + Tyr213Leu + Leu221Ala + Gly223AspSer207Asp + Thr212Gly + Pro214Asn + Thr224AsnAla204Pro + Ser207Glu + Ile209Val + Ala219ThrGly206Gln + Trp208Ile + Tyr218Ile + Gly223AsnAla204His + Trp208Leu + Ile209Gly + Ser216AspAla204Gln + Thr212Gly + Pro214Glu + Ala219SerAla204Asn + Pro205Gln + Ser207Asp + Thr212ProPro205Gly + Trp208Leu + Ser216Glu + Ala219GlnTrp208Tyr + Thr217Gln + Leu221Gln + Thr224GluThr212Gly + Thr217Ser + Tyr218Ala + Gly223AspAla204Gly + Gly206Asn + Thr215Asn + Gly223AspGly206Ser + Tyr210Cys + Tyr213Thr + Thr215GlyAla204Gln + Pro205Ser + Gly206Asn + Ala219AspAla204Ser + Ile209Gly + Leu221Val + Thr224ProGly206Asn + Ser207Glu + Trp208His + Thr217GlyGly206Ser + Trp208Pro + Ala219Thr + Ser222AspAla204Thr + Tyr213Gln + Thr217Ser + Thr224ProPro205Ser + Thr212Ser + Tyr213Ile + Gly223AspGly206Gln + Ile209Asn + Thr215Pro + Ser220GluTrp208Cys + Thr212Gln + Thr215Gly + Ser222GluGly206Asn + Trp208Tyr + Ser222Glu + Gly223AsnPro205Gly + Gly206Asn + Leu221Cys + Ser222AspPro205Gln + Tyr210His + Tyr213Ile + Ser220AspAla204His + Trp208Asn + Thr212Asn + Ala219AspAla204His + Gly206Pro + Leu221Met + Gly223AspAla204Ser + Tyr213Thr + Leu221Met + Ser222AspTyr213Ile + Pro214Gln + Ser216Glu + Thr217ProPro205Gln + Tyr213Cys + Ser220Glu + Thr224GlyGly206Asn + Tyr213Asn + Tyr218Pro + Ser220GluPro205Ser + Gly206Pro + Tyr218Asp + Gly223ProAla204Pro + Pro205Ser + Thr217Asp + Thr224SerPro205Asn + Trp208Ile + Thr212Gln + Thr217GlyIle209Cys + Pro214Ser + Ser220Asp + Gly223GlnAla204His + Pro205Asn + Trp208Glu + Tyr210HisIle209Gly + Thr215Asn + Thr217Asn + Ala219ProPro205Gly + Pro214Asn + Tyr218Ile + Ser222AspAla204Ser + Pro205Ser + Thr212Asn + Tyr213IlePro205Gln + Ser207Glu + Tyr210Pro + Thr215SerPro205Gln + Ser207Glu + Thr212Gln + Tyr213SerAla204Ser + Ile209Asn + Tyr213Cys + Ser216AspTrp208Ala + Ile209Gln + Thr217Glu + Gly223AsnTrp208Pro + Pro214Gly + Tyr218Leu + Leu221AlaGly206Pro + Thr215Ser + Ser216Glu + Leu221CysAla204Ser + Trp208Ile + Ile209Val + Thr224AspTyr210Pro + Thr212Gln + Thr215Asn + Thr217AspTyr210Ser + Tyr213Cys + Pro214Asn + Thr217AsnThr212Gln + Tyr218Met + Ser222Asp + Gly223GlnPro205Ser + Trp208Met + Ala219Gln + Leu221AspAla204Asn + Tyr210Cys + Tyr213Asn + Tyr218LeuAla204His + Pro205Gly + Tyr218Ser + Ser222AspGly206Pro + Tyr213Asn + Pro214Asn + Tyr218SerAla204Pro + Ile209His + Thr215Gln + Leu221ThrAla204Asn + Gly206Gln + Thr212Gly + Gly223GluThr215Gly + Tyr218Ala + Gly223Glu + Thr224ProPro205Gln + Tyr213Gly + Ser220Asp + Leu221ThrPro205Gly + Trp208His + Tyr210His + Leu221GlnTyr210Leu + Thr212Pro + Tyr218Gln + Ala219GluGly206Gln + Thr212Gln + Tyr218His + Gly223AspTrp208Ile + Tyr213His + Thr215Ser + Ser222GluTrp208Val + Pro214Asn + Thr215Gly + Leu221AspPro205Asn + Tyr213Met + Pro214Ser + Thr224SerPro205Gly + Trp208Met + Ile209Leu + Tyr213MetPro205Gly + Thr217Glu + Tyr218Leu + Ala219HisTrp208Val + Tyr213Gly + Ser216Asp + Leu221ThrTyr210Gln + Thr212Pro + Tyr218Gly + Gly223GluPro205Gly + Trp208Asp + Ile209His + Tyr210AlaTrp208Ala + Ile209Leu + Tyr210Glu + Tyr213CysGly206Asn + Trp208Leu + Tyr213Val + Ser216AspAla204Pro + Pro205Gly + Thr217Glu + Leu221AlaTrp208Asn + Thr212Asn + Tyr213His + Ser216GluAla204His + Thr212Gly + Ala219Gln + Leu221ThrGly206Gln + Thr212Pro + Tyr213Met + Thr224ProTrp208Thr + Ile209Gln + Tyr213Ser + Ser222AspAla204Asn + Ile209Asn + Tyr213Pro + Leu221ProPro205Gly + Tyr210His + Tyr218Ile + Leu221AsnAla204Pro + Tyr210Cys + Pro214Gln + Thr215ProPro205Ser + Tyr213Ser + Ser216Asp + Thr217GlyPro205Asn + Trp208Ser + Ala219Glu + Ser220AspTyr210Ala + Tyr218Met + Ala219Glu + Ser220AspAla204Gly + Pro205Gln + Pro214Asp + Thr215AspTyr210Ser + Thr215Asn + Ser222Glu + Gly223GluGly206Pro + Tyr213Val + Ser222Glu + Gly223AspPro214Ser + Leu221Glu + Ser222Asp + Gly223GlnPro205Gln + Ser216Asp + Thr217Glu + Ala219ThrTrp208Leu + Thr212Gln + Ser220Glu + Leu221GluGly206Gln + Ser220Asp + Leu221Asp + Gly223AsnThr217Asp + Tyr218Asp + Gly223Ser + Thr224GlyPro205Gln + Ile209Ala + Thr217Glu + Tyr218GluPro214Gln + Thr215Asp + Ser216Glu + Thr224ProAla204His + Ser207Glu + Tyr213Cys + Ser222AspPro205Gly + Ser207Glu + Thr212Gln + Ser222AspSer207Glu + Tyr210Ser + Tyr218Leu + Ser222GluSer207Glu + Tyr218Cys + Ser222Glu + Thr224ProPro205Ser + Trp208Glu + Leu221Glu + Ser222GluTrp208Glu + Ile209Asn + Thr217Gly + Ser222GluSer216Glu + Thr217Asp + Tyr218Glu + Thr224GlyTrp208Asp + Tyr218Leu + Ala219Thr + Leu221GluAla204His + Thr212Gly + Thr215Glu + Thr217GluPro214Asp + Ser216Asp + Ala219His + Gly223AsnTyr210Pro + Pro214Asp + Ser216Glu + Tyr218IlePro205Gln + Tyr210Leu + Pro214Glu + Thr217AspAla204Ser + Ser207Glu + Pro214Ser + Gly223AspSer207Asp + Thr212Pro + Thr217Gly + Gly223AspSer207Glu + Pro214Asn + Ser220Glu + Ser222AspPro205Ser + Thr217Pro + Ala219Glu + Leu221GluTrp208Gln + Tyr210Glu + Thr212Asn + Leu221GluTrp208Gln + Thr215Pro + Ser220Glu + Ser222GluGly206Pro + Thr217Gly + Ser220Asp + Ser222GluGly206Gln + Tyr210Gly + Ser220Glu + Ser222AspSer207Glu + Thr212Asn + Tyr218Pro + Leu221AspAla204Asn + Ser207Glu + Trp208His + Leu221AspTyr213Ile + Tyr218Leu + Ser222Asp + Thr224GluGly206Gln + Tyr210Met + Ser222Asp + Thr224GluIle209Ser + Tyr210His + Ser222Glu + Thr224AspAla204Ser + Tyr218Asp + Ala219Pro + Ser220GluGly206Gln + Thr217Ser + Tyr218Glu + Ser220GluPro214Asp + Tyr218Asp + Gly223Ser + Thr224GlyGly206Gln + Tyr210Gly + Pro214Glu + Tyr218GluTrp208Asp + Thr217Gln + Ser220Asp + Gly223AspSer207Glu + Tyr210Ala + Thr215Asn + Ser220AspSer207Glu + Thr217Gly + Ser220Asp + Leu221GlnSer207Glu + Thr217Gly + Tyr218Asn + Ser220AspPro205Asn + Ser207Asp + Thr212Gly + Ser220AspSer207Glu + Thr212Asn + Ala219Gly + Ser220AspGly206Pro + Ser207Glu + Trp208Asp + Thr224AspSer207Asp + Trp208Thr + Tyr218Gln + Thr224GluAla204Thr + Ser207Asp + Leu221Ala + Thr224GluSer207Asp + Ala219Glu + Ser220Asp + Gly223SerSer207Glu + Tyr210Glu + Thr215Gln + Thr224AsnAla204Gln + Tyr210Glu + Thr212Asn + Ser222Asp
表26
环6-五取代变异体Gly206Asn + Tyr210Gly + Thr212Gln + Tyr218Ile +
Thr224GlyAla204Asn + Pro205Gly + Trp208Glu + Thr215Asn +
Gly223ProPro205Gln + Gly206Pro + Trp208Asn + Thr212Gln +
Thr224SerAla204Ser + Pro205Ser + Tyr213Ser + Thr215Ser +
Ala219GlyAla204Gly + Gly206Gln + Thr212Pro + Ser220Asp +
Leu221SerPro214Ser + Thr215Gln + Tyr218Val + Ser222Asp +
Thr224AsnAla204Pro + Ser207Asp + Tyr210Cys + Thr212Pro +
Thr215ProGly206Asn + Pro214Gln + Ala219Gly + Gly223Asn +
Thr224AspAla204Gln + Pro205Ser + Gly206Ser + Tyr210Gln +
Ala219AspGly206Pro + Ser207Asp + Trp208Met + Thr215Pro +
Leu221ProAla204Asn + Pro205Gln + Tyr210Cys + Thr212Asn +
Leu221SerAla204Asn + Gly206Gln + Trp208Thr + Ile209Ser +
Ser222AspPro205Gln + Tyr213Leu + Thr217Glu + Tyr218Met +
Leu221GlnAla204Pro + Trp208Met + Ile209Val + Tyr210Leu +
Ser216GluTrp208Ser + Ile209Ser + Tyr218His + Ala219His +
Gly223ProTrp208Pro + Ile209Thr + Thr212Gly + Thr215Gly +
Thr224GluIle209Val + Pro214Ser + Ser216Asp + Tyr218Met +
Leu221MetGly206Gln + Ile209Gln + Ser220Asp + Leu221Gly +
Thr224SerPro205Gln + Trp208Cys + Thr212Asn + Tyr213Cys +
Pro214AspPro205Gln + Trp208Gln + Thr217Asp + Gly223Ser +
Thr224GlySer207Glu + Thr212Ser + Thr217Gly + Tyr218Gln +
Leu221HisTyr210His + Thr212Ser + Leu221Glu + Gly223Ser +
Thr224ProTrp208Phe + Tyr213Met + Thr215Pro + Ser220Glu +
Thr224GlyGly206Ser + Trp208Phe + Ile209Ser + Tyr213Cys +
Ser216GluAla204Gln + Pro205Ser + Tyr210His + Tyr218Pro +
Thr224GluPro205Gln + Gly206Gln + Leu221His + Ser222Asp +
Gly223GlnAla204Gly + Ile209His + Tyr210Met + Ser216Glu +
Thr217ProGly206Pro + Ile209Ala + Tyr210Gly + Leu221Asp +
Gly223GlnGly206Asn + Tyr210Met + Thr215Glu + Gly223Asn +
Thr224GlyGly206Gln + Ile209Pro + Ser216Asp + Leu221Cys +
Gly223ProTrp208Thr + Tyr210Val + Thr212Ser + Pro214Asp +
Gly223GlnThr212Gly + Pro214Gln + Thr215Ser + Leu221Gly +
Thr224GluAla204Thr + Thr212Ser + Thr217Ser + Gly223Asp +
Thr224ProPro205Ser + Ile209Pro + Tyr213Ile + Ala219Pro +
Ser222GluTrp208Gly + Thr212Gln + Tyr218Gly + Ser222Glu +
Gly223SerGly206Pro + Ile209Pro + Tyr213Ser + Thr217Gly +
Tyr218ValPro205Gln + Tyr213Cys + Pro214Gln + Ser220Glu +
Thr224GlyGly206Asn + Thr212Gly + Tyr213Asn + Tyr218Pro +
Ser220GluAla204Pro + Pro205Ser + Thr212Gln + Thr217Asp +
Thr224SerPro205Asn + Trp208Ile + Thr212Gln + Pro214Ser +
Thr217GlyIle209Cys + Thr217Gly + Ser220Asp + Leu221Gln +
Gly223GlnIle209Thr + Thr215Gly + Ser216Glu + Tyr218Leu +
Ala219ProIle209Met + Pro214Gly + Thr217Gly + Tyr218Cys +
Leu221MetPro205Gly + Pro214Asn + Tyr218Ile + Ala219Gly +
Ser222AspTyr210Thr + Thr212Ser + Pro214Ser + Leu221Asn +
Ser222AspGly206Asn + Tyr210Ile + Pro214Gln + Ala219Asn +
Leu221ValPro205Asn + Thr215Ser + Ala219Ser + Leu221Ala +
Thr224AspGly206Pro + Pro214Asn + Thr215Gln + Tyr218Ser +
Leu221ThrAla204Pro + Gly206Gln + Ile209His + Thr212Gly +
Gly223GluPro205Gln + Tyr213Gly + Tyr218Ala + Gly223Glu +
Thr224ProPro205Ser + Pro214Gly + Thr217Pro + Ala219Thr +
Leu221GluAla204Pro + Pro205Asn + Ser207Asp + Tyr210Ala +
Pro214SerGly206Ser + Tyr213Thr + Ala219Gln + Ser220Glu +
Gly223SerPro205Gly + Thr212Asn + Thr215Pro + Ser216Glu +
Leu221AsnSer207Glu + Ile209Ala + Thr212Asn + Thr215Gln +
Tyr218MetTyr213His + Thr215Gln + Thr217Asn + Ala219Glu +
Leu221SerGly206Gln + Tyr213Asn + Thr215Ser + Tyr218Ala +
Leu221AlaTrp208Met + Tyr210Met + Tyr218Gln + Ser220Glu +
Thr224SerAla204Thr + Pro205Ser + Ile209Leu + Thr217Asn +
Thr224ProPro205Gly + Ile209Leu + Thr215Asn + Ser216Glu +
Gly223SerIle209Ser + Tyr213Leu + Thr217Asp + Tyr218Asn +
Ala219HisAla204Ser + Pro205Ser + Ser207Glu + Leu221Val +
G1y223ProAla204His + Thr212Gly + Ala219Gln + Leu221Thr +
Gly223GluPro205Ser + Gly206Gln + Thr212Pro + Gly223Pro +
Thr224ProAla204Asn + Trp208Thr + Tyr213Pro + Leu221Pro +
Ser222AspPro205Gly + Ile209Met + Tyr218Met + Leu221Val +
Gly223AsnThr212Gly + Tyr218Ile + Ala219Asn + Ser220Glu +
Thr224ProAla204Gln + Pro205Gln + Gly206Gln + Ile209Met +
Ser216AspAla204Thr + Thr212Gly + Ala219Asn + Gly223Asp +
Thr224AsnAla204Pro + Trp208His + Tyr213Leu + Thr217Ser +
Ser222GluPro205Ser + Ser207Asp + Ile209Asn + Tyr213Met +
Thr217GlnGly206Pro + Trp208Ser + Thr212Gly + Tyr213Cys +
Gly223GluAla204Asn + Gly206Pro + Trp208Val + Pro214Asn +
Tyr218GluIle209Gly + Tyr210Ala + Tyr218Pro + Gly223Ser +
Thr224SerPro205Gln + Tyr213His + Tyr218Met + Ser220Glu +
Leu221GlyAla204Asn + Tyr210Cys + Thr212Gln + Tyr218Leu +
Ser222AspGly206Ser + Ile209Leu + Thr212Gly + Pro214Gln +
Thr215GluSer207Asp + Pro214Ser + Thr215Asn + Thr217Asn +
Leu221SerPro205Gly + Trp208Thr + Thr217Gly + Ser220Glu +
Gly223SerGly206Ser + Trp208Glu + Tyr210Gln + Thr217Gln +
Leu221ProGly206Gln + Ser207Glu + Thr212Asn + Pro214Ser +
Leu221AlaAla204Ser + Trp208Leu + Ile209Asn + Thr215Asp +
Thr224AsnGly206Ser + Ile209Pro + Thr217Ser + Ser220Glu +
Gly223GlnPro205Gln + Trp208Pro + Thr212Pro + Pro214Glu +
Ala219SerGly206Pro + Ser207Glu + Tyr210Met + Thr212Gly +
Tyr213LeuAla204His + Tyr210Pro + Tyr213Ala + Thr217Glu +
Thr224SerTrp208Cys + Ile209Met + Tyr210Ala + Tyr213Met +
Ala219AsnTrp208Thr + Tyr210Ala + Tyr218Met + Ala219Glu +
Ser220AspTrp208Asn + Ile209Pro + Ala219Asp + Ser220Asp +
Gly223SerPro205Ser + Tyr213Thr + Pro214Asp + Thr215Asp +
Tyr218ValIle209Ser + Tyr213Pro + Leu221Met + Ser222Asp +
Gly223AspAla204Thr + Thr217Gln + Tyr218Ile + Ser222Glu +
Gly223AspPro205Ser + Tyr210Ala + Thr215Asn + Ser222Glu +
Gly223GluTyr210Pro + Thr212Pro + Leu221Asn + Ser222Asp +
Gly223AspTrp208Pro + Ile209Pro + Tyr213Ile + Ser222Asp +
Gly223AspGly206Gln + Tyr213Gly + Thr217Gly + Ser222Glu +
Gly223GluTrp208Ser + Ile209Pro + Thr212Pro + Ser216Glu +
Thr217AspGly206Asn + Tyr218Met + Ala219His + Ser220Glu +
Leu221AspIle209Ala + Thr215Glu + Ser216Glu + Gly223Gln +
Thr224AsnIle209Gly + Pro214Ser + Thr215Glu + Ser216Asp +
Tyr218IleAla204Ser + Trp208Val + Ile209Ser + Thr215Glu +
Ser216AspThr212Pro + Thr215Asp + Ser216Glu + Thr217Glu +
Thr224SerAla204His + Ser207Glu + Trp208His + Tyr210His +
Ser222AspSer207Glu + Tyr213Asn + Leu221Thr + Ser222Asp +
Thr224GlyGly206Ser + Ser207Glu + Trp208Pro + Ala219Thr +
Ser222AspPro205Gly + Ser207Glu + Thr212Gln + Tyr213Gln +
Ser222AspPro205Ser + Ser207Glu + Tyr213His + Thr217Pro +
Ser222GluGly206Pro + Ser207Asp + Tyr213Ile + Tyr218Asn +
Ser222GluGly206Pro + Trp208Glu + Tyr210Cys + Pro214Gln +
Ser220AspTyr210Glu + Tyr213Gly + Tyr218Leu + Ala219Asp +
Thr224SerGly206Pro + Ser207Asp + Leu221Asp + Ser222Glu +
Thr224GlnTyr213Ser + Pro214Ser + Thr215Glu + Thr217Asp +
Tyr218AlaAla204Asn + Tyr210Gln + Thr212Asn + Pro214Glu +
Ser216AspPro205Gln + Gly206Pro + Pro214Glu + Ser216Asp +
Thr224SerSer207Asp + Thr212Pro + Thr215Gly + Thr217Gly +
Gly223AspSer207Glu + Tyr210Val + Pro214Asn + Leu221Thr +
Gly223GluSer207Asp + Ile209Thr + Tyr210Met + Thr212Pro +
Gly223AspSer207Glu + Tyr210Ser + Tyr218Leu + Ser220Asp +
Ser222GluSer207Asp + Thr217Gln + Ser220Asp + Ser222Asp +
Thr224AsnTrp208Thr + Thr217Glu + Tyr218Ser + Ala219Glu +
Leu221CysPro205Gly + Thr212Ser + Ala219Gln + Ser220Glu +
Ser222GluGly206Pro + Trp208His + Tyr210Gly + Ser220Glu +
Ser222AspAla204Gln + Gly206Ser + Trp208Gln + Ser220Glu +
Ser222GluAla204Ser + Tyr213Thr + Ser220Asp + Leu221Met +
Ser222AspThr212Gly + Thr215Asp + Ser216Asp + Thr217Gly +
Tyr218AspAla204Asn + Tyr210Glu + Tyr213Ala + Ser220Glu +
Ser222GluAla204His + Gly206Pro + Ser216Glu + Tyr218Asp +
Leu221SerGly206Pro + Ile209Ser + Tyr210His + Ser222Glu +
Thr224AspTrp208Met + Tyr218Leu + Ala219Pro + Ser222Glu +
Thr224GluTrp208Val + Tyr210Leu + Thr212Asn + Tyr218Asp +
Ser220GluAla204Ser + Tyr213Gln + Tyr218Asp + Ala219Pro +
Ser220GluThr212Asn + Tyr213Gln + Thr217Ser + Tyr218Glu +
Ser220GluPro214Ser + Leu221Glu + Ser222Asp + Gly223Ser +
Thr224AspPro205Gly + Tyr210Cys + Ala219Asp + Ser220Glu +
Ser222AspGly206Gln + Tyr210Gly + Pro214Glu + Thr217Pro +
Tyr218GluTrp208Asp + Pro214Ser + Thr217Gln + Ser220Asp +
Gly223AspSer207Glu + Tyr210Ala + Thr215Asn + Ser220Asp +
Leu221AlaSer207Asp + Trp208Cys + Ile209Ala + Thr215Asn +
Ser220GluSer207Asp + Tyr210Met + Tyr218Asn + Ser220Glu +
Leu221CysSer207Glu + Tyr210Pro + Thr215Gln + Tyr218Cys +
Ser220GluSer207Glu + Thr212Ser + Pro214Gln + Ser220Asp +
Leu221ProPro205Asn + Ser207Asp + Thr215Pro + Ser220Asp +
Leu221ThrTrp208Glu + Ile209Asn + Thr217Gly + Ala219Asp +
Ser222GluPro205Asn + Ser207Glu + Tyr213Leu + Ser220Asp +
Gly223GluAla204Ser + Pro205Gly + Ser207Asp + Thr212Ser +
Thr224AspAla204Pro + Gly206Gln + Ser207Glu + Tyr218Asn +
Thr224GluGly206Ser + Ser207Glu + Tyr210Met + Tyr213Cys +
Thr224AspPro205Asn + Ser207Glu + Ile209Val + Tyr213Pro +
Thr224GluSer207Asp + Ile209Val + Tyr213Ala + Gly223Gln +
Thr224GluPro205Asn + Ser207Glu + Ala219Glu + Ser220Glu +
Gly223GlnGly206Asn + Ser207Glu + Trp208Asp + Ala219Asp +
Thr224ProAla204Gly + Gly206Ser + Ser207Asp + Tyr210Asp +
Leu221GlyAla204Pro + Ser207Glu + Ile209Thr + Tyr210Glu +
Tyr218LeuIle209Thr + Tyr213Gly + Ala219Asp + Ser222Glu +
Gly223AspPro205Gln + Tyr213Gln + Tyr218Glu + Leu221Glu +
Ser222AspGly206Gln + Ser207Glu + Thr212Asn + Ser220Glu +
Thr224GluSer207Glu + Thr217Gly + Tyr218Asn + Ser220Asp +
Thr224GluAla204Thr + Tyr210Cys + Ser220Glu + Leu221Ser +
Gly223GluIle209Pro + Ser216Glu + Ala219Glu + Ser220Asp +
Gly223AsnGly206Gln + Tyr210Met + Thr212Pro + Ala219Glu +
Ser222AspPro205Asn + Tyr210Glu + Thr217Asp + Tyr218Ala +
Thr224ProAla204Gly + Trp208Gln + Thr212Gln + Ser216Asp +
Ala219GluAla204Asn + Trp208Ala + Ser216Asp + Ala219Glu +
Thr224GlyAla204Asn + Tyr210Asp + Thr215Glu + Thr217Glu +
Ala219GlyTrp208Leu + Ile209Gly + Thr215Asp + Thr217Glu +
Ser220AspThr212Pro + Tyr213Leu + Ser216Glu + Tyr218Glu +
Leu221GluThr217Asp + Ala219Glu + Leu221Asn + Ser222Glu +
Thr224SerPro205Ser + Trp208Glu + Tyr218Glu + Leu221Gly +
Ser222GluSer207Glu + Thr215Pro + Thr217Asp + Ser220Glu +
Gly223ProThr217Asp + Ala219Asn + Leu221Glu + Ser222Glu +
Gly223AsnTrp208Tyr + Ile209Asn + Thr217Glu + Leu221Glu +
Ser222AspAla204Gly + Tyr210Glu + Pro214Glu + Ser216Glu +
Thr217AsnPro214Asp + Thr215Gln + Ser216Glu + Ser220Glu +
Leu221MetAla204Gln + Tyr210Glu + Thr212Asn + Thr217Glu +
Ser222AspTrp208Leu + Ile209Val + Thr215Asp + Thr217Glu +
Leu221GluGly206Pro + Ile209Val + Tyr213Pro + Thr215Asp +
Ala219GluPro205Asn + Ser207Glu + Tyr218Asp + Ser222Asp +
Thr224GlyGly206Ser + Tyr210Asp + Pro214Asp + Thr215Pro +
Leu221AspTyr210Ser + Pro214Asp + Tyr218Cys + Ser220Glu +
Ser222GluSer207Asp + Ile209Ser + Thr212Ser + Pro214Asp +
Thr215Asp
表27
环6-六取代变异体Gly206Asn + Tyr210Gly + Thr212Gln + Tyr213Gly +Tyr218Ile + Thr224GlyPro205Asn + Gly206Ser + Tyr213Gly + Ser216Asp +Thr217Ser + Thr224AsnAla204Ser + Pro205Ser + Tyr213Ser + Tyr218His +Ala219Gln + Leu221AsnSer207Glu + Thr212Gln + Thr215Pro + Tyr218Gln +Leu221Met + Gly223GlnPro205Ser + Ser207Asp + Tyr210Val + Tyr213Leu +Leu221Gln + Thr224GlyAla204Ser + Trp208Cys + Ile209His + Thr215Glu +Thr217Pro + Gly223SerAla204Gly + Ile209Asn + Tyr210Ile + Ser216Asp +Ala219Asn + Leu221MetAla204Gly + Pro205Gln + Pro214Glu + Thr215Ser +Tyr218Asn + Thr224SerGly206Asn + Trp208Cys + Thr212Gly + Tyr213Gly +Ser216Asp + Leu221HisTrp208Pro + Ile209Gly + Thr215Ser + Ala219Thr +Leu221Thr + Thr224AspPro205Gly + Gly206Gln + Ile209Gly + Thr212Asn +Pro214Asn + Ser220GluPro205Gly + Trp208Leu + Thr217Gln + Leu221Gln +Gly223Gln + Thr224GluAla204Gly + Gly206Asn + Tyr210Cys + Tyr213Thr +Gly223Asp + Thr224AsnGly206Asn + Trp208His + Ile209Gly + Thr217Gly +Leu221Val + Thr224ProAla204Thr + Thr212Gln + Tyr213Gln + Thr217Ser +Ser222Asp + Thr224ProTrp208Gly + Thr212Gln + Tyr213Leu + Tyr218Gly +Ser222Glu + Gly223SerPro205Ser + Trp208Ser + Tyr213Asn + Tyr218Gln +Ser222Asp + Thr224GlnAla204His + Pro205Gly + Ile209Asn + Tyr213Gln +Thr215Ser + Thr217SerAla204Thr + Gly206Asn + Trp208Leu + Thr215Ser +Ser216Asp + Ala219HisGly206Ser + Thr212Gln + Tyr213Val + Tyr218Cys +Ser220Glu + Thr224GlnGly206Asn + Trp208Ile + Ile209Ser + Tyr218Gln +Gly223Glu + Thr224GlyGly206Asn + Ser207Glu + Ile209Cys + Thr212Ser +Thr217Pro + Ala219HisAla204Asn + Tyr210Cys + Tyr213Asn + Tyr218Leu +Leu221His + Ser222GluGly206Pro + Tyr213Asn + Pro214Asn + Thr215Gln +Tyr218Ser + Leu221ThrAla204Pro + Pro205Asn + Gly206Gln + Ile209His +Thr212Gly + Gly223GluPro205Gln + Tyr213Gly + Thr215Gly + Tyr218Ala +Gly223Glu + Thr224ProAla204His +Ser207Glu + Trp208Gly + Ile209His +Tyr213Cys + Thr215SerGly206Ser + Trp208His + Tyr213Thr + Ala219Gln +Ser220Glu + Gly223SerTyr213His + Thr215Gln + Thr217Asn + Tyr218Ala +Ala219Glu + Leu221SerAla204Asn + Pro205Ser + Gly206Asn + Ile209Leu +Thr215Asp + Thr224ProPro205Ser + Ile209Gly + Tyr213Leu + Thr217Asp + Tyr218Asn + Ala219HisAla204Asn + Pro205Gln + Ser207Asp + Tyr210Val +Thr212Gly + Leu221ThrAla204Asn + Trp208Thr + Ile209Gln + Tyr213Pro +Leu221Pro + Ser222AspAla204Ser + Gly206Ser + Tyr213Gly + Pro214Gln +Tyr218Val + Ser222AspAla204Pro + Tyr210Cys + Tyr213Ser + Pro214Gln +Thr215Pro + Thr217GlyThr212Ser + Pro214Asp + Thr217Gly + Ala219Thr +Leu221Pro + Thr224GlnPro205Gln + Gly206Gln + Ser207Glu + Tyr210Gly +Thr212Pro + Tyr218GlyPro205Gln + Gly206Pro + Trp208Asn + Ile209Met +Tyr213Ser + Ala219AspAla204Asn + Ser207Glu + Trp208Ala + Tyr213Asn +Leu221Thr + Thr224GlyAla204Pro + Trp208His + Ile209Asn + Tyr213Leu +Thr217Ser + Ser222GluGly206Pro + Trp208Ser + Tyr210Val + Thr212Gly +Tyr213Cys + Ala219AspPro205Gly + Gly206Gln + Ile209Cys + Tyr210His +Thr215Gln + Ala219ThrTrp208Gly + Ile209Ser + Thr212Gln + Tyr218Met +Leu221Ser + Gly223SerPro205Asn + Ile209Asn + Thr212Asn + Thr217Glu +Gly223Pro + Thr224SerPro205Gly + Trp208Thr + Thr212Ser + Ser216Glu +Thr217Gly + Gly223SerGly206Ser + Trp208Glu + Tyr210Gln + Thr215Gly +Thr217Gln + Leu221ProGly206Ser + Ser207Glu + Trp208Leu + Thr212Asn +Leu221Ala + Thr224AsnAla204Asn + Pro205Gln + Gly206Gln + Trp208Ile +Ser216Asp + Tyr218CysAla204Thr + Tyr210Cys + Tyr213Ser + Thr215Ser +Ser216Asp + Ala219HisTrp208Leu + Ile209Asn + Thr212Gly + Thr215Ser +Tyr218Pro + Leu221GlyPro205Gly + Gly206Gln + Tyr218Gly + Ala219Asn +Leu221Gln + Thr224SerPro205Gln + Trp208Val + Tyr210Pro + Thr215Gly +Ala219Ser + Thr224GluPro205Gly + Trp208Met + Ile209Ser + Thr212Asn +Tyr218Ala + Ala219ThrThr212Gly + Tyr213Asn + Pro214Ser + Ser216Glu +Thr217Pro + Leu221ThrAla204Gly + Trp208Pro + Ile209Pro + Tyr213Gln +Tyr218Ser + Ser220GluAla204Gly + Gly206Asn + Ile209Met + Thr212Pro +Ala219Glu + Leu221MetPro205Asn + Gly206Pro + Trp208Leu + Tyr210Pro +Thr217Gln + Tyr218GluPro205Asn + Gly206Gln + Tyr213Val + Ser220Asp +Gly223Ser + Thr224AsnAla204Thr + Pro205Gly + Gly206Gln + Pro214Gln +Leu221Val + Gly223GlnPro205Ser + Ile209Gln + Thr212Pro + Ala219Gly +Ser220Glu + Gly223GlnAla204Gly + Gly206Gln + Ile209Leu + Thr212Asn +Pro214Gln + Thr215SerGly206Asn + Trp208Ser + Thr212Ser + Pro214Asn +Ser220Glu + Gly223SerAla204His + Tyr213Leu + Pro214Asn + Thr217Pro +Ser220Glu + Gly223GlnGly206Pro + Ile209Cys + Pro214Gln + Thr217Ser +Tyr218Cys + Ala219GlnAla204Thr + Tyr210Ala + Thr215Gly + Thr217Gln +Ala219Pro + Ser220AspSer207Asp + Ile209Thr + Thr212Gly + Thr217Pro +Ala219Gly + Thr224ProAla204Gly + Ser207Glu + Thr212Asn+ Pro214Ser +Tyr218Ser + Ala219SerPro205Gln + Gly206Gln + Thr212Ser + Tyr213Ala +Thr215Ser + Gly223GluPro205Ser + Tyr210Ala + Pro214Ser + Thr215Asn +Ser222Asp + Gly223GluIle209Pro + Tyr210Pro + Thr212Pro + Leu221Asn +Ser222Asp + Gly223AspPro205Ser + Thr212Ser + Tyr213Ile + Ala219Pro +Ser222Glu + Gly223AspAla204Asn + Ile209Cys + Tyr213Cys + Tyr218Ala +Leu221Asp + Ser222GluIle209Val + Thr212Asn + Tyr213Met + Thr217Asn +Gly223Asp + Thr224AspPro205Gly + Ile209Asn + Thr212Asn + Ser216Glu +Thr217Glu + Tyr218GlnAla204His + Trp208Ser + Thr212Pro + Tyr213Pro +Ser216Glu + Thr217AspPro205Ser + Ile209Ala + Thr212Gly + Ser216Asp +Thr217Glu + Ala219ThrPro205Ser + Trp208Met + Pro214Asn + Ala219Gln +Ser220Glu + Leu221AspAla204Pro + Gly206Ser + Tyr213Ile + Ser220Glu +Leu221Asp + Gly223SerGly206Pro + Tyr210Gly + Tyr213His + Thr217Glu +Tyr218Asp + Leu221AsnThr212Pro + Tyr213Ser + Thr215Asp + Ser216Asp +Tyr218Pro + Gly223GlnSer207Glu + Ile209Asn + Thr212Ser + Thr217Gly +Ser222Glu + Thr224GlnPro205Gly + Ser207Glu + Tyr210Ser + Pro214Gln +Ser222Asp + Gly223AsnGly206Pro + Ser207Asp + Trp208Ser + Pro214Ser +Leu221Asn + Ser222AspGly206Gln + Ser207Asp + Ile209Cys + Tyr210Met + Thr217Asn + Ser222GluGly206Pro + Ser207Glu + Ile209Pro + Tyr210Ile +Leu221Asn + Ser222GluPro205Ser + Ser207Glu + Trp208His + Thr215Pro +Leu221Asn + Ser222GluPro205Asn + Ser207Asp + Thr215Gln + Tyr218Ile +Leu221Gly + Ser222GluAla204Pro + Ser207Asp + Tyr210Cys + Thr212Pro +Thr215Pro + Ser222GluPro205Gly + Pro214Asp + Thr215Glu + Ser216Asp +Thr217Pro + Gly223GlnIle209Thr + Thr217Gly + Ala219Glu + Ser220Asp +Leu221Glu + Gly223SerAla204Gln + Ser207Asp + Ile209Thr + Tyr218Asn +Ser222Glu + Gly223AspPro205Ser + Ser207Glu + Pro214Gly + Thr215Asn +Ser222Asp + Gly223GluPro205Asn + Gly206Pro + Trp208Asp + Thr212Gln +Ser222Asp + Gly223SerGly206Gln + Pro214Asp + Thr215Asp + Thr217Asp +Gly223Ser + Thr224GlyAla204His + Ile209Thr + Ser216Asp + Thr217Asp +Tyr218Asp + Leu221SerThr212Asn + Pro214Gln + Tyr218Met + Ser222Glu +Gly223Asp + Thr224GluAla204Gln + Gly206Pro + Tyr210Glu + Pro214Gly +Ala219Asp + Leu221GlnAla204Gly + Pro205Gln + Trp208Asp + Ile209Gln +Pro214Gly + Leu221GluAla204Asn + Gly206Gln + Ser207Glu + Trp208His +Leu221Asp + Ser222AspTrp208Gly + Tyr213Ser + Pro214Ser + Thr215Glu +Thr217Asp + Tyr218AlaAla204Asn + Pro205Ser + Tyr210Pro + Pro214Asp +Ser216Glu + Tyr218IleGly206Gln + Ile209Leu + Thr212Gly + Pro214Asp +Ser216Glu + Leu221HisPro205Gln + Trp208Glu + Tyr210Asp + Thr212Gln +Tyr213Asn + Thr215SerSer207Asp + Thr212Pro + Thr215Gly + Thr217Gln +Leu221Met + Gly223AspAla204Asn + Pro205Gln + Ser207Glu + Trp208Met +Ala219Pro + Gly223AspAla204Thr + Ser207Asp + Thr212Gly + Ala219Asn +Gly223Asp + Thr224AsnSer207Asp + Pro214Asn + Thr215Gly + Tyr218Ala +Leu221Ser + Gly223AspSer207Glu + Thr212Asn + Tyr213Gln + Tyr218Pro +Ser220Glu + Leu221AspAla204Asn + Gly206Ser + Ser207Asp + Tyr210Thr +Ser220Asp + Leu221AspGly206Ser + Thr212Asn + Thr215Pro + Tyr218Val +Leu221Asp + Gly223AspTrp208Met + Tyr210Thr + Thr212Asn + Ala219Gln +Leu221Asp + Gly223AspGly206Ser + Ser207Glu + Pro214Ser + Ala219Asn +Ser220Glu + Ser222GluAla204His + Ser207Glu + Ala219Ser + Ser220Glu +Leu221His + Ser222AspPro205Ser + Ser207Glu + Tyr213His + Thr217Pro +Ser220Glu + Ser222GluSer207Glu + Ile209Met + Tyr210Gly + Tyr213Leu +Ser220Glu + Ser222AspSer207Asp + Tyr213Pro + Pro214Gln + Ser220Glu +Ser222Glu + Gly223ProGly206Ser + Trp208Gln + Tyr210Glu + Thr212Asn +Thr215Gly + Leu221GluPro205Gly + Thr212Ser + Tyr218Leu + Ala219Gln +Ser220Glu + Ser222GluTrp208Phe + Thr212Asn + Pro214Ser + Ser220Glu +Ser222Asp + Gly223GlnAla204Gly + Gly206Pro + Tyr210Ser + Tyr218Cys +Ser220Glu + Ser222AspPro205Ser + Trp208Leu + Tyr213Ala + Thr217Ser +Ser220Asp + Ser222AspPro205Gly + Trp208His + Tyr210His + Ser220Glu +Leu221Gln + Ser222AspPro205Gly + Thr212Gln + Ala219Thr + Ser220Glu +Ser222Asp + Gly223ProAla204Ser + Gly206Pro + Tyr210Gly + Ser220Glu +Leu221Ser + Ser222AspIle209Ser + Tyr210Ser + Thr212Asn + Tyr213Cys +Ser220Asp + Ser222GluAla204Asn + Pro205Gly + Ser207Asp + Ile209Ser +Tyr213Val + Leu221GluAla204Asn + Gly206Pro + Thr215Glu + Ser216Glu +Thr217Asn + Tyr218GluGly206Pro + Ile209Ser + Tyr210His + Thr217Gly +Ser222Glu + Thr224AspGly206Gln + Thr217Gly + Ser220Asp + Ser222Glu +Gly223Glu + Thr224SerGly206Gln + Trp208Gin + Ile209Gln + Ser216Asp +Tyr218Asp + Ala219GluGly206Asn + Thr212Pro + Pro214Ser + Thr217Glu +Tyr218Glu + Ser220GluGly206Gln + Trp208Ser + Tyr210Asp + Pro214Gln +Thr217Glu + Ala219AspGly206Ser + Ser207Glu + Trp208Pro + Tyr210Asp +Ala219Thr + Ser222AspTrp208Gly + Tyr210His + Thr212Ser + Pro214Glu +Tyr218Asp + Leu221IleAla204Gln + Gly206Gln + Tyr210Gly + Pro214Glu +Thr217Pro + Tyr218GluAla204His + Pro214Glu + Thr215Gln + Thr217Gln +Tyr218Glu + Leu221ProSer207Asp + Ile209Cys + Thr215Pro + Tyr218His + Ala219Thr + Ser220GluPro205Asn + Ser207Glu + Trp208Tyr + Ile209Asn +Thr217Gln + Ser220GluSer207Asp + Pro214Ser + Thr215Asn + Thr217Asn +Ser220Glu + Leu221SerPro205Gly + Ser207Asp + Tyr210Ser + Ser220Asp +Gly223Pro + Thr224SerGly206Gln + Ser207Glu + Ile209Gln + Ser220Asp +Leu221Ala + Thr224SerAla204Ser + Pro205Gly + Ser207Asp + Thr212Ser +Thr215Gln + Ser220GluPro205Ser + Ser207Asp + Thr217Gln + Ala219His +Ser220Glu + Gly223GluSer207Glu + Thr215Gln + Tyr218Val + Ser220Asp +Gly223Glu + Thr224AsnPro205Gly + Ile209Pro + Tyr210Asp + Ala219Asp +Ser222Asp + Gly223SerGly206Asn + Tyr210Gln + Pro214Asn + Thr215Glu +Tyr218Glu + Leu221AlaIle209Asn + Tyr210Asp + Thr212Gln + Ser222Glu +Gly223Pro + Thr224AsnAla204Gly + Gly206Gln + Ser220Asp + Leu221Asp +Gly223Asn + Thr224GluGly206Pro + Thr212Pro + Thr217Pro + Tyr218Asn +Leu221Glu + Thr224GluIle209His + Tyr210Leu + Tyr213Leu + Pro214Asp +Thr217Asp + Ser220GluIle209Asn + Tyr210Ile + Thr212Asn + Thr215Gly +Ser220Asp + Gly223GluGly206Gln + Tyr210Ala + Thr215Asn + Ser216Glu +Thr217Asp + Ser220AspTrp208Thr + Tyr213Pro + Tyr218His + Ala219Glu +Leu221Cys + Ser222AspAla204Gly + Trp208Gln + Thr212Gln + Ser216Asp +Ala219Glu + Gly223SerGly206Ser + Pro214Gly + Ser216Asp + Tyr218Pro +Ala219Glu + Leu221ProPro205Asn + Tyr210Asn + Thr217Glu + Ala219Gln +Ser220Glu + Gly223GlnAla204Thr + Tyr213Ala + Pro214Gln + Thr215Gln +Thr217Asp + Ser220GluTrp208Gln + Tyr210His + Thr217Asp + Tyr218Met +Ser220Asp + Thr224SerAla204Asn + Gly206Pro + Tyr210Asp + Pro214Glu +Ser220Glu + Thr224AsnTyr213Ile + Ser216Glu + Tyr218His + Ala219Glu +Leu221Asp + Thr224SerPro205Ser + Trp208Glu + Tyr218Glu + Leu221Gly +Ser222Glu + Thr224GlySer207Asp + Tyr213Met + Pro214Gly + Thr217Gln +Tyr218Asp + Ala219GluTrp208Phe + Ala219Asp + Leu221Ala + Ser222Asp +Gly223Asn + Thr224GluGly206Pro + Ile209His + Pro214Glu + Ser220Asp +Leu221Glu + Thr224ProTrp208Tyr + Ile209Asn + Thr212Asn + Thr217Glu +Leu221Glu + Ser222AspPro205Gly + Ser216Glu + Thr217Glu + Ala219Gln +Leu221Glu + Gly223GlnPro214Asn + Thr217Pro + Ala219Asp + Ser220Asp +Leu221Gln + Thr224AspAla204His + Gly206Asn + Ile209His + Pro214Glu +Ser216Asp + Ser220GluSer207Asp + Tyr210Glu + Thr217Asp + Tyr218Ala +Leu221Ile + Thr224ProThr212Gly + Tyr213Ala + Pro214Glu + Leu221Glu +Ser222Asp + Thr224SerPro205Gln + Gly206Asn + Thr212Gln + Tyr218Glu +Ser222Asp + Gly223AspPro205Gln + Thr212Gly + Tyr213Ala + Thr217Glu +Tyr218Glu + Ser222GluIle209Gln + Tyr213Ala + Pro214Glu + Ala219Asp +Ser222Asp + Thr224ProAla204Gly + Tyr210Pro + Pro214Glu + Ala219Glu +Leu221Asn + Ser222AspGly206Pro + Thr212Asn + Tyr213Thr + Pro214Asp +Ser220Asp + Ser222GluAla204Thr + Tyr213Ser + Pro214Asp + Ala219Asn +Ser220Asp + Ser222GluAla204His + Gly206Asn + Tyr210Pro + Pro214Glu +Ser220Glu + Ser222GluSer207Asp + Trp208Met + Pro214Glu + Thr215Asp +Thr217Ser + Gly223ProTrp208Cys + Ile209Ser + Pro214Glu + Ser222Asp +Gly223Asp + Thr224GlyPro205Asn + Thr212Asn + Tyr213Leu + Thr215Asp +Ser222Asp + Gly223Glu
表28
环6-七取代突变变异体Gly206Asn + Tyr210Gly + Thr212Gln + Thr215Gln +Tyr218Ile + Leu221Ile + Thr224GlyAla204Ser + Pro205Ser + Ile209Met + Tyr213Ser +Tyr218His + Ala219Gln + Leu221AsnAla204Gly + Trp208Gln + Thr212Gln + Ser216Asp +Tyr218Gln + Leu221Met + Gly223SerGly206Gln + Ile209Leu + Tyr210Ala + Thr212Gly +Tyr213Gly + Ser216Asp + Gly223GlnAla204Gln + Pro205Asn + Gly206Asn + Trp208Ile +Tyr210Gln + Thr215Gln + Ala219AspAla204Asn + Pro205Gln + Ile209Ala + Tyr213Leu +Thr217Glu + Tyr218Met + Leu221GlnPro205Gly + Ile209Gln + Thr212Ser + Ala219Gln +Ser220Glu + Leu221Cys + Thr224SerGly206Asn + Trp208Cys + Thr212Gly + Tyr213Gly +Ser216Asp + Thr217Gly + Leu221HisAla204Gln + Pro205Ser + Tyr210His + Thr212Pro +Tyr218Pro + Ala219Ser + Thr224GluAla204Gly + Gly206Asn + Tyr210Cys + Tyr213Thr +Thr215Gly + Gly223Asp + Thr224AsnAla204Gln + Pro205Ser + Gly206Gln + Thr212Asn +Tyr218Cys + Ala219His + Gly223GluAla204Ser + Gly206Asn + Ile209Gly + Pro214Gly +Thr217Gly + Leu221Val + Thr224ProPro205Gly + Ile209Gln + Pro214Asn + Tyr218Ile +Ala219Gly + Leu221Met + Ser222AspAla204Asn + Trp208Ala + Ile209Gln + Pro214Gly +Thr217Glu + Tyr218Leu + Gly223AsnGly206Asn + Tyr210Glu + Tyr213Gly + Thr217Gln +Tyr218Leu + Ala219Thr + Thr224SerGly206Pro + Ile209His + Tyr213Asn + Pro214Asn +Thr215Gln + Tyr218Ser + Leu221ThrPro205Gly + Gly206Ser + Trp208His + Tyr213Thr +Ala219Gln + Ser220Glu + Gly223SerPro205Gln + Gly206Ser + Trp208Val + Tyr210Leu +Thr212Asn + Tyr218Met + Ser220GluAla204His + Gly206Asn + Tyr210Ser + Pro214Ser +Thr217Gln + Tyr218His + Ser222AspGly206Gln + Tyr213Asn + Thr215Gly + Ser216Glu +Thr217Gly + Tyr218His + Gly223AsnGly206Gln + Ser207Glu + Tyr210Gly + Thr212Pro +Tyr213Met + Gly223Pro + Thr224ProAla204Asn + Trp208Thr + Ile209Gln + Tyr213Pro +Thr217Ser + Leu221Pro + Ser222AspAla204Ser + Gly206Gln + Trp208Asn + Thr212Ser +Ala219Gln + Ser220Glu + Leu221AsnPro205Gly + Gly206Gln + Ile209Asn + Tyr213Val +Thr215Gln + Thr217Ser + Ala219ThrAla204Ser + Pro205Gln + Ile209Asn + Thr212Asn +Thr217Glu + Gly223Pro + Thr224SerThr212Gly + Tyr213Ile + Pro214Gly + Tyr218Gly +Ala219Pro + Leu221Asn + Thr224GluAla204Asn + Tyr210Asp + Thr212Pro + Tyr213Asn +Thr217Asn + Tyr218Cys + Leu221HisAla204Gln + Ile209Cys + Tyr210Gln + Thr212Gly +Tyr213Gln + Gly223Pro + Thr224AspPro205Gln + Gly206Gln + Trp208Thr + Tyr210Leu +Thr215Gly + Thr217Gly + Tyr218LeuTyr210Ala + Tyr213Val + Pro214Gln + Ala219Ser +Leu221Cys + Ser222Glu + Thr224AsnAla204Asn + Gly206Pro + Ser207Asp + Trp208His +Ile209His + Pro214Gln + Leu221GlnAla204Asn + Gly206Asn + Trp208Ile + Ile209Asn +Ser216Glu + Ala219Pro + Gly223AsnAla204Ser + Gly206Ser + Ile209Ala + Thr212Ser +Pro214Gln + Tyr218Asp + Leu221HisGly206Asn + Ile209Pro + Tyr213Cys + Thr215Gln + Ser216Glu + Thr217Pro + Ala219HisAla204Gln + Trp208His + Tyr210Gly + Thr212Gln +Tyr218Met + Ala219Asn + Ser220GluAla204Pro + Ser207Asp + Tyr213Val + Pro214Gly +Thr215Gly + Tyr218Ala + Leu221GlnPro205Asn + Gly206Pro + Trp208Leu + Tyr210Pro +Pro214Asn + Thr217Gln + Tyr218GluPro205Asn + Gly206Gln + Tyr213Val + Ala219Gln +Ser220Asp + Gly223Ser + Thr224AsnGly206Pro + Ile209Cys + Tyr210Ser + Thr212Asn +Tyr213Cys + Pro214Gln + Tyr218CysAla204His + Trp208Asn + Thr212Pro + Pro214Gln +Thr215Asn + Thr217Gly + Thr224AspPro205Ser + Gly206Ser + Tyr210Ser + Thr215Pro +Ala219Asn + Leu221Met + Ser222AspAla204Ser + Gly206Gln + Trp208Pro + Thr212Asn +Thr215Glu + Tyr218Ile + Gly223AsnAla204Ser + Pro205Asn + Trp208Tyr + Thr215Gly +Tyr218Pro + Ser220Glu + Thr224GlyAla204Asn + Tyr210His + Pro214Asn + Tyr218Pro +Ala219Gln + Ser220Glu + Leu221IleAla204Gly + Gly206Asn + Ser207Asp + Thr212Asn +Tyr213Leu + Thr215Pro + Gly223ProAla204Asn + Tyr210Met + Thr212Gln + Pro214Gly +Ala219Gly + Ser222Asp + Gly223GlnTyr210Pro + Thr212Gly + Tyr213Leu + Thr215Gly +Thr217Gly + Ala219Glu + Ser220AspGly206Ser + Trp208Asn + Ile209Pro + Ala219Asp +Ser220Asp + Gly223Ser + Thr224GlyPro205Ser + Tyr210Cys + Tyr213Thr + Pro214Asp +Thr215Asp + Tyr218Val + Leu221SerTrp208His + Ile209Val + Thr212Pro + Tyr213Leu +Thr217Ser + Ser222Glu + Gly223GluPro205Gly + Gly206Gln + Tyr210Asn + Tyr213Gly +Thr217Gly + Ser222Glu + Gly223GluAla204Thr + Thr212Gln + Tyr213Gln + Thr217Ser +Ser222Asp + Gly223Asp + Thr224ProPro205Gly + Ile209Cys + Tyr213Val + Pro214Gln +Thr215Pro + Ser222Glu + Gly223AspTrp208Ser + Ile209Ser + Ser216Glu + Thr217Glu +Tyr218Gly + Ala219His + Gly223ProGly206Pro + Tyr210Gln + Thr212Pro + Tyr213Leu +Ser216Glu + Thr217Asp + Leu221ProTrp208Met + Ile209Gln + Tyr210Met + Tyr218Gln +Ser220Glu + Leu221Glu + Thr224SerIle209Gln + Thr212Ser + Tyr213Val + Thr215Asp +Ser216Glu + Tyr218Met + Ala219ProTyr213Pro + Pro214Gln + Thr215Asp + Ser216Glu +Thr217Asp + Tyr218Met + Leu221AlaAla204His + Pro205Gly + Ser207Glu + Trp208His +Tyr210His + Tyr213Cys + Ser222AspGly206Pro + Ser207Asp + Trp208Ser + Pro214Ser +Leu221Asn + Ser222Asp + Gly223ProSer207Glu + Trp208Tyr + Ile209Gly + Tyr213Thr +Pro214Ser + Tyr218Met + Ser222GluPro205Ser + Ser207Asp + Trp208Ser + Thr212Gly +Tyr213Asn + Tyr218Gln + Ser222AspAla204Thr + Gly206Asn + Trp208Thr + Thr212Gln +Pro214Glu + Thr215Asp + Ser216AspAla204Thr + Gly206Asn + Ser207Glu + Tyr210Ile +Leu221Thr + Ser222Glu + Gly223GluAla204His + Ser207Glu + Thr215Gln + Thr217Gln +Tyr218Asn + Ser222Glu + Gly223GluAla204Gln + Trp208Asp + Tyr210Thr + Thr212Ser +Thr217Asn + Ser222Asp + Gly223AsnIle209Thr + Ser216Asp + Thr217Asp + Tyr218Asp +Leu221Ser + Gly223Ser + Thr224GlyAla204Asn + Gly206Gln + Ser207Glu + Trp208His +Thr212Ser + Leu221Asp + Ser222AspGly206Ser + Ser207Asp + Tyr210Val + Thr212Gln +Tyr218Ile + Leu221Asp + Ser222AspAla204Ser + Gly206Pro + Pro214Gly + Thr215Asp +Thr217Asp + Tyr218Pro + Gly223ProGly206Gln + Ile209His + Pro214Asp + Thr215Pro +Ser216Asp + Leu221Gly + Thr224SerGly206Asn + Ser207Glu + Trp208Asp + Ile209Gly +Tyr213Gly + Ser220Glu + Ser222GluPro205Ser + Ser207Asp + Ile209Asn + Tyr213Met +Thr217Gln + Ala219His + Gly223GluPro205Gly + Gly206Gln + Ser207Glu + Ala219Pro +Ser220Asp + Leu221Glu + Thr224SerTrp208Met + Tyr210Thr + Thr212Asn + Ala219Gln +Leu221Asp + Gly223Asp + Thr224GlnPro205Ser + Ser207Glu + Tyr213His + Thr217Pro +Ala219Ser + Ser220Glu + Ser222GluSer207Asp + Tyr210Met + Pro214Ser + Tyr218Asn +Ser220Glu + Leu221Cys + Ser222GluAla204Pro + Ser207Glu + Pro214Gln + Ser220Asp +Leu221His + Ser222Asp + Thr224SerSer207Glu + Ile209Met + Tyr210Gly + Tyr213Leu +Thr215Gln + Ser220Glu + Ser222AspGly206Pro + Ser207Asp + Ile209Ser + Tyr210His +Thr217Gly + Ser222Glu + Thr224AspPro205Asn + Tyr210Cys + Tyr213Gly + Thr217Asn +Tyr218Met + Ser220Glu + Ser222AspIle209Gly + Thr212Pro + Thr215Asn + Thr217Gly +Ala219His + Ser220Glu + Ser222GluAla204Gln + Gly206Gln + Thr212Pro + Ser220Glu +Leu221Val + Ser222Asp + Thr224GlnAla204Gln + Pro205Ser + Gly206Ser + Trp208Gln +Thr215Pro + Ser220Glu + Ser222GluSer207Glu + Trp208Leu + Tyr210Ile + Thr212Gly +Tyr218Cys + Leu221Asp + Gly223AsnPro205Gly + Gly206Pro + Ser207Asp + Ile209Met +Leu221Asp + Ser222Glu + Thr224AspPro205Gly + Thr215Asn + Thr217Pro + Tyr218Val + Leu221Thr + Ser222Glu + Thr224AspTrp208Phe + Tyr213Met + Thr215Asn + Tyr218Asp +Ala219Pro + Ser220Glu + Thr224GlyAla204Pro + Pro205Gly + Trp208Leu + Thr217Gln +Leu221Asp + Ser222Glu + Thr224AspIle209Ala + Thr212Gln + Thr217Gln + Tyr218Thr +Ser220Glu + Leu221Asp + Gly223GluAla204Asn + Ile209Cys + Tyr213Cys + Tyr218Ala +Ala219Glu + Leu221Asp + Ser222GluGly206Ser + Ser207Glu + Trp208His + Tyr210Asp +Tyr218Val + Ala219Thr + Ser222AspPro205Gly + Ser207Asp + Trp208Thr + Thr212Ser +Thr217Gly + Ser220Glu + Gly223SerSer207Glu + Trp208Asn + Tyr210Pro + Pro214Asn +Tyr218Ser + Ser220Asp + Gly223SerAla204Gln + Ser207Asp + Trp208Gly + Ile209Ala +Thr212Asn + Ser220Glu + Gly223ProPro205Gln + Ser207Asp + Ile209Gln + Pro214Asn +Thr215Gln + Ser220Asp + Thr224GlyPro205Asn + Ser207Glu + Ile209Val + Thr212Gly +Tyr213Pro + Ala219Asn + Thr224GluAla204Gly + Ser207Glu + Ile209Ala + Thr212Pro +Tyr213Cys + Ala219Glu + Ser220AspPro205Ser + Trp208Glu + Thr212Asn + Tyr218Glu +Ala219His + Leu221Glu + Ser222GluPro205Ser + Ile209Pro + Tyr210Asp + Thr212Ser +Tyr213Ile + Ala219Pro + Ser222GluGly206Ser + Trp208Thr + Tyr210Ala + Pro214Asp +Tyr218Met + Ala219Glu + Ser220AspIle209Leu + Pro214Glu + Thr215Pro + Ala219Glu +Ser220Glu + Gly223Pro + Thr224GlyTrp208Tyr + Thr212Asn + Thr217Glu + Ala219Gly +Ser220Asp + Leu221Glu + Ser222AspAla204Thr + Tyr210Pro + Tyr213Asn + Pro214Glu +Tyr218Asp + Ser220Asp + Thr224GlyGly206Pro + Thr212Pro + Pro214Gly + Thr215Pro +Ser220Asp + Leu221Asp + Thr224GluThr212Asn + Pro214Gln + Tyr218Met + Ala219Asp +Ser222Glu + Gly223Asp + Thr224GluTrp208Gly + Tyr210Pro + Thr212Gly + Ser220Glu +Leu221Met + Gly223Asp + Thr224GluGly206Gln + Tyr213Asn + Pro214Asn + Thr217Asp +Ala219His + Ser220Glu + Leu221AspPro205Gln + Ile209Pro + Tyr210Asn + Tyr218Thr +Ala219Glu + Ser220Asp + Gly223AspAla204Thr + Ser207Asp + Tyr210Asp + Tyr213Met +Ala219Asn + Ser220Glu + Thr224AspPro205Ser + Trp208Pro + Ile209Pro + Tyr213Ile +Ala219Asp + Ser222Asp + Gly223AspAla204Asn + Thr212Pro + Thr217Glu + Tyr218Asp +Ser220Glu + Ser222Asp + Thr224GlnGly206Pro + Ser207Asp + Trp208Val + Tyr210Thr +Tyr213Ala + Ser220Asp + Thr224AspPro205Gly + Ser207Glu + Tyr210Ile + Pro214Ser +Thr217Gly + Ser220Asp + Thr224GluAla204Thr + Tyr210Cys + Thr212Pro + Tyr218Ile +Ser220Glu + Leu221Ser + Gly223GluAla204Gln + Trp208Pro + Pro214Asn + Ala219Thr +Ser220Glu + Gly223Asp + Thr224GlnPro205Gln + Tyr213Gly + Thr215Gly + Tyr218Ala +Ser220Asp + Gly223Glu + Thr224ProPro205Gly + Gly206Ser + Thr217Pro + Ala219His +Ser220Asp + Leu221Cys + Gly223AspPro205Ser + Tyr210Thr + Thr212Asn + Tyr213Cys +Pro214Ser + Ala219Asp + Ser222AspTrp208Asn + Thr217Glu + Tyr218Cys + Ala219Glu +Ser220Asp + Gly223Glu + Thr224SerPro205Ser + Gly206Pro + Trp208Pro + Ile209Ala +Thr215Asp + Tyr218Asp + Ser220AspTrp208Thr + Thr212Gly + Tyr213Ile + Thr215Asp +Leu221Asp + Ser222Glu + Gly223GluAla204Gly + Pro205Gly + Tyr210Glu + Thr217Glu +Tyr218Cys + Ala219Gly + Leu221ValSer207Asp + Thr215Pro + Thr217Asp + Tyr218Ile +Leu221Ala + Ser222Glu + Gly223AspGly206Pro + Trp208Glu + Tyr210Cys + Pro214Gln +Thr217Asp + Ser220Asp + Gly223GlnTrp208Thr + Tyr213Pro + Pro214Ser + Ser216Asp +Tyr218Met + Ala219Asp + Thr224ProAla204Asn + Trp208Ala + Tyr213Asn + Ser216Asp +Ala219Glu + Leu221Cys + Thr224GlyGly206Ser + Tyr210Ser + Pro214Gly + Ser216Asp +Tyr218Cys + Ala219Glu + Leu221ProAla204Thr + Tyr210Gln + Thr212Gln + Tyr213Ala +Pro214Gln + Thr217Asp + Ser220GluPro205Gln + Gly206Ser + Ser207Glu + Tyr210Ser +Ser216Asp + Thr217Glu + Tyr218GluTyr210Met + Tyr213Met + Ser216Asp + Ala219Glu +Ser220Glu + Leu221Cys + Ser222GluAla204Gln + Ile209His + Ser216Glu + Thr217Ser +Ala219Glu + Ser220Asp + Ser222GluIle209Ser + Tyr210Leu + Tyr213Leu + Pro214Asp +Thr217Asp + Ser220Glu + Leu221GluAla204His + Pro205Gln + Pro214Asn + Tyr218Gln +Ala219Asp + Leu221Glu + Thr224AspAla204Gly + Ser207Glu + Pro214Glu + Tyr218Pro +Ser220Glu + Ser222Asp + Thr224AsnAla204Pro + Pro205Gly + Trp208Glu + Ile209Ser +Tyr210Asp + Ala219Thr + Thr224GluAla204Gly + Ser207Glu + Trp208Glu + Tyr210Asp +Ser216Glu + Thr217Gln + Thr224GlyTrp208Ala + Tyr210Cys + Thr212Ser + Thr217Glu +Ala219Gln + Ser220Asp + Ser222GluGly206Asn + Ser207Glu + Trp208Glu + Thr212Ser +Ser216Glu + Leu221Met + Gly223GluAla204Thr + Ser207Asp + Ile209Cys + Thr215Asp + Tyr218Ile + Ser220Asp + Leu221AspSer207Asp + Thr212Pro + Pro214Gln + Ser216Glu +Tyr218Val + Ser220Asp + Ser222AspIle209Pro + Thr212Gly + Tyr213Ser + Pro214Glu +Thr215Glu + Ala219Gln + Ser220GluPro205Asn + Ser207Glu + Ile209Met + Pro214Ser +Ser216Asp + Ala219Glu + Leu221AspAla204Thr + Ser207Glu + Tyr210Leu + Ser216Glu +Ala219Asp + Leu221Asp + Gly223AsnGly206Asn + Ser207Glu + Pro214Gly + Thr215Glu +Thr217Glu + Tyr218Thr + Ala219GluGly206Gln + Ser207Glu + Tyr210Ala + Thr215Asn +Thr217Asp + Ser220Asp + Leu221AlaSer207Glu + Trp208Leu + Tyr213Val + Pro214Asp +Ala219Glu + Ser222Glu + Thr224ProAla204Thr + Ser207Asp + Trp208Leu + Tyr210Asp +Thr212Pro + Thr215Asp + Leu221GluPro205Gln + Ser207Asp + Ile209Ala + Thr217Asn +Tyr218Glu + Ala219Asp + Gly223AspSer207Glu + Ile209Thr + Tyr213Gly + Ser216Asp +Ala219Asp + Ser222Glu + Gly223ProGly206Ser + Ser207Asp + Ile209Ala + Thr212Gln +Thr217Glu + Ala219Glu + Leu221AsnPro205Asn + Tyr210Glu + Thr217Asp + Tyr218Ala +Leu221Ile + Ser222Glu + Thr224ProSer216Asp + Thr217Asp + Tyr218Cys + Ala219His +Leu221Glu + Ser222Glu + Gly223AsnAla204Gln + Trp208Asp + Pro214Gln + Ser216Glu +Thr217Gln + Ser220Asp + Gly223AspPro205Gln + Tyr210Leu + Tyr213Thr + Ser216Glu +Tyr218Glu + Ser220Glu + Gly223GluPro214Gly + Thr217Asp + Tyr218Gly + Ala219Glu +Leu221Asn + Ser222Glu + Thr224GluGly206Pro + Ser207Asp + Trp208Asp + Thr212Gln +Thr217Asp + Gly223Ser + Thr224GluAla204Pro + Pro205Ser + Gly206Pro + Ile209Val +Tyr213Pro + Thr215Asp + Ala219GluAla204Pro + Gly206Pro + Tyr210Val + Pro214Glu +Ser220Glu + Ser222Glu + Thr224ProAla204Gly + Gly206Pro + Thr212Gln + Pro214Glu +Ser220Glu + Leu221Val + Ser222AspAla204Asn + Gly206Gln + Ile209Ser + Tyr210Ala +Pro214Glu + Ser220Glu + Ser222AspPro205Gln + Trp208Gln + Pro214Glu + Thr217Pro +Leu221Met + Ser222Asp + Gly223GluAla204Thr + Pro205Asn + Pro214Asp + Thr215Pro +Leu221Cys + Ser222Asp + Gly223AspIle209Pro + Tyr210Pro + Thr212Pro + Thr217Asp +Leu221Asn + Ser222Asp + Gly223AspGly206Gln + Trp208Thr + Ile209Ser + Tyr213Met +Thr215Glu + Ser222Asp + Gly223GluPro205Asn + Trp208Ser + Thr212Gly + Tyr213Asn +Thr215Asp + Ser222Asp + Gly223GluTyr210Gly + Thr212Pro + Pro214Gly + Ser216Asp +Ala219His + Leu221Glu + Ser222AspPro205Gly + Gly206Pro + Ile209His + Thr215Asp +Ala219Gly + Leu221Glu + Ser222GluPro205Gln + Tyr210Gly + Thr212Asn + Ser216Asp +Ala219Pro + Ser220Glu + Leu221AspGly206Ser + Trp208Glu + Tyr210Gln + Thr215Gly +Ser216Glu + Thr217Glu + Leu221ProPro205Gln + Gly206Gln + Ser216Glu + Thr217Asp +Leu221Ala + Ser222Glu + Thr224ProGly206Ser + Ser207Glu + Trp208Asp + Ile209Leu +Thr215Asp + Ser216Glu + Tyr218GlnTyr210His + Thr212Gln + Thr215Asp + Ser216Glu +Leu221Glu + Gly223Ser + Thr224ProAla204Ser + Pro205Ser + Gly206Asn + Thr215Asp +Ser216Glu + Tyr218Val + Gly223AspAla204Gln + Gly206Asn + Ser207Glu + Trp208Pro +Pro214Asp + Thr215Asp + Ser222AspSer207Asp + Tyr210Gln + Thr212Gln + Thr215Asp +Ser216Asp + Leu221Gly + Ser222AspIle209Met + Tyr210Leu + Thr217Glu + Tyr218Thr +Ser220Glu + Gly223Glu + Thr224AsnPro205Ser + Ser207Glu + Trp208His + Thr215Pro +Thr217Asp + Leu221Asn + Ser222GluAla204Thr + Ser207Glu + Thr212Gly + Tyr213Leu +Pro214Asn + Ser216Glu + Ser222GluAla204Pro + Ser207Asp + Tyr210Leu + Thr212Gln +Tyr213Thr + Thr215Glu + Ser222Glu
表29
多环双突变变异体Leu104Gly + Tyr210ProAsn 66Gln + Thr217GluGly 67Ser + Gly 69SerVal103Gln + Gly223AspTrp112Cys + Thr217GlyAla164Glu + Trp208GlnLeu134Ile + Trp208GlnAla219Gly + Ser220GluAsn 66Asp + Leu104GlyAla168Thr + Ser216GluLeul04Ala + Ser109GluThr111Gln + Val115AlaSer109Asp + Tyr210ProGly 69Ser + Serl91AspVal103Asp + Gly206GlnGly 67Glu + Pro214AsnTrp208Ile + Thr215AspSer107Glu + Thr113GlyGly108Ser + Leu221GluSer 70Glu + Pro169AsnGly165Gln + Leu221AspGly108Glu + Ala164GlyLeu104His + Thr167ProAla164His + Ser220GluThr195Gln + Ser222AspTrp208His + Ala219ProGly 69Glu + Asn106GlnTrp112Cys + Thr113ProGly 67Gln + Trp208AspAla114Glu + Leu134IleSer109Glu + Gly136SerGly110Ser + Ser207GluVal103Ala + Thr212SerAla114Glu + Thr167ProThr195Glu + Ala204ThrGly 67Ser + Thr217GluTrp208Tyr + Ser220GluAla168His + Thr217AspAla168Glu + Tyr213AsnGly110Pro + Ser220AspTyr171Asn + Leu221MetThr 73Asp + Leu134SerThr137Asn + Thr217ProSer 70Asp + Ala168ThrThr167Glu + Tyr210ProThr195Glu + Tyr210GlyTyr210Gln + Thr217GluGly 67Glu + Val115CysSer 70Glu + Ala219ThrGly136Pro + Leu221AlaGly 67Ser + Thr195SerPhe193His + Ser222AspAsn 68Glu + Tyr210SerGly165Asp + Pro169SerTrp208Pro + Thr215GlyThr 73Gly + Leu104GlyThr113Asn + Ser216GluGly 72Gln + Gly139AspTyr210Ile + Leu221GlyThr 73Pro + Tyr171AspLeu104Gln + Thr137GlyGly 69Pro + Tyr210AlaThr217Gly + Gly223GluThr195Asp + Tyr213SerGly 72Asn + Ala219AsnVal138Pro + Tyr210AlaTyr218Met + Gly223AsnSer220Glu + Leu221AsnTrp112Ala + Gly139AsnGly135Glu + Leu221GlyThr113Asn + Thr217SerThr195Asn + Thr217AspTyr210Val + Ser220GluVal138Gly + Ala219AspAsn166Glu + Leu221HisGly165Gln + Leu221HisThr113Gln + Ser192AspAsn 66Asp + Thr 73SerThr167Gln + Thr217GlyVal115Pro + Gly135SerAla219Gln + Gly223AspTrp208His + Thr217AsnTrp208Leu + Tyr210ValGly108Asn + Ser191GluSer107Glu + Tyr210AlaAsn 68Gln + Ser220GluTrp112Cys + Leu221AsnGly108Gln + Ser109AspVal103Met + Ala168HisAla164Pro + Ser192GluPro214Ser + Ser220GluThr215Asn + Thr217GlnSer220Glu + Leu221ProGly136Ser + Trp208PheThr195Asp + Tyr210GlnLeu104Gln + Ala219AspGly 69Pro + Leu221ProGly 69Asn + Ala204GlyGly135Glu + Ala219AsnGly136Asn + Ser220GluGly136Ser + Trp208ValGly139Asp + Asn140GlnTrp208Cys + Tyr210AsnGly165Ser + Ser220AspGly110Ser + Trp208ThrAla114Asp + Gly162AsnSer192Asp + Leu221CysVal103Ser + Gly135GluAsn140Glu + Trp208AlaThr113Asp + Leu221SerSer109Asp + Gly110SerSer109Glu + Thr113GlnThr 73Gly + Gly136AspAla164His + Leu221AsnGly 67Pro + Va1103HisAla114Asn + Thr212GlnAsn140Asp + Gly223ProGly139Gln + Pro214AsnAsp105Glu + Thr195GlyGly135Pro + Ser207GluLeu104Glu + Ala114ProAsn 66Glu + Gly 69ProVal115Cys + Ser207GluAsn 68Glu + Leu221AlaAsn 66Ser + Gly 69AspSer109Glu + Phe193ProTrp208Glu + Thr212ProGly136Gln + Trp208CysSer 70Asp + Leu221HisSer 70Glu + Phe193SerThr217Asn + Ser222GluTyr213Thr + Tyr218GlyTrp208Ser + Leu221CysAsn163Asp + Leu221SerAsn 66Gln + Gly223ProGly139Asn + Ser220AspThr 73Ser + Pro214AspSer 70Asp + Leu221IleGly165Gln + Ser220AspAsn166Asp + Thr217AsnAsp105Glu + Thr111ProAsn140Asp + Trp208PheAsn166Glu + Pro214AsnTrp208Ala + Thr217SerGly108Ser + Gly110GluVal138Asn + Asn170AspGly110Glu + Thr224GlnTrp208Met + Ser220AspAsn 68Glu + Val115SerAsn170Glu + Tyr210GlyGly110Asn + Ser192GluTrp208Gln + Ser220GluAsn140Gln + Pro214GlnThr217Ser + Ser220GluSer141Glu + Ala168ThrGly162Glu + Tyr210HisLeu104Gly + Thr224AspTrp112Pro + Ser191AspTrp208Thr + Tyr210GluGly108Gln + Thr111AspLeu134Val + Thr217ProAsn170Asp + Tyr210HisVal115Ser + Ser220GluGly 69Pro + Ser 70AspThr 73Gly + Tyr210GluGly 67Asp + Gly108GlnGly 69Ser + Gly110AspTyr210Thr + Ser220GluTrp208Phe + Leu221MetGly136Gln + Leu221AspThr113Ser + Trp208ValTyr171Leu + Tyr210ThrLeu134Asn + Thr167GlnAsn140Ser + Tyr210AspAsn 66Gln + Gly 67GluLeu134Cys + Pro214AsnGly136Ser + Leu221AlaThr111Pro + Trp208ValThr113Asp + Tyr171GlyTrp208Ile + Ser220GluPro214Ser + Thr224GluGly165Pro + Ser220GluThr137Gln + Tyr171GluTrP208Glu + Pro214GlySer216Asp + Leu221MetGly136Ser + Thr217GlnSer141Glu + Tyr210LeuTrp208Thr + Gly223SerAsn 66Ser + Asn170GlnAsn 66Gln + Asn170GluPro169Glu + Tyr210ThrTyr171Leu + Leu221AspGly139Glu + Leu221ProThr 73Pro + Leu134GlyTyr210Glu + Leu221AlaGly139Ser + Tyr210GlyAsn163Asp + Ala164SerAsn163Gln + Thr217AspAsn140Asp + Tyr218SerGly108Ser + Ala164GlnVal138Ser + Trp208LeuThr217Ser + Thr224AspSer109Asp + Tyr218AlaAsn 66Glu + Phe193ValAsn140Asp + Ala204GlyVal138Ser + Ser220AspGly108Asn + Tyr210ThrGly139Asn + Tyr171ProThr113Pro + Trp208ThrSer141Glu + Leu221GlyAsn106Glu + Thr167GlyTrp112Ser + Ala219SerAsp105Glu + Asn166GlnAla114Pro + Tyr171AsnThr217Gln + Ser220AspThr113Gly + Tyr210AspTyr171Cys + Tyr210GluAsn166Asp + Leu221ValAla114His + Ser191AspGly 67Glu + Thr217AsnAsn 66Glu + Thr215GlnThr111Asn + Ser220GluTrp208Gln + Leu221SerGly 69Gln + Ala114ThrAsn163Asp + Leu221AlaPro214Gly + Leu221IleThr217Gly + Ser220AspAla219Thr + Ser220AspTyr210Cys + Ser220GluVal138Asp + Gly223ProThr195Asn + Leu221GlyLeu104Thr + Thr215SerGly 69Gln + Leu134ThrGly165Pro + Leu221GlyThr167Ser + Thr217GluThr212Pro + Thr217GluAsn166Ser + Tyr218AspGly108Glu + Tyr171GlnGly 67Asn + Tyr210IleVal115Cys + Pro214GlyThr137Asp + Tyr210ValLeu104Met + Trp208GlyLeu134Ala + Ser192AspTrp112Gln + Ala219GlnGly108Glu + Val115HisThr113Asn + Gly139ProTrp208Gln + Gly223AsnSer207Glu + Trp208GlnAsn 66Ser + Tyr210AsnSer207Glu + Tyr210ProVal103Ala + Thr215GluVal103Asn + Tyr210AlaAsn 66Ser + Trp208PheThr215Asn + Ala219ThrGly136Asn + Gly139AsnTyr171Thr + Ser216GluLeu104Glu + Tyr210AsnSer109Glu + Thr217AsnLeu134Ile + Asn166SerTyr171Gly + Thr217GlnVal115Cys + Thr224AspPro169Ser + Thr215AspGly 72Gln + Gly135AsnTyr210Thr + Ser220AspAsn 68Glu + Thr113SerAsn106Gln + Thr113AspSer109Glu + Trp208AsnLeu134Gln + Thr137GluAla114Thr + Asn170GluAla219Pro + Ser220GluTyr171Val + Trp208ProGly139Ser + Tyr213SerSer107Asp + Thr113AsnThr167Gly + Leu221MetTyr171Gly + Ser207GluGly 67Ser + Thr217SerVal115Gly + Thr217SerAsn 66Ser + Trp208IleSer191Glu + Trp208CysThr137Asp + Trp208SerLeu221Gly + Ser222GluSer216Glu + Leu221ThrGly162Pro + Ser207GluThr217Asp + Ala219ThrLeu104Cys + Ser207GluGly 69Gln + Ser220GluGly 69Gln + Val138AspPro169Gly + Tyr210LeuThr 73Asp + Gly110SerThr215Gly + Thr217SerGly 69Ser + Gly 72AsnSer191Glu + Thr217AsnGly135Ser + Tyr171SerThr111Asn + Phe193LeuTrp112Leu + Ser207AspLeu104Glu + Trp208AlaLeu221Ile + Thr224AspGly136Glu + Gly165AsnTyr210Glu + Thr217ProTrp112Phe + Pro214AspGly110Gln + Trp112MetAsn 68Ser + Trp208AspGly 69Ser + Leu104CysThr137Glu + Thr215ProAsn166Gln + Thr217GluTyr210Thr + Thr212SerLeu134His + Trp208AspSer141Glu + Tyr210ValSer191Glu + Tyr210LeuGly139Pro + Ser207AspGly162Ser + Tyr171MetThr 73Gln + Val103AlaTyr171Val + Phe193AspLeu221Thr + Ser222GluGly 69Asp + Ala114GlnGly108Glu + Trp208MetGly 72Ser + Tyr210GlnThr137Asn + Trp208AspAsn140Asp + Phe193ThrSer192Glu + Trp208AlaAsn170Glu + Pro205SerTyr210Ala + Tyr213SerTyr171Gln + Tyr218IleAsn 66Asp + Leu221CysAsn163Gln + Gly223AsnVal103Thr + Leu134SerThr167Ser + Asn170AspSer 70Glu + Gly162SerTyr171Gln + Ser192GluTyr210Val + Gly223SerThr137Glu + Leu221ProGly 69Asn + Thr217AspPro205Ser + Tyr218GluTyr210Met + Leu221AsnThr215Asp + Thr217ProLeu134Val + Gly136GluThr 73Pro + Leu221GluTyr213Val + Ser220GluThr 73Ser + Ser107GluTrp208Ser + Leu221ProThr113Asp + Leu221HisAla114Thr + Thr217AspAla168Ser + Tyr218GluTrp208Asp + Leu221AsnThr137Asp + Trp208IleGly135Gln + Ser191GluVal103Glu + Trp208GlyAsn140Asp + Leu221HisSer192Asp + Tyr210CysGly108Asn + Tyr210HisAsp105Glu + Trp208AsnVal103Pro + Tyr210ProThr 73Gln + Trp112TyrLeu134Cys + Asn170SerAsn166Glu + Tyr210CysAsn106Ser + Leu221MetSer109Glu + Leu221CysSer107Asp + Leu221AlaAla114Ser + Leu221AsnGly162Asn + Leu221Val
表33
多环三突变变异体Leu104Gly + Tyr210Pro + Thr217GluAsn 66Gln + Gly 67Ser + Gly 69SerVal103Gln + Trp112Cys + Gly223AspAla164Glu + Trp208Gln + Thr217GlyLeu134Ile + Trp208Gln + Ser220GluAsn 66Asp + Leu104Gly + Ala219GlyLeu104Ala + Thr111Gln + Val115AlaGly 69Ser + Ser109Asp + Tyr210ProGly139Asn + Thr167Asn + Thr217SerGly108Glu + Ala164Gly + Gly165GlnLeu104His + Thr167Pro + Ser220AspGly 69Glu + Asn106Gln + Trp208HisTrp112Cys + Thr113Pro + Trp208AspGly110Ser + Ser207Glu + Thr212SerAla168His + Trp208Tyr + Ser220GluTyr171Asn + Thr217Asp + Leu221MetSer 70Asp + Thr137Asn + Thr217ProThr167Glu + Ala168Thr + Tyr210ProSer 70Glu + Gly136Pro + Leu221AlaGly 67Ser + Thr195Ser + Ser222AspPro169Ser + Trp208Pro + Thr215GlyThr 73Gly + Leu104Gly + Tyr171GluGly 72Gln + Tyr210Ile + Leu221GlyLeu104Gln + Thr137Gly + Tyr210AlaTyr213Ser + Thr217Gly + Gly223GluGly 72Asn + Thr195Asp + Ala219AsnThr137Asp + Val138Pro + Tyr210AlaSer109Glu + Val115Met + Trp208CysGly139Asn + Ser220Glu + Leu221AsnTrp112Ala + Gly135Glu + Leu221GlyThr113Asn + Thr195Asn + Thr217SerAla164His + Ser220Asp + Leu221HisAsn 66Asp + Thr 73Ser + Thr113GlnGly135Ser + Thr167Gln + Thr217GlyTyr171Asp + Trp208His + Thr217AsnGly108Asn + Trp208Leu + Tyr210ValTrp112Cys + Ser220Glu + Leu221AsnVal103Met + Ala168His + Ser191AspPro214Ser + Thr217Gln + Ser220GluThr215Asn + Ser220Glu + Leu221ProGly136Ser + Trp208Phe + Tyr210GlnGly 69Pro + Ala219Asp + Leu221ProGly 69Asn + Ala204Gly + Ser220AspGly136Ser + Gly139Asp + Trp208ValAsn140Gln + Trp208Cys + Tyr210AsnGly110Ser + Gly165Ser + Trp208ThrVal103Ser + Gly135Glu + Leu221CysAsn140Glu + Trp208Ala + Leu221SerGly 67Pro + Val103His + Ala164HisAsn140Asp + Thr212Gln + Gly223ProGly139Gln + Thr195Gly + Pro214AsnGly 69Pro + Leu104Glu + Ala114ProPhe193Pro + Trp208Glu + Thr212ProGly136Gln + Trp208Cys + Leu221HisSer 70Asp + Thr 73Gly + Phe193SerTyr213Thr + Tyr218Gly + Gly223AspThr195Glu + Trp208Ser + Leu221CysAsn163Asp + Leu221Ser + Gly223ProAsn 66Gln + Gly139Asn + Ser220AspThr 73Ser + Pro214Asp + Leu221IleThr111Pro + Asn140Asp + Trp208PheAsn166Glu + Pro214Asn + Thr217SerGly108Ser + Gly110Glu + Trp208AlaLeu134Asp + Gly165Asn + Tyr210ThrPhe193Thr + Thr217Gln + Ser220AspGly 67Pro + Trp112Gln + Ser192AspTyr210Ile + Thr217Gln + Ser222AspVal103Ser + Leu104Ile + Ala164AsnAla114Asp + Thr167Gly + Thr217AsnThr111Asp + Thr195Gly + Tyr210LeuTyr210Thr + Ser220Glu + Leu221MetGly136Gln + Trp208Phe + Leu221AspThr113Ser + Trp208Val + Tyr210ThrLeu134Asn + Asn140Ser + Thr167GlnGly136Ser + Trp208Val + Leu221AlaGly165Pro + Pro214Ser + Ser220GluGly136Ser + Tyr210Leu + Thr217GlnSer141Glu + Trp208Thr + Gly223SerAsn 66Ser + Thr113Pro + Asn170GluThr 73Pro + Tyr210Thr + Ser216GluThr 73Pro + Tyr210Glu + Leu221AlaGly139Ser + Ala164Ser + Tyr210GlyGly108Ser + Val138Ser + Ala164GlnTrp208Leu + Thr217Ser + Thr224AspAsn 66Glu + Phe193Val + Tyr218AlaGly108Asn + Tyr171Pro + Tyr210ThrThr113Pro + Gly139Asn + Trp208ThrAsp105Glu + Asn166Gln + Tyr171AsnAla114Pro + Thr217Gln + Ser220AspGly 67Glu + Ala114His + Thr217AsnSer141Glu + Trp208Gln + Leu221SerGly 69Gln + Ala114Thr + Asn170AspAsn163Asp + Ala219Thr + Leu221AlaSer 70Asp + Thr217Gly + Ala219ThrThr195Asn + Leu221Gly + Gly223ProGly 69Gln + Leu104Thr + Leu134ThrGly139Asp + Gly165Pro + Leu221GlyGly 67Asn + Gly108Glu + Tyr210IleVal115Cys + Tyr210Val + Pro214GlyLeu104Met + Thr137Asp + Trp208GlyTrp112Gln + Leu134Ala + Ser192AspGly108Glu + Val115His + Ala219GlnThr113Asn + Gly139Pro + Thr167AspThr137Asp + Trp208Gln + Gly223AsnSer207Glu + Trp208Gln + Tyr210AsnAsn 66Ser + Ser207Glu + Tyr210ProVal103Asn + Tyr210Ala + Thr215GluAsn 66Ser + Trp208Phe + Ala219ThrGly136Asn + Gly139Asn + Thr215AsnSer109Glu + Asn166Ser + Thr217AsnLeu134Ile + Tyr171Gly + Thr217GlnGly 72Gln + Gly110Asp + Gly135AsnAsn 68Glu + Asn106Gln + Thr113SerAla114Thr + Leu134Gln + Thr137GluSer107Asp + Gly139Ser + Tyr213SerThr113Asn + Thr167Gly + Leu221MetGly 67Ser + Ser207Glu + Thr217SerAsn 66Ser + Trp208Ile + Thr217SerTrp208Ser + Leu221Gly + Ser222GluGly162Pro + Ser216Glu + Leu221ThrLeu104Cys + Asn106Gln + Ser207GluGly 69Gln + Pro169Gly + Tyr210LeuThr 73Asp + Gly110Ser + Thr217SerGly 69Ser + Gly 72Asn + Thr215GlyGly135Ser + Tyr171Ser + Ser191GluThr111Asn + Phe193Leu + Thr217GluTrp208Ala + Leu221Ile + Thr224AspTrp112Phe + Pro214Asp + Thr217ProAsn 68Ser + Gly110Gln + Trp112MetGly 69Ser + Leu104Cys + Trp208AspThr137Glu + Asn166Gln + Thr215ProGly108Glu + Tyr210Thr + Thr212SerThr 73Gln + Gly162Ser + Tyr171MetTyr171Val + Phe193Asp + Leu221ThrGly 72Ser + Gly108Glu + Trp208MetThr137Asn + Trp208Asp + Tyr210GlnAsn140Asp + Phe193Thr + Trp208AlaGly 69Glu + Tyr210Ala + Tyr213SerAsn 66Asp + Tyr218Ile + Leu221CysVal103Thr + Leu134Ser + Asn163GlnTyr171Gln + Ser192Glu + Gly223SerGly 69Asn + Thr137Glu + Leu221ProPro205Ser + Tyr218Glu + Leu221AsnTyr210Met + Thr215Asp + Thr217ProThr 73Pro + Leu134Val + Gly136GluThr 73Ser + Ser107Glu + Trp208SerGly162Gln + Ser220Asp + Leu221ProVal103Glu + Gly135Gln + Trp208GlyAsn140Asp + Tyr210Cys + Leu221HisGly108Asn + Ser192Asp + Tyr210HisAsp105Glu + Trp208Asn + Tyr210ProThr 73Gln + Val103Pro + Trp112TyrLeu134Cys + Asn170Ser + Tyr210CysAsn106Ser + Ser109Glu + Leu221CysSer107Asp + Ala114Ser + Leu221AlaGly 69Asn + Gly162Asn + Leu221AsnGly 67Ser + Gly135Asp + Gly162AsnVal115Gly + Leu134Ser + Ala164GlnAsn 66Glu + Leu104Asn + Ala168GlyGly 69Ser + Asn166Glu + Tyr218AsnThr113Asp + Ala204Ser + Tyr218SerAsn166Gln + Trp208Thr + Thr217AspGly108Glu + Thr217Ser + Tyr218GlyAsn106Asp + Trp208Met + Thr217GlnGly 69Glu + Tyr171Val + Trp208HisGly139Asn + Pro169Gly + Trp208AsnVal115Ala + Thr215Gln + Leu221GluThr111Pro + Pro169Gln + Ala219GluThr 73Glu + Thr137Asn + Trp208HisAsn 66Asp + Asn106Ser + Leu221ThrGly 67Pro + Gly 69Ser + Thr224SerVal115His + Ser191Asp + Tyr218ValThr137Glu + Thr195Asn + Trp208GlyLeu134Glu + Trp208Ser + Leu221SerVal103Ser + Trp208Glu + Thr217AsnGly139Asn + Phe193Pro + Leu221CysAsn106Gln + Ala164Pro + Asn166AspAsp105Glu + Val138Ser + Thr217ProGly135Pro + Tyr171Thr + Ser222GluSer216Asp + Thr217Asn + Thr224GlyAla114Gly + Asn140Gln + Tyr218AspSer107Asp + Pro214Gly + Leu221SerAsp105Glu + Asn170Ser + Thr195ProSer 70Glu + Tyr171Leu + Pro214SerGly 67Asp + Leu104Thr + Gly223AsnGly108Asp + Ala114Asn + Tyr210CysGly110Asn + Pro214Gly + Ser220AspGly 69Asn + Pro169Gln + Ser220GluGly162Glu + Trp208Ala + Thr217AsnTrp112Tyr + Val115Pro + Ser220GluTrp208Gly + Tyr210Ile + Pro214AspLeu134Asn + Pro169Ser + Leu221AspThr113Ser + Trp208Leu + Leu221GluAsn166Ser + Tyr218Ile + Ser220GluTrp112Pro + Phe193His + Pro214AspLeu104Glu + Val115Ser + Ala164SerGly 69Pro + Pro169Asp + Leu221HisGly108Gln + Val138Ser + Ser220GluGly110Asn + Thr137Gln + Ser220GluAla164Thr + Asn166Ser + Tyr210AspTrp112Pro + Val115Pro + Pro214GluVal115Glu + Gly139Asn + Tyr210GlnGly 69Ser + Thr 73Ser + Tyr210ProAla114Ser + Ser207Asp + Thr224ProThr 73Pro + Asn166Glu + Thr212GlyGly 72Gln + Gly110Asp + Leu221HisTyr210Cys + Pro214Asn + Tyr218AspGly 69Asn + Gly136Pro + Asn166SerThr 73Gly + Gly108Ser + Ser220GluThr 73Gly + Pro205Ser + Thr224SerVal103Met + Ala164Asp + Leu221MetThr111Asn + Thr167Glu + Phe193MetThr 73Asp + Tyr210Asn + Thr217GlnAsn 66Glu + Thr 73Gly + Leu221GlnThr113Asp + Asn166Ser + Thr167ProAsn 66Gln + Tyr171Gly + Tyr210ProAsn 68Ser + Gly 69Pro + Asn170GlnAsn 68Ser + Val115Glu + Tyr210AsnThr113Gln + Ser191Asp + Leu221HisThr167Pro + Trp208Val + Tyr210LeuGly162Glu + Trp208Ala + Thr217ProLeu104Met + Ser207Asp + Thr212GlyThr137Gln + Asn163Asp + Pro205GlnVal115Thr + Thr217Gln + Ser220AspSer 70Asp + Val138Ser + Tyr210ValPro214Gly + Leu221Ile + Thr224AspLeu134Val + Gly165Gln + Trp208ThrSer109Asp + Ala114Asn + Tyr210SerAsn 66Ser + Val115Asn + Trp208GlnThr 73Pro + Phe193Glu + Gly206GlnLeu134Ser + Leu221Pro + Ser222AspVal115Cys + Ala164Glu + Asn170GlnAla164Asn + Trp208Cys + Thr217GluAsn 66Glu + Ala168Pro + Thr215ProGly135Ser + Ser207Asp + Thr217GlnAsn140Gln + Thr167Asp + Trp208MetGly165Gln + Trp208Thr + Leu221AsnThr 73Pro + Asn106Glu + Leu221CysThr113Ser + Leu221Ile + Gly223AspAsn 66Asp + Gly136Gln + Gly162GlnSer191Glu + Thr217Asn + Thr224ProLeu104Gln + Gly162Glu + Tyr210GlnTrp112Gln + Asn163Asp + Thr217GlyLeu134Pro + Thr217Asn + Leu221GluGly165Pro + Tyr210Met + Thr217GluAla164Gly + Ser191Glu + Trp208CysVal115Cys + Gly139Asn + Pro205AsnGly135Pro + Trp208Ala + Tyr210ProPro169Asn + Thr195Glu + Tyr210HisSer107Asp + Trp208Met + Tyr210ProThr 73Gly + Ala164Gly + Tyr210GluLeu104Val + Pro205Gln + Leu221AspThr111Gln + Ser141Asp + Leu221AsnTrp112Cys + Thr167Gln + Thr217ProVal138Thr + Asn170Glu + Leu221ProSer 70Asp + Leu134Ser + Thr224AsnThr137Asn + Ser207Asp + Tyr218ThrGly135Glu + Ala164Asn + Ala203SerLeu134Gly + Trp208Glu + Thr212SerAsn163Asp + Trp208Asn + Tyr210ThrVal115Thr + Ser192Glu + Thr217SerGly 72Pro + Ser109Asp + Tyr210GlnSer109Glu + Asn163Gln + Phe193ProGly136Pro + Pro169Glu + Leu221SerGly110Asn + Ser220Glu + Leu221MetTrp112Gly + Tyr210Asp + Thr217ProTrp208Ser + Tyr210Ser + Leu221ProTrp112Met + Ser192Glu + Leu221AlaThr137Gly + Thr167Ser + Thr217GluSer109Asp + Gly162Pro + Tyr210AlaAla114Thr + Gly162Asn + Ser191GluThr195Asn + Tyr218Pro + Leu221GluThr 73Glu + Ala164Gln + Pro214SerAsn 68Glu + Trp208Tyr + Thr217AsnGly165Pro + Thr212Ser + Ser220GluGly 67Asn + Gly139Gln + Ala168AspSer141Glu + Pro214Gln + Leu221GlnGly206Ser + Trp208Tyr + Thr215GlnLeu134Pro + Gly162Asn + Phe193IleTrp112Ala + Thr217Asn + Ser220AspThr111Gly + Gly139Glu + Thr212AsnSer107Glu + Tyr213Ala + Thr217ProGly165Ser + Thr217Glu + Gly223ProTyr210His + Thr217Asp + Leu221AlaTrp208Cys + Thr217Asp + Tyr218AsnGly 69Gln + Val103Ala + Ser220GluAsn106Ser + Tyr210Gly + Gly223SerTrp208Cys + Tyr210Asn + Thr217AspAsn 66Gln + Gly139Gln + Thr217GlyThr 73Glu + Leu104Ile + Tyr210IleThr113Ser + Pro169Asn + Thr217SerTyr210Gly + Pro214Gly + Leu221GluLeu134Ile + Ala219Glu + Ser220AspAsn140Asp + Ser141Glu + Tyr218SerTyr213Val + Ser220Glu + Leu221GluTyr210Val + Ser220Glu + Leu221AspAla164His + Ser220Glu + Leu221GluTyr171His + Ser220Asp + Leu221GluTyr210Ile + Ser220Glu + Leu221AspTrp112Leu + Ser220Asp + Leu221GluGly136Pro + Ser191Asp + Ser192GluAla168Ser + Thr217Asp + Tyr218GluAsn166Ser + Thr217Glu + Tyr218AspGly162Asp + Asn163Glu + Leu221MetTrp208Ser + Tyr210Glu + Ser220AspAla114Thr + Gly165Asp + Phe193GluSer207Glu + Thr217Asn + Ser222GluAsn140Ser + Trp208Glu + Ser220AspTrp208Asp + Leu221Asn + Ser222GluGly139Gln + Trp208Glu + Leu221AspAsn 68Glu + Asp105Glu + Asn106AspThr137Gln + Gly139Asp + Tyr171GluTrp208Asp + Tyr210Glu + Leu221AlaGly 69Pro + Asn166Glu + Ala168GluTyr171Cys + Tyr210Glu + Leu221AspThr137Glu + Pro169Asp + Asn170SerGly162Glu + Tyr171Asp + Thr217SerLeu134Ile + Ser191Glu + Gly223GluVal115Pro + Ser207Glu + Ser220AspGly139Pro + Ser207Asp + Ser220GluSer 70Glu + Val103Pro + Asn106AspGly 69Asp + Thr 73Asp + Tyr218IleAsn 66Asp + Trp208Gly + Pro214GluAsn 66Asp + Ser 70Glu + Gly136SerAsn163Asp + Ser191Asp + Ser207GluAsn 66Ser + Gly 69Asp + Ser109GluGly 67Glu + Trp208Ile + Thr215AspSer 70Glu + Tyr210Gln + Thr217GluSer 70Glu + Gly135Pro + Thr217GluVal138Glu + Ser141Asp + Tyr210GlnVal138Glu + Ser141Glu + Pro214AsnSer107Asp + Gly110Asp + Ala114GluSer109Glu + Ala114Glu + Gly136SerGly110Glu + Gly136Glu + Tyr210GlnGly135Ser + Tyr210Asp + Thr217GluSer 70Glu + Pro169Asn + Leu221AspVal115Gly + Gly162Asp + Thr167AspGly110Pro + Thr217Asp + Ser220AspAla164His + Thr217Asp + Ser220AspTyr210Cys + Thr217Asp + Ser220AspVal103Ser + Thr217Asp + Ser220AspThr217Glu + Tyr218Val + Ser220GluGly 69Ser + Thr217Glu + Ser220GluTyr210Val + Thr217Asp + Ser220GluGly 67Glu + Ser107Glu + Thr111AsnAsn166Asp + Ser191Asp + Leu221ValLeu134Asp + Phe193Glu + Gly223AspAsn 66Asp + Ser216Asp + Thr217GluVal103Glu + Asn106Glu + Thr113GluSer 70Asp + Gly139Ser + Ser220AspLeu104Asp + Asp105Glu + Ser191GluAla114Glu + Ser216Asp + Thr217GluThr195Glu + Ser216Asp + Thr217GluVal115Glu + Ser220Asp + Leu221GluSer109Asp + Ser220Asp + Leu221GluThr215Glu + Ser216Asp + Ser220AspSer192Asp + Phe193Glu + Thr217AspGly165Asp + Tyr210Glu + Ser220AspSer 70Glu + Asp105Glu + Tyr218AspGly 69Glu + Ala168Glu + Thr195Glu
表34
多环四突变变异体Asn 66Gln + Leu104Gly + Tyr210Pro + Thr217GluGly 67Ser + Gly 69Ser + Val103Gln + Gly223AspTrp112Cys + Ala164Glu + Trp208Gln + Thr217GlyLeu134Ile + Trp208Gln + Ala219Gly + Ser220GluLeu104Ala + Ser109Glu + Thr111Gln + Val115AlaGly108Glu + Ala164Gly + Gly165Gln + Thr167ProGly 69Glu + Asn106Gln + Trp112Cys + Thr113ProThr 73Asp + Leu134Ser + Thr137Asn + Thr217ProGly 67Ser + Gly136Pro + Thr195Ser + Leu221AlaThr 73Gly + Leu104Gly + Trp208Pro + Thr215GlyGly110Glu + Gly136Pro + Phe193Met + Thr217ProAla164Gln + Tyr171Ile + Phe193Ile + Thr224GlyGly136Pro + Ala164Thr + Ser207Asp + Trp208LeuGly108Gln + Trp112Pro + Trp208Thr + Tyr210GluThr111Asp + Leu134Val + Tyr210His + Thr217ProGly136Gln + Trp208Phe + Ala219Asp + Leu221MetThr113Ser + Tyr171Leu + Trp208Val + Tyr210ThrGly136Ser + Ser141Glu + Trp208Thr + Tyr210LeuAsn 66Ser + Thr 73Pro + Thr113Pro + Asn170GluThr 73Pro + Gly139Ser + Tyr210Glu + Leu221AlaGly108Ser + Val138Ser + Trp208Leu + Thr217SerAsn 68Ser + Gly110Gln + Trp112Met + Ser192AspThr 73Gln + Val103Ala + Gly162Ser + Ala164GluThr137Asn + Asn140Asp + Phe193Thr + Trp208AlaLeu134Ser + Asn163Gln + Ser220Glu + Gly223AsnAsn140Glu + Tyr171Gln + Tyr210Val + Gly223SerThr 73Pro + Leu134Val + Gly136Glu + Thr217ProSer107Glu + Gly162Gln + Trp208Ser + Leu221ProGly108Asn + Gly162Asn + Ser192Asp + Tyr210CysVal103Pro + Ser109Glu + Trp208Asn + Tyr210ProThr 73Gln + Trp112Tyr + Leu134Cys + Asn170SerGly 69Asn + Gly162Asn + Asn166Ser + Leu221AsnGly 67Ser + Leu134Ser + Gly162Asn + Ala164GlnLeu134Ile + Gly135Pro + Ala168Thr + Trp208MetTrp112Ser + Ala168His + Thr217Ser + Leu221AlaGly110Ser + Thr111Gln + Thr215Ser + Ser222AspThr111Pro + Thr137Asn + Pro169Gln + Ala219GluThr 73Glu + Asn106Ser + Trp208His + Leu221ThrAsn 66Asp + Gly 67Pro + Gly 69Ser + Thr224SerLeu104Cys + Val115His + Ser191Asp + Tyr218ValLeu134Glu + Thr195Asn + Trp208Ser + Leu221SerVal103Ser + Gly139Asn + Thr217Asn + Leu221CysAsn106Gln + Ala164Pro + Asn166Asp + Phe193ProAsp105Glu + Gly135Pro + Val138Ser + Thr217ProGly 67Asp + Thr113Asn + Ala114Asn + Tyr210CysAsn166Asp + Thr195Gly + Tyr210Gly + Thr224GlnTrp112Pro + Asn166Ser + Phe193His + Tyr218IleGly 69Pro + Leu104Glu + Val115Ser + Ala164SerTrp112Pro + Val115Glu + Gly139Asn + Tyr210GlnGly 69Ser + Thr 73Ser + Tyr210Pro + Thr224ProGly 72Gln + Thr 73Pro + Gly110Asp + Leu221HisThr111Asn + Gly136Pro + Tyr210Thr + Tyr218AspGly108Asp + Gly136Asn + Asn166Ser + Leu221ThrVal103Met + Pro205Ser + Leu221Met + Thr224SerGly 69Asn + Thr 73Asp + Trp208Gln + Tyr210LeuThr 73Gly + Asn166Ser + Thr167Pro + Leu221GlnAsn 66Gln + Thr113Asp + Tyr171Gly + Tyr210ProGly162Glu + Thr167Pro + Trp208Val + Tyr210LeuSer207Asp + Trp208Ala + Thr212Gly + Thr217ProLeu104Met + Thr137Gln + Asn163Asp + Pro205GlnSer109Asp + Leu134Val + Gly165Gln + Trp208ThrAla114Asn + Val115Asn + Trp208Gln + Tyr210SerAsn 66Glu + Ala164Asn + Trp208Cys + Thr215ProGly135Ser + Ala168Pro + Ser207Asp + Thr217GlnThr167Asp + Trp208Met + Thr217Gln + Leu221AsnThr 73Pro + Asn106Glu + Thr113Ser + Leu221CysGly162Gln + Ser191Glu + Thr217Asn + Thr224ProGly 69Gln + Leu104Gln + Gly162Glu + Tyr210GlnLeu104Gln + Tyr171Pro + Thr217Gly + Leu221GluGly110Glu + Phe193Asn + Trp208Cys + Gly223ProAla164Gly + Ser191Glu + Pro205Asn + Trp208CysGly135Pro + Ser207Glu + Trp208Ala + Tyr210CysThr 73Glu + Leu104Val + Pro205Gln + Leu221CysTrp112Cys + Val138Thr + Thr167Gln + Thr217ProSer109Asp + Ala164His + Gly223Pro + Thr224AsnAsn 68Gln + Tyr210Pro + Thr212Ser + Ser220AspLeu134Gly + Trp208Glu + Tyr210Met + Thr212SerAsn163Asp + Asn170Gln + Trp208Asn + Tyr210ThrTrp112Gly + Trp208Ser + Tyr210Asp + Thr217ProTrp112Met + Ser192Glu + Trp208Ser + Tyr210ValTrp112Leu + Ser220Asp + Leu221Ala + Thr224AsnLeu104Met + Thr111Asp + Ala164Gln + Pro214SerGly139Gln + Gly165Pro + Thr212Ser + Ser220GluThr113Asp + Leu134His + Trp208Ile + Leu221AlaAsp105Glu + Pro214Gln + Thr215Gln + Leu221GlnLeu134Pro + Gly162Asn + Gly206Ser + Trp208TyrTrp112Ala + Phe193Ile + Thr217Asn + Ser220AspSer107Glu + Thr212Asn + Tyr213Ala + Thr217ProGly 67Gln + Gly 69Gln + Thr113Asn + Ser220GluAsn 66Gln + Trp208Cys + Tyr210Asn + Thr217AspThr 73Glu + Leu104Ile + Gly139Gln + Thr217GlyThr113Ser + Pro169Asn + Tyr210Ile + Thr217SerLeu134Pro + Thr167Gln + Tyr210Met + Ser220AspThr 73Glu + Asn166Gln + Tyr171His + Leu221AlaVal138Met + Asn170Gln + Thr217Asn + Ala219HisGly136Gln + Ala219Ser + Ser220Asp + Thr224GlnGly 69Asn + Asn166Gln + Tyr210Glu + Thr217GlyThr 73Ser + Gly165Gln + Thr212Pro + Ser222GluThr137Pro + Asn140Gln + Tyr171Gly + Ser220GluTyr210Pro + Tyr213Ile + Ser220Asp + Leu221ProVal103Asp + Asn106Ser + Phe193His + Thr217GlyAsn 68Asp + Thr113Asn + Val138Ala + Trp208HisAsp105Glu + Leu134Met + Gly165Ser + Leu221GlnAsn 68Gln + Trp208Thr + Thr217Ser + Ser220AspTyr171Asn + Thr195Asn + Tyr210Asn + Tyr218AsnAsn 66Ser + Pro169Ser + Tyr210Cys + Gly223GluAla114Gln + Phe193Glu + Tyr210Gly + Thr224ProAsn 66Ser + Gly136Ser + Ala164Asp + Ala168GlnLeu104Gly + Gly162Pro + Ser192Asp + Leu221AsnGly108Asp + Gly162Pro + Trp208Thr + Gly223AsnAsn 66Glu + Thr113Gly + Ala164Pro + Gly223ProGly110Pro + Trp112Ala + Ser216Glu + Leu221HisVal115Met + Pro169Ser + Trp208His + Tyr210AspLeu104Ser + Ser109Asp + Gly110Pro + Thr195GlyLeu104Asn + Thr137Glu + Trp208Ser + Leu221ValAsn106Gln + Ala114Asp + Tyr171Thr + Leu221ProPro205Asn + Tyr210Asn + Thr217Gly + Tyr218LeuLeu104Pro + Asn106Ser + Thr137Asp + Thr217ProGly110Ser + Thr137Gln + Asn170Glu + Thr217AsnThr113Gly + Phe193Met + Trp208His + Tyr213IleAsn166Gln + Ser207Asp + Thr217Ser + Tyr218MetGly162Asp + Phe193Pro + Trp208Tyr + Thr217GlnGly 69Ser + Ser107Glu + Trp208Met + Tyr210LeuVal103Asp + Thr167Pro + Ile209Val + Tyr210GlnThr111Asn + Asn170Ser + Trp208Tyr + Thr212GlySer107Glu + Thr137Pro + Gly162Ser + Trp208CysVal103Thr + Ala164Thr + Ala168Glu + Thr217SerGly139Pro + Tyr210Asn + Leu221Pro + Thr224ProLeu134Ser + Ser141Glu + Tyr210Ala + Thr217GlnVal103Asn + Leu134Ala + Tyr218Met + Thr224AsnAsn 66Asp + Gly135Gln + Asn163Ser + Leu221ValGly 69Glu + Val115Pro + Thr137Pro + Trp208IleGly 69Pro + Gly165Pro + Tyr210Ser + Leu221IleAsn 66Gln + Gly110Glu + Trp112Gly + Trp208ProTrp112Ala + Ala114Pro + Gly135Ser + Asn163AspGly 67Asn + Ser207Glu + Tyr210Thr + Thr217ProAsn166Asp + Thr167Pro + Ile209His + Tyr210ValThr 73Ser + Asn106Gln + Tyr171Glu + Thr217AsnVal115Glu + Tyr210Ser + Thr217Gln + Ala219GlyLeu104Pro + Thr195Gln + Trp208Tyr + Ser222AspSer141Asp + Ala204Gln + Trp208Val + Tyr210LeuGly 67Ser + Gly 69Glu + Val103Gly + Tyr210GlnTyr171Met + Phe193Leu + Thr215Asp + Thr217GlnAsn 68Gln + Asp105Glu + Trp208Phe + Leu221AlaLeu104Asn + Val115Gln + Leu134Gln + Pro214GluGly165Ser + Ser191Asp + Pro214Gly + Leu221ThrGly 67Ser + Asn 68Gln + Tyr210Val + Ser220AspSer109Asp + Val138Gly + Trp208Ser + Thr217ProThr111Gly + Gly136Pro + Ser141Asp + Leu221IleTrp112Asn + Gly162Ser + Ile209Cys + Thr224GlnVal115Ala + Pro169Glu + Tyr210Val + Leu221CysGly135Ser + Tyr210Leu + Thr217Glu + Leu221ProAsn 66Gln + Ser109Asp + Gly110Gln + Tyr218ThrThr 73Gly + Trp208Ile + Thr217Glu + Leu221AsnLeu134Ser + Trp208His + Tyr210Leu + Ser220GluGly135Pro + Thr137Gln + Ala219Ser + Leu221GlnAsn 68Gln + Asn106Glu + Pro169Gln + Thr215GlnVal138Gln + Gly165Glu + Tyr218Leu + Leu221CysPhe193Gln + Ser207Asp + Trp208Cys + Thr217GlnGly 67Asn + Gly165Asn + Phe193Tyr + Pro205SerAsn 66Ser + Ala168Ser + Tyr210Cys + Thr217AspPro214Gly + Ser216Asp + Thr217Gln + Leu221HisAsn 66Gln + Val115Gln + Asn166Asp + Tyr218MetThr111Glu + Gly165Ser + Ala219His + Leu221ThrSer141Asp + Asn163Ser + Phe193Tyr + Ala204SerGly108Pro + Asn163Ser + Gly165Asp + Tyr213SerTrp208His + Tyr210Thr + Tyr218Gln + Ser220AspGly 69Glu + Ala164Pro + Trp208Val + Thr224SerSer109Asp + Ala203Pro + Trp208His + Thr217SerThr 73Asp + Tyr171Cys + Tyr210Pro + Leu221ProGly162Pro + Tyr218Val + Ala219Thr + Ser220GluAsn170Gln + Ser192Glu + Trp208Pro + Tyr210GlyThr113Ser + Thr137Gln + Phe193Ser + Pro205SerTrp112Pro + Thr137Gly + Ser220Asp + Leu221AsnGly162Glu + Ala164Ser + Ala168His + Pro214AsnAla114Pro + Leu134Glu + Asn140Ser + Phe193IleAla164Gln + Ala168Glu + Trp208Asn + Tyr210LeuVal103Pro + Gly139Pro + Asn163Asp + Gly165GlnGly162Ser + Trp208Glu + Tyr210Ser + Leu221GlnSerl41Asp + Tyr171His + Trp208Ala + Leu221MetAsn 66Ser + Gly139Gln + Asn163Gln + Tyr210ThrLeu134Asp + Gly136Ser + Asn163Gln + Trp208ValSer191Asp + Phe193Leu + Pro214Gln + Thr217SerGly 67Asn + Gly108Asp + Thr195Asn + Thr215ProAla114Thr + Asn140GIu + Asn166Ser + Ala203ProSer191Glu + Ala203Gly + Trp208Asn + Tyr210AlaAsn140Asp + Ser141Glu + Ala164Gln + Tyr218SerThr 73Ser + Tyr213Val + Ser220Glu + Leu221GluPhe193Met + Thr215Gln + Ser220Glu + Leu221GluThr113Ser + Trp208Leu + Ser220Glu + Leu221GluThr 73Pro + Val115Ala + Ser192Asp + Phe193GluAla114Asn + Trp208Pro + Tyr210Glu + Ser220GluTrp208Cys + Tyr210Glu + Ala219Asn + Ser220AspAla114Pro + Gly139Pro + Tyr210Asp + Ser220GluTyr210Glu + Thr217Gln + Tyr218Gln + Ser220GluAsn140Ser + Trp208Glu + Ser220Asp + Leu221AspVal103Pro + Trp208Glu + Tyr210Ser + Ser220GluThr 73Gln + Ser107Asp + Ser109Glu + Asn163GlnGly 67Gln + Ser107Asp + Ser109Glu + Leu221CysAsp105Glu + Asn106Ser + Ser109Glu + Thr217ProLeu104Glu + Asp105Glu + Ser107Asp + Leu221SerGly206Pro + Pro214Asp + Thr217Glu + Leu221AsnSer207Asp + Trp208Glu + Ser220Asp + Leu221SerGly135Asp + Gly162Glu + Tyr171Asp + Thr217SerGly 69Glu + Leu104Asp + Asp105Glu + Tyr210AlaAla168Asn + Ser220Asp + Leu221Pro + Ser222AspPhe193Gln + Ser207Glu + Thr217Ser + Leu221GluGly110Gln + Gly165Gln + Ser222Asp + Thr224AspAsn106Glu + Ser109Asp + Phe193Ile + Trp208LeuGly110Asn + Thr137Glu + Pro169Asp + Asn170SerGly136Glu + Gly162Glu + Trp208Ala + Thr217AsnLeu134Ile + Ser191Glu + Leu221His + Gly223GluGly139Pro + Tyr171Met + Ser207Asp + Ser220GluVal103Pro + Thr111Gly + Ser207Asp + Ser220AspGly108Asn + Thr195Gly + Ser207Asp + Ser220AspSer 70Glu + Val103Pro + Asn106Asp + Ala168ProThr113Gly + Gly162Ser + Asn163Asp + Asn170GluLeu104Ile + Ser107Glu + Gly110Asp + Asn170GlnThr 73Gly + Gly135Asp + Pro169Asp + Trp208ThrTyr210Met + Thr215Asp + Tyr218Glu + Leu221AsnLeu104Thr + Gly136Glu + Pro169Asp + Leu221ValAsn 66Asp + Thr 73Glu + Tyr218Ile + Leu221CysAsp105Glu + Thr111Asp + Ala114Pro + Val138MetAsn 68Glu + Thr 73Glu + Trp208Tyr + Thr217AsnSer207Glu + Tyr213Ile + Ala219Asp + Ser222AspAsn163Asp + Ala164Ser + Ser191Asp + Ser207GluThr 73Glu + Ala168Ser + Tyr210Glu + Tyr218GluGly 67Glu + Trp208Ile + Pro214Asn + Thr215AspAsp105Glu + Thr111Glu + Thr113Glu + Asn163GlnSer 70Glu + Tyr210His + Thr217Asp + Leu221AlaGly 67Glu + Gly108Asp + Thr137Gln + Thr167ProSer107Asp + Gly110Asp + Ala114Glu + Tyr218AsnSer 70Asp + Tyr210Glu + Thr217Asn + Ser220GluThr113Gly + Trp208Glu + Ser220Asp + Thr224AspVal138Met + Ala219Glu + Ser222Asp + Thr224GlnSer 70Glu + Trp112Tyr + Tyr218Cys + Leu221GluGly 67Ser + Ser 70Glu + Pro169Asn + Leu221AspVal115Cys + Gly139Asn + Thr217Asp + Ser220AspGly 69Ser + Val115Thr + Thr217Glu + Ser220GluAla168His + Trp208Tyr + Thr217Asp + Ser220GluThr111Gln + Gly135Pro + Thr217Asp + Ser220GluAla164Thr + Ser191Glu + Trp208Glu + Thr215AsnAsn 68Glu + Val103Glu + Ser109Glu + Asn166GlnSer 70Asp + Gly139Ser + Thr217Glu + Ser220AspLeu134Asp + Phe193Glu + Ala203Ser + Gly223AspAsn 66Asp + Asn 68Ser + Ser216Asp + Thr217GluAla164Glu + Trp208Cys + Leu221Glu + Ser222AspSer 70Glu + Ser207Glu + Ala219Ser + Ser220GluGly 67Asp + Ser 70Asp + Thr 73Gly + Tyr210GluTrp208Ile + Thr217Glu + Tyr218Asp + Ser222AspLeu104Ile + Thr111Glu + Gly135Asp + Asn163GlnAla114Glu + Leu134Ile + Ala219Glu + Ser220AspAsn166Glu + Thr167Gly + Ser222Asp + Gly223AspGly139Glu + Tyr210Ala + Ser222Asp + Gly223GluVal103Gly + Gly110Glu + Thr111Glu + Ser222GluAla114Glu + Asn170Ser + Ser216Asp + Thr217GluGly108Asp + Ser109Asp + Tyr210Gln + Thr217GluAsn 68Ser + Ser109Asp + Gly110Glu + Ser220GluAsn 68Glu + Asn163Ser + Ser207Asp + Trp208GluSer207Asp + Trp208Glu + Thr217Glu + Leu221CysGly 69Pro + Asp105Glu + Asn106Asp + Asn166AspSer109Asp + Tyr210Gln + Ser220Glu + Leu221AspTyr171Gly + Ser192Glu + Ser220Asp + Leu221GluAsn170Glu + Ser191Glu + Ser192Glu + Pro205SerAsn 66Asp + Tyr210Gln + Thr217Glu + Tyr218GluAsn106Asp + Ser107Glu + Thr137Asp + Tyr210GlnSer192Asp + Phe193Glu + Pro205Ser + Thr217AspSer 70Asp + Gly162Asp + Asn163Glu + Leu221SerGly 69Asn + Asn106Glu + Tyr210Asp + Ser220GluVal103Ser + Asn140Asp + Tyr210Glu + Ser220AspGly 69Glu + Phe193His + Ser207Glu + Ser222AspGly162Glu + Asn163Gln + Trp208Asp + Ser220GluVal115Cys + Asn170Asp + Trp208Asp + Ser220AspGly110Glu + Trp208Asp + Leu221Asn + Ser222GluPro169Asp + Tyr171Glu + Pro214Asn + Gly223GluAsn163Glu + Ser207Glu + Trp208Met + Tyr210ThrGly 67Glu + Ser 70Glu + Val115Cys + Gly162GluGly162Glu + Ala164Glu + Ser220Glu + Leu221GlyAsn 66Glu + Val103Asp + Gly108Asn + Leu221MetAsp105Glu + Ser109Glu + Tyr218Asp + Leu221AlaGly 67Asp + Ala164Asp + Ala168Glu + Tyr213AsnGly 69Pro + Ser141Asp + Pro214Asp + Thr217AspThr 73Glu + Trp112Ile + Trp208Gly + Ser220AspGly 72Ser + Ser107Asp + Thr111Asp + Asn166GlnGly165Glu + Ser192Asp + Thr217Asp + Leu221IleGly135Glu + Asn140Glu + Ala204His + Pro214SerAsp105Glu + Gly110Asp + Asn170Asp + Ala219GlyAsn 66Asp + Asn 68Asp + Leu134Gly + Asn163GluLeu104Thr + Ala168Asp + Asn170Asp + Ser220GluThr 73Asp + Ser109Glu + Pro214Glu + Thr217AsnThr 73Asp + Val115Cys + Pro214Asp + Thr224GluSer109Asp + Thr113Asp + Leu134Asn + Phe193ProGly 69Ser + Gly108Asp + Gly110Asp + Ser220GluGly139Asp + Ser141Asp + Ala164Asp + Leu221ValAsn 66Asp + Trp208Met + Ser220Asp + Ser222GluTrp112Asp + Tyr210Thr + Ser220Glu + Ser222GluSer109Asp + Ser207Glu + Tyr210Gly + Leu221GluGly 69Asp + Asp105Glu + Thr137Asp + Trp208AsnSer109Asp + Thr111Glu + Ser191Glu + Tyr210LeuGly139Gln + Pro169Asp + Ser191Asp + Ser207GluGly 67Ser + Asn140Asp + Ser222Asp + Thr224GluVal138Asp + Asn170Glu + Trp208His + Thr215GluAsn 68Asp + Thr111Gly + Val138Asp + Asn170AspLeu104Glu + Ser107Asp + Gly135Gln + Tyr171AspLeu104Pro + Trp112Asp + Asn140Glu + Ser222GluGly139Glu + Asn170Asp + Gly206Pro + Ser220GluAla164Asp + Asn170Asp + Ser216Asp + Thr217ProThr113Glu + Ala114His + Ser207Glu + Ser220GluGly110Glu + Val115Gln + Ser207Asp + Thr224GluAsn106Glu + Asn163Glu + Asn170Glu + Leu221HisGly 69Asp + Thr 73Glu + Thr113Asn + Ser141GluGly 69Asp + Thr 73Asp + Thr111Gly + Gly139GluSer107Asp + Asn163Glu + Ser192Asp + Trp208MetAsn 68Asp + Gly108Asp + Ser207Asp + Ile209AlaGly135Asp + Gly139Pro + Pro169Asp + Trp208GluAsn 66Glu + Thr167Gly + Trp208Glu + Pro214AspAsn 66Asp + Ser109Asp + Trp208Gly + Pro214GluGly 69Glu + Asn140Ser + Ser192Glu + Pro214AspLeu104Gly + Gly139Glu + Ser191Asp + Thr224AspAsn106Gln + Thr113Asp + Asn163Asp + Asn166GluLeu134Glu + Ser207Asp + Trp208Pro + Tyr210GluSer191Glu + Thr195Asn + Tyr218Pro + Leu221GluAsn170Asp + Phe193Ser + Ser220Glu + Thr224AspAsp105Glu + Ala114Glu + Ser192Glu + Thr224AsnGly 67Asp + Gly108Asp + Ser207Asp + Tyr210CysAsn 68Asp + Gly110Glu + Thr113Gly + Leu221AlaLeu104Ile + Ala114Glu + Ser220Glu + Gly223GluSer107Asp + Ser220Glu + Leu221Pro + Gly223AspVa1103Asp + Val138Glu + Ser141Glu + Gly206GlnVal138Glu + Ser141Asp + Tyr210Gln + Ser220AspAsn 68Asp + Val115Gly + Thr217Asp + Ser220GluAsn 66Asp + Leu104Gly + Ala168Thr + Ser216GluVal103Glu + Asn106Glu + Thr113Pro + Ser220AspAsp105Glu + Thr113Asp + Gly139Asn + Ser141GluSer 70Glu + Asp105Glu + Gly139Asp + Tyr210AlaAsn 66Glu + Tyr210Asp + Thr217Glu + Leu221AlaGly108Glu + Gly135Ser + Tyr210Asp + Thr217GluGly 69Asp + Gly108Gln + Trp112Asp + Ser141GluSer107Glu + Gly110Pro + Thr217Asp + Ser220AspGly135Glu + Thr217Glu + Tyr218Val + Ser220GluAsn163Asp + Tyr171Thr + Thr217Asp + Ser220AspAla114Thr + Thr137Glu + Thr217Asp + Ser220AspAsn166Glu + Thr212Pro + Thr217Asp + Ser220AspGly 67Asp + Ser107Asp + Tyr210Asn + Ser222GluAsn140Ser + Trp208Gly + Thr217Glu + Leu221AspAsn166Gln + Thr215Pro + Thr217Glu + Leu221AspVal115Gln + Ala168Gln + Phe193Asp + Leu221AspTrp208Ile + Pro214Ser + Ser220Glu + Thr224GluGly108Glu + Thr137Glu + Asn163Asp + Trp208SerGly139Pro + Ala164Glu + Ser191Asp + Pro214GluThr 73Asp + Thr111Pro + Asn166Asp + Ser191AspSer 70Glu + Ala168His + Thr195Glu + Ser216AspSer109Glu + Thr137Asp + Gly139Ser + Ala164GlnAsn170Gln + Tyr210Asp + Pro214Glu + Gly223SerSer 70Glu + Gly139Asn + Ala164Glu + Ser220AspAsn 66Asp + Thr113Glu + Ser141Asp + Trp208GlyAsn 66Asp + Gly108Glu + Thr215Gly + Leu221SerAsn 68Ser + Thr 73Glu + Asn166Glu + Leu221AspThr 73Asp + Ser192Asp + Ser220Asp + Leu221MetSer107Asp + Thr111Asp + Ser192Glu + Thr217Ser
表35
多环五突变变异体Asn 66Gln + Gly 67Ser + Leu104Gly + Tyr210Pro +
Thr217GluGly 69Ser + Val103Gln + Trp112Cys + Thr217Gly +
Gly223AspVal115Pro + Leu134Ile + Ala164Glu + Phe193Asn +
Trp208GlnLeu104Gly + Thr113Ser + Ala168Glu + Asn170Ser +
Ala219GlyGly 69Ser + Ser109Asp + Thr111Gln + Val115Ala +
Tyr210ProVal103Thr + Gly108Glu + Val115Pro + Tyr171Ser +
Trp208AsnLeu134Ala + Val138Cys + Asn163Gln + Tyr171Ser +
Ser220AspAsn 66Ser + Val115Gly + Ser191Glu + Trp208Ile +
Thr217SerGly 72Asn + Trp112Phe + Val115Ala + Asn166Gln +
Trp208GlyLeu134His + Gly165Gln + Trp208Pro + Tyr210Val +
Thr217GluLeu104Pro + Tyr171Leu + Trp208Leu + Ala219Glu +
Leu221SerAsn166Glu + Thr167Pro + Tyr210Pro + Tyr213Ile +
Pro214AsnGly139Gln + Asn170Glu + Trp208Cys + Thr215Ser +
Leu221AsnAla114Glu + Gly135Ser + Ala168Thr + Ala203Thr +
Trp208MetAla164His + Ala168His + Phe193Ala + Thr217Ser +
Ser220AspGly 67Pro + Gly 69Ser + Ser191Asp + Tyr218Val +
Thr224SerGly139Asn + Ala164Pro + Phe193Pro + Thr217Asn +
Leu221CysLeu104Met + Ser207Asp + Trp208Ala + Thr212Gly +
Thr217ProLeu134Val + Trp208Thr + Pro214Gly + Leu221Ile +
Thr224AspSerl09Asp + Ala114Asn + Val115Asn + Gly165Gln +
Tyr210SerLeu104Ser + Gly110Pro + Gly136Glu + Ala168Thr +
Ala219GlnGly110Pro + Leu134Pro + Asn166Asp + Asn170Ser +
Trp208AsnThr113Gln + Pro169Gly + Asn170Gln + Tyr218Gln +
Leu221SerThr137Ser + Thr195Gln + Ser207Glu + Trp208Gln +
Thr217ProGly 69Gln + Val103Gly + Trp112Met + Trp208Asn +
Pro214GlyGly136Pro + Gly162Ser + Trp208Ile + Tyr210Gly +
Pro214GlnThr137Asp + Gly165Gln + Thr195Ser + Trp208Pro +
Thr212SerAsn 66Ser + Pro169Ser + Tyr210Asn + Tyr218Asn +
Gly223GluSer 70Glu + Asn106Gln + Val138Ala + Ala168Gln +
Leu221ValGly 69Ser + Thr113Asp + Ala114Gln + Trp208Thr +
Ala219SerThr113Gln + Ala168Asp + Pro169Ser + Asn170Ser +
Ala219ThrGly139Ser + Asn170Gln + Tyr171Leu + Trp208Cys +
Thr215SerGly 67Ser + Ala114Asn + Gly139Ser + Tyr210Thr +
Tyr218SerTrp112Glu + Ala164Pro + Tyr171Pro + Thr195Asn +
Thr217SerGly 67Glu + Val115Asn + Gly162Gln + Gly165Gln +
Phe193ValGly135Ser + Tyr171Cys + Trp208His + Tyr210Met +
Leu221ValGly 69Asn + Asn106Ser + Asn166Glu + Tyr210Cys +
Leu221GlyGly135Ser + Tyr171Asn + Trp208His + Ser216Glu +
Leu221GlyGly 69Asn + Ser109Asp + Gly165Asn + Phe193Asn +
Trp208HisPro169Ser + Ser207Glu + Pro214Asn + Thr215Asn +
Thr217GlySer 70Asp + Asn163Ser + Tyr210Val + Tyr213Asn +
Leu221AlaGly108Gln + Gly110Ser + Gly135Gln + Thr137Asn +
Trp208GluAsn 68Ser + Ala164Gln + Asn170Glu + Thr217Ser +
Gly223SerGly 69Ser + Trp112Cys + Ala164Gly + Leu221Ser +
Gly223GluGly 69Glu + Gly 72Gln + Thr137Pro + Trp208Ile +
Tyr218ProVal103Gln + Val138His + Gly162Ser + Asn166Asp +
Trp208HisVal115Gln + Leu134Cys + Ala168Ser + Pro169Asp +
Tyr210SerGly 67Gln + Trp112Leu + Phe193His + Thr195Glu +
Thr217SerGly 67Gln + Leu104Asn + Ala164Glu + Ala204Ser +
Pro205SerGly135Ser + Val138Ser + Asn140Glu + Tyr210Leu +
Thr217GlyGly 67Asn + Thr 73Ser + Tyr171Leu + Phe193Tyr +
Tyr210ProGly135Pro + Trp208His + Tyr210Leu + Ala219Ser +
Leu221GlnAsn 68Gln + Asn106Glu + Thr137Gln + Pro169Gln +
Thr215GlnThr137Asp + Val138Gln + Trp208Ile + Tyr218Leu +
Leu221CysGly 67Asn + Gly165Asn + Phe193Tyr + Pro205Ser +
Trp208CysAsn 66Ser + Thr 73Ser + Ser107Asp + Ala168Ser +
Tyr210CysThr137Gly + Tyr210Pro + Thr215Pro + Ser216Asp +
Thr217SerThr 73Pro + Trp112Asp + Gly139Pro + Thr215Pro +
Leu221HisLeu104Cys + Thr111Pro + Trp208Pro + Leu221Cys +
Ser222AspThr113Gln + Ala204His + Trp208Phe + Thr217Asp +
Leu221CysThr111Gly + Gly139Gln + Tyr171Leu + Tyr210Met +
Thr217GluGly110Asn + Val115Gln + Pro169Gln + Thr195Gly +
Ser220AspTyr171Asp + Gly206Ser + Thr217Gln + Ala219Gly +
Leu221ThrSer109Glu + Thr111Ser + Val115Cys + Val138Cys +
Tyr171SerLeu104His + Asn163Asp + Trp208Thr + Tyr210Cys +
Thr217AsnGly136Asn + Gly139Pro + Ser141Glu + Pro169Gln +
Ala219HisGly 69Ser + Gly136Ser + Thr137Asn + Thr195Pro +
Ala219GluAsn106Asp + Gly162Ser + Asn163Gln + Pro169Gly +
Leu221ProAla168Gly + Phe193Met + Pro205Asn + Tyr210Asn +
Leu221AsnGly 69Pro + Gly139Asp + Ala164Gly + Tyr213Ser +
Thr217SerGly 72Ser + Leu104Gln + Asn166Glu + Pro169Asn +
Thr215GlyLeu134Cys + Trp208Asn + Tyr210Ala + Tyr218Gly +
Thr224GlyLeu104Gln + Thr195Asn + Trp208Val + Tyr210Pro +
Leu221CysGly 67Pro + Thr 73Asn + Trp208Pro + Ala219Asn +
Ser220AspThr 73Gln + Gly108Pro + Gly139Pro + Ser192Glu +
Gly223GlnAsn 68Glu + Thr 73Gln + Pro169Gln + Tyr210Pro +
Leu221MetLeu104His + Thr113Gln + Asn140Gln + Thr167Asn +
Phe193AspGly 67Pro + Asn 68Ser + Gly139Asp + Thr215Gly +
Thr217ProGly136Ser + Val138Met + Thr167Gln + Tyr218Asn +
Ser220AspAsp105Glu + Gly110Ser + Trp112His + Tyr210Gln +
Ala219ThrAsn 68Asp + Asn106Gln + Ala164Ser + Thr195Gln +
Trp208CysLeu104Val + Leu134Ala + Thr137Asn + Asn170Asp +
Leu221IleGly 67Ser + Leu134Met + Trp208Ala + Tyr210Ser +
Thr217ProAla114Gly + Gly135Asp + Gly139Ser + Asn170Ser +
Thr217SerThr111Asn + Phe193Ser + Pro205Gln + Trp208Thr +
Leu221GluSer109Asp + Val138Asn + Phe193Ala + Trp208Cys +
Thr212GlnSer 70Asp + Asn166Ser + Ile209Pro + Tyr210Met +
Leu221SerAsn106Ser + Leu134Cys + Gly136Ser + Ala168Thr +
Tyr210GluGly 67Gln + Leu134Gln + Gly135Asp + Phe193His +
Thr224GlyAsn 68Ser + Leu104Ala + Gly162Asp + Tyr171Met +
Thr217GlyAsn163Glu + Thr212Gln + Thr215Asn + Thr217Asn +
Leu221MetGly 69Gln + Asn106Gln + Ser207Glu + Trp208Thr +
Thr217GlyGly139Glu + Ala168His + Thr195Gly + Leu221His +
Thr224AsnGly 72Ser + Ala114Asn + Leu134His + Pro169Glu +
Trp208TyrVal138Ser + Ala164Asn + Tyr210Cys + Tyr218Leu +
Ser220AspLeu104Gln + Tyr171Glu + Trp208Gly + Thr217Gln +
Ala219HisAsn106Ser + Tyr171Gly + Phe193Gln + Thr215Gln +
Ala219GlnGly 72Ser + Gly108Asn + Trp112Ile + Asn166Glu +
Leu221CysAsn 68Ser + Leu104Thr + Thr215Ser + Ala219Thr +
Thr224AspVal138Ser + Asn163Asp + Phe193Cys + Pro205Asn +
Thr217SerGly 67Ser + Tyr210Pro + Tyr218Thr + Ser220Asp +
Leu221SerGly135Asn + Gly136Gln + Gly162Ser + Tyr210Asn +
Pro214AsnThr195Ser + Trp208Pro + Tyr210Gly + Ala219Ser +
Leu221IleAsn 68Glu + Thr111Pro + Asn163Ser + Phe193Tyr +
Leu221ValGly 67Ser + Thr 73Pro + Asn170Glu + Tyr210Leu +
Leu221ValGly135Asn + Gly139Ser + Phe193Asp + Thr217Gly +
Leu221GlyGly136Gln + Gly139Gln + Gly165Ser + Tyr210Asp +
Thr217ProSer141Glu + Gly162Pro + Thr167Gly + Tyr210Ile +
Thr217SerLeu104Pro + Gly110Pro + Gly136Pro + Pro205Asn +
Thr217GluVal115Ala + Ala164Thr + Ser192Asp + Trp208Leu +
Tyr210MetThr 73Asn + Val103Asn + Thr215Asn + Thr217Gln +
Ser222AspVal103Pro + Trp208Tyr + Ile209Cys + Tyr210Thr +
Thr217GlnTrp112Phe + Ala114Ser + Pro169Gln + Ser220Glu +
Leu221HisGly108Asn + Leu134Met + Tyr210Ser + Tyr213Ile +
Pro214GluAla168Pro + Trp208His + Tyr210Ala + Ser220Glu +
Leu221GlnGly139Pro + Asn140Glu + Ser141Asp + Asn166Ser +
Trp208ThrVal103Asn + Thr111Gly + Tyr210Gln + Ser216Asp +
Thr217GluVal138His + Gly139Asn + Ser207Asp + Trp208Glu +
Leu221GlnAla168Asn + Pro169Gly + Thr217Pro + Ser220Asp +
Leu221AspTyr210Leu + Ala219Gly + Ser220Asp + Leu221Asp +
Gly223SerVal103Ala + Pro169Asn + Thr217Gln + Ser220Asp +
Leu221AspThr 73Asn + Asn170Asp + Tyr171Glu + Tyr210Ile +
Leu221SerGly136Ser + Ser191Asp + Ser192Glu + Tyr218Cys +
Leu221AsnThr 73Pro + Val115Ala + Ser192Asp + Phe193Glu +
Trp208GlnThr137Gln + Gly162Asp + Asn163Asp + Ala203His +
Pro205GlnAsn163Gln + Tyr210Asp + Thr215Pro + Ser220Glu +
Gly223ProAsp105Glu + Asn106Glu + Ser107Asp + Trp208Phe +
Tyr210SerTrp112Gly + Phe193Gln + Ser207Asp + Trp208His +
Ser222AspGly108Asn + Ser207Glu + Tyr213Thr + Thr215Pro +
Ser222AspLeu134Met + Ser207Asp + Trp208Val + Tyr210His +
Ser222AspGly 69Glu + Asn106Asp + Ala114His + Ala168Ser +
Thr217GlnLeu104Pro + Trp208Glu + Thr212Asn + Thr215Ser +
Ser222AspThr111Pro + Thr137Asn + Pro169Gln + Tyr210Glu +
Ala219GluThr 73Gly + Val115Gly + Trp208Glu + Tyr210Asp +
Ser220GluGly135Gln + Gly136Glu + Asn170Ser + Tyr171Glu +
Trp208GlnThr 73Ser + Ser107Asp + Ser109Asp + Gly136Pro +
Tyr210GlyLeu134Pro + Pro169Gln + Pro214Glu + Ser216Glu +
Thr217GlyAsp105Glu + Ser109Glu + Pro169Gly + Tyr171Gln +
Thr212SerGly135Pro + Val138Ala + Ser207Asp + Tyr210Thr +
Gly223GluGly135Pro + Ser207Glu + Trp208Ala + Tyr210Pro +
Gly223GluPro169Gln + Phe193Val + Tyr210Glu + Thr215Asn +
Leu221AspAla114Gln + Gly136Ser + Ala219Asn + Ser220Asp +
Ser222AspLeu134Ser + Thr167Gly + Ser220Asp + Leu221Pro +
Ser222AspGly108Gln + Trp112Leu + Tyr218Glu + Ser220Asp +
Leu221GluGly108Pro + Ala114Asn + Ser207Asp + Tyr210Glu +
Ser220GluThr137Glu + Gly162Glu + Pro169Asn + Asn170Ser +
Tyr171AspAsn 68Gln + Gly162Glu + Ala164Thr + Gly165Asp +
Tyr171AlaThr137Pro + Gly139Asp + Asn170Glu + Trp208Met +
Tyr210ThrAsn 68Gln + Val138Ser + Gly162Pro + Ala164Glu +
Asn170AspAsn 68Gln + Ser207Glu + Trp208Thr + Thr217Ser +
Ser220AspVal138Ala + Phe193Thr + Ser207Asp + Tyr210Met +
Ser220AspPhe193Ala + Trp208Glu + Ile209Ser + Leu221Cys +
Gly223GluThr111Gly + Phe193Gln + Ser207Asp + Ser220Asp +
Gly223GluSer 70Asp + Asp105Glu + Gly108Glu + Thr111Ser +
Tyr210ValGly136Glu + Gly162Asp + Gly165Ser + Ala168Pro +
Thr195GluLeu104Gly + Gly165Glu + Pro169Glu + Thr217Asn +
Leu221AlaThr195Asn + Tyr210Asp + Thr217Glu + Ser220Glu +
Gly223ProTrp208Phe + Tyr210Asp + Tyr213Cys + Thr217Glu +
Ser220GluSer 70Asp + Gly108Glu + Tyr171Gly + Thr217Gln +
Leu221ProThr195Gly + Ser207Glu + Tyr210Glu + Tyr213Cys +
Ala219SerAsn 68Glu + Asp105Glu + Gly110Asp + Thr111Pro +
Asn140SerSer 70Asp + Val103Gln + Pro214Gln + Ser220Asp +
Leu221GluAsn 66Asp + Thr 73Glu + Asn106Ser + Trp208His +
Leu221ThrAla164Asp + Thr167Asp + Tyr171Pro + Ala219Gln +
Leu221ThrAla114His + Ala219Gly + Ser220Glu + Ser222Glu +
Thr224GluAsn106Asp + Ser109Asp + Thr111Asp + Thr137Ser +
Pro205AsnLeu104Cys + Asn163Glu + Ser192Asp + Ala219Pro +
Ser222GluGly162Asp + Ala164Gln + Ser207Asp + Trp208Tyr +
Gly223AspSer 70Asp + Ser107Glu + Ser109Glu + Val115Cys +
Gly135SerAla114Gly + Ala219Asp + Ser220Glu + Leu221Asn +
Gly223GluAsn 66Asp + Asp105Glu + Val115Gln + Thr137Ser +
Trp208HisGly 69Glu + Thr 73Glu + Val103His + Ser107Asp +
Leu221AlaGly108Pro + Trp112Gln + Gly162Asn + Ser220Glu +
Gly223AspAsn 68Asp + Asn170Gln + Tyr171Pro + Thr215Asp +
Ser216AspSer 70Asp + Tyr171Ser + Thr195Gly + Tyr210Asp +
Ser220GluThr113Gly + Gly162Ser + Trp208Glu + Ser220Asp +
Thr224AspSer 70Glu + Asn140Ser + Ala203Gln + Ala219Glu +
Gly223ProTrp112Pro + Asn170Ser + Ser216Asp + Thr217Asp +
Ser220AspThr 73Asn + Val138Asp + Pro169Glu + Asn170Ser +
Thr217GlySer 70Asp + Asp105Glu + Gly162Pro + Trp208Val +
Thr217SerGly 67Asn + Asn140Ser + Ala219Glu + Leu221Met +
Ser222GluGly 72Ser + Gly110Pro + Trp112Ala + Tyr210Asp +
Thr217GluGly108Pro + Trp112Leu + Leu134Cys + Gly135Asp +
Ala164AspAsn 66Gln + Trp208Ala + Thr217Glu + Ser220Glu +
Leu221ThrThr 73Gly + Leu134Ser + Thr217Glu + Ser220Glu +
Leu221AsnGly 67Ser + Ala168Gly + Pro214Gln + Thr217Glu +
Ser220AspVal115Cys + Glyl39Asn + Tyr210Cys + Thr217Asp +
Ser220AspLeu134Glu + Thr137Glu + Thr195Asn + Trp208Ser +
Leu221SerAla164Gln + Tyr210Val + Thr215Asp + Thr217Glu +
Ser220AspGly 67Asp + Ser 70Asp + Thr217Gly + Tyr218Asp +
Leu221IleSer 70Asp + Thr111Asn + Trp208Cys + Thr217Glu +
Ser220GluLeu134Glu + Gly162Ser + Asn166Gln + Ser207Asp +
Gly223AspGly162Pro + Thr167Asn + Pro169Glu + Ser191Glu +
Phe193AspSer 70Glu + Trp112Tyr + Ser207Glu + Tyr218Cys +
Leu221GluAsn 66Glu + Leu104Glu + Ser107Asp + Trp208Ser +
Thr215ProLeu104Ala + Gly135Asp + Thr195Asp + Tyr213Ile +
Thr217GlyThr167Gln + Ser192Glu + Ser207Glu + Trp208Cys +
Ser220AspGly165Pro + Ser191Asp + Trp208Gly + Ser220Asp +
Leu221GluGly135Glu + Asn163Asp + Tyr213Met + Leu221His +
Ser222AspThr111Asp + Gly136Glu + Gly162Glu + Ala164Asn +
Pro214SerThr113Gln + Tyr213Thr + Ser216Glu + Thr217Glu +
Leu221GluSer 70Glu + Glyl08Glu + Ala164Gly + Gly165Gln +
Leu221AspSer 70Glu + Leu104Gln + Ser220Asp + Leu221His +
Gly223ProGly165Asp + Pro169Ser + Ser207Glu + Trp208Gly +
Ser222AspAla168Pro + Pro169Asn + Thr215Asp + Thr217Asp +
Leu221GluGly110Pro + Thr113Ser + Gly165Glu + Trp208Asp +
Ser222AspGly110Glu + Thr113Asp + Gly139Glu + Trp208Tyr +
Tyr210AlaAsn 68Glu + Asn106Glu + Tyr171His + Tyr210Gln +
Ser216GluAla164Glu + Ala168His + Ala219Glu + Ser220Asp +
Leu221CysAsn 68Asp + Tyr210Gln + Ala219Glu + Ser220Glu +
Leu221SerThr 73Asp + Asn163Ser + Ala219His + Ser222Glu +
Gly223GluGly 67Asp +Asn 68Glu + Ala168Gln + Pro169Glu +
Phe193AsnGly110Asp + Gly139Gln + Asn140Glu + Ser141Glu +
Tyr218AsnSer109Glu + Ser216Glu + Thr217Asp + Tyr218Asn +
Leu221HisGly135Pro + Gly136Ser + Phe193Asp + Ser216Asp +
Thr217GluGly165Gln + Ser192Glu + Ala203Ser + Ser216Asp +
Thr217AspGly 67Gln + Leu134His + Gly136Asp + Ser207Asp +
Trp208GluVal103Ser + Ser207Asp + Trp208Glu + Thr217Glu +
Leu221CysGly 69Pro + Asp105Glu + Asn106Asp + Asn166Asp +
Ile209ThrTrp112Tyr + Asn170Glu + Trp208Gln + Ser220Glu +
Leu221GluThr137Gly + Gly139Gln + Gly162Asp + Ser220Glu +
Leu221AspThr113Asp + Ala164Gly + Ser191Glu + Ser192Glu +
Thr195GlySer107Glu + Thr113Glu + Ala114Asp + Tyr210Pro +
Leu221AlaAsn 66Asp + Gly 67Asp + Leu134Pro + Tyr210Asn +
Ser222AspAsn 68Gln + Leu104Ala + Pro169Asp + Asn170Glu +
Leu221AspAsn106Glu + Ser107Asp + Gly139Asp + Ala203Asn +
Thr217SerThr 73Gly + Thr137Glu + Tyr210Asp + Thr217Gln +
Ser220GluTrp112Ile + Gly136Glu + Phe193Thr + Tyr210Glu +
Ser220GluTrp112Ala + Asn163Ser + Ser192Glu + Tyr210Glu +
Ser220GluSer 70Glu + Thr 73Ser + Val103Asp + Pro214Glu +
Tyr218GlyAsn106Glu + Ser109Glu + Thr195Gln + Trp208Pro +
Pro214GluAsn 68Gln + Val103Asp + Thr137Gln + Ser207Asp +
Ser222AspThr137Asp + Ser207Glu + Thr217Gly + Leu221Pro +
Ser222AspGly 69Asp + Thr 73Glu + Thr111Pro + Gly139Asn +
Ser220AspAsn 66Glu + Ser 70Asp + Pro214Gly + Ser220Glu +
Leu221SerSer 70Glu + Leu104Cys + Tyr210Ser + Thr215Glu +
Ser220GluThr167Pro + Ser216Glu + Ala219Glu + Leu221Met +
Ser222AspVal138Asp + Asn166Glu + Thr167Asp + Ala168Thr +
Leu221AlaLeu134Pro + Serl41Glu + Trp208Glu + Thr217Ser +
Ser220GluGly135Glu + Tyr171Asn + Pro205Gln + Trp208Glu +
Ser220AspTrp112Asn + Thr167Glu + Ala168Gly + Trp208Asp +
Ser220GluAsn140Asp + Pro169Gln + Trp208Glu + Thr215Gln +
Ser220GluAsn106Gln + Gly136Asp + Trp208Glu + Thr217Ser +
Ser220GluAsn106Asp + Thr111Asn + Thr167Gly + Tyr210Asp +
Ala219AspAsn 66Gln + Pro169Asp + Tyr171Glu + Pro214Asn +
Gly223GluTrp112Gly + Asn163Asp + Thr167Glu + Ser191Glu +
Gly223SerTrp112Asp + Thr113Ser + Ala114Glu + Thr217Glu +
Gly223GlnAsn 66Asp + Thr 73Gly + Val103Asp + Thr111Pro +
Thr195AsnAsn 68Asp + Trp112Met + Trp208Asn + Pro214Asp +
Ser220AspAsp105Glu + Ser109Glu + Asn163Glu + Asn166Ser +
Tyr218GlyGly110Gln + Trp112Asp + Leu134Pro + Pro214Asp +
Thr217AspGly 67Pro + Thr113Glu + Gly135Asp + Gly162Asp +
Thr215GlnSer 70Glu + Thr195Ser + Pro214Ser + Ser220Glu +
Gly223GluGly110Pro + Val138Glu + Trp208Glu + Tyr210Glu +
Thr217GlyGly108Asn + Asn163Glu + Ala219Asp + Leu221Val +
Thr224AspThr 73Asp + Gly108Asn + Ser109Glu + Pro214Glu +
Thr217AsnThr137Asp + Gly139Glu + Asn170Ser + Trp208Cys +
Tyr210AspTrp112Phe + Gly139Glu + Tyr213Thr + Ala219Glu +
Leu221GluLeul04Pro + Pro169Glu + Tyr210Glu + Leu221Asp +
Thr224GlyAsp105Glu + Gly165Glu + Thr167Gln + Ala168Glu +
Tyr210ProThr137Asp + Trp208Asn + Tyr218Ser + Ser220Asp +
Ser222AspGly 69Glu + Thrll3Gln + Thr217Ser + Ser220Glu +
Ser222GluLeu104His + Thr113Asp + Ser207Asp + Thr217Ser +
Leu221GluAla114Thr + Gly135Asp + Ser207Asp + Thr217Ser +
Leu221AspGly 69Ser + Leu104Asp + Asn106Glu + Ser191Glu +
Thr217GlyVal103Ala + Tyr171Val + Ser207Asp + Ser216Asp +
Tyr218AspGly 69Ser + Leu134Ile + Gly162Asp + Ser207Glu +
Ser220GluGly 72Pro + Thr113Glu + Asn166Asp + Thr167Asp +
Thr217GlnAsn166Glu + Thr167Asp + Ser207Glu + Thr217Gly +
Leu221ValThr111Asn + Trp112Glu + Val138Gly + Asn163Asp +
Tyr171GluGly 67Pro + Asn106Glu + Leu134Pro + Thr137Asp +
Pro169GluThr195Glu + Ser207Asp + Ser220Asp + Leu221Val +
Thr224GlyAsn 68Asp + Gly 69Gln + Gly162Asp + Pro169Ser +
Gly223AspThr 73Ser + Gly165Asn + Ser207Asp + Tyr210Asp +
Ser216GluVal103Glu + Ala164Asp + Asn170Glu + Trp208Ala +
Tyr210IleAla114Thr + Ala164Asp + Asn170Glu + Tyr210Ala +
Thr215AspAsn 68Glu + Gly139Pro + Ala164Pro + Ser191Asp +
Pro214GluAsn 68Glu + Ala164Glu + Gly165Ser + Tyr210Gly +
Pro214AspGly 67Ser + Thr167Gly + Ser207Glu + Ser216Asp +
Ser220AspAsp105Glu + Gly110Asn + Ser207Asp + Ala219Gly +
Ser220GluVal103Gly + Asn140Asp + Ser207Asp + Tyr213Asn +
Ser220AspSer109Glu + Gly165Asn + Ser207Glu + Tyr210Cys +
Ser220AspGly110Asn + Asn140Glu + Ser207Glu + Thr217Ser +
Ser220GluGly110Asp + Gly136Pro + Gly206Gln + Ser207Glu +
Ser220GluThr113Gln + Leu134Ile + Ser192Glu + Thr195Asp +
Ser220AspGly136Glu + Trp208Glu + Thr217Gly + Leu221Asn +
Gly223AspSer107Asp + Gly110Asp + Leu134Ser + Asn140Glu +
Tyr210ThrThr167Glu + Trp208Asp + Thr217Asn + Ala219Asp +
Gly223AsnGly 69Glu + Ala114Asp + Thr195Ser + Trp208Ser +
Pro214GluVal103Asp + Thr113Glu + Phe193Tyr + Trp208Asp +
Ala219ProAsn106Asp + Gly136Ser + Val138Met + Ser191Asp +
Ser222AspGly135Asp + Gly139Glu + Tyr210Ala + Thr217Ser +
Ser222AspThr113Asp + Asn140Ser + Ala203Gln + Ser207Glu +
Tyr210GluGly108Glu + Thr113Gln + Ser207Glu + Tyr210Asp +
Pro214AsnGly135Glu + Asn163Asp + Tyr210Met + Tyr218Pro +
Ser220AspAsn163Asp + Ala164Thr + Ser191Glu + Thr217Asp +
Thr224GlnAsn 68Glu + Leu104Asp + Val115Thr + Ala164Asp +
Pro214GlnThr 73Gly + Ser107Glu + Gly108Pro + Leu221Glu +
Thr224AspGly 67Ser + Gly162Asp + Thr167Asp + Ser207Glu +
Thr217SerGly 67Gln + Ser109Glu + Val138Ser + Pro214Glu +
Ala219GluSer107Glu + Gly135Glu + Trp208His + Thr217Ser +
Thr224GluAsn 66Glu + Asp105Glu + Val138His + Ser192Glu +
Tyr210IleAsn 66Glu + Asp105Glu + Ala219Gln + Ser220Glu +
Leu221AsnGly 69Ser + Ser 70Glu + Gly110Glu + Thr217Asp +
Leu221AlaTyr171Glu + Ser192Asp + Ser207Glu + Trp208Pro +
Tyr210MetGly 67Gln + Asp105Glu + Ala164Asp + Pro169Asn +
Gly223AspThr 73Asp + Ser141Asp + Trp208Leu + Ser216Glu +
Thr217ProAsn 66Asp + Trp208Gln + Ile209Thr + Tyr210Val +
Ser216GluAsn 66Ser + Val103Glu + Asn106Asp + Ser191Glu +
Tyr210AlaAsn 68Gln + Leu134Glu + Ser192Glu + Leu221Gly +
Ser222GluSer 70Asp + Asp105Glu + Gly108Ser + Gly139Glu +
Thr217GlyGly 67Ser + Asn106Asp + Gly165Gln + Ala219Asp +
Ser222GluAsn106Gln + Leu134Met + Asn140Asp + Ala219Asp +
Ser222AspGly 69Glu + Val103Gln + Leu134Asp + Thr167Gln +
Trp208GlyThr137Glu + Gly165Glu + Tyr210Ser + Thr217Ser +
Leu221ProSer107Asp + Gly162Asp + Thr167Asp + Tyr171Cys +
Trp208IleAsn106Glu + Trp112Asp + Leu134Gly + Ser141Glu +
Phe193SerAla114Thr + Asn140Asp + Phe193Thr + Thr217Glu +
Ser220GluThr113Ser + Val115Asp + Leu134Val + Thr217Asp +
Ser220GluGly110Asp + Asn170Gln + Thr217Glu + Tyr218Ala +
Ser220AspThr167Asp + Phe193Thr + Thr217Asp + Ala219Gln +
Ser220AspGly108Glu + Pro214Asn + Thr217Glu + Ser220Asp +
Leu221ValAsn106Gln + Gly135Glu + Val138His + Thr217Glu +
Ser220GluGly110Glu + Tyr210Met + Thr217Glu + Ser220Asp +
Leu221HisGly 67Glu + Ser107Glu + Gly110Ser + Thr113Glu +
Thr215SerGly 67Glu + Ser107Glu + Thr111Pro + Ala168His +
Ser220GluThr111Asp + Gly136Asp + Trp208Glu + Tyr210Met +
Thr217GlnAsp105Glu + Gly162Ser + Asn166Asp + Ser191Asp +
Trp208PheAsp105Glu + Asn166Glu + Ser191Glu + Trp208Ala +
Leu221HisGly 67Glu + Val103Glu + Ala164Asn + Ser207Asp +
Tyr218AlaGly 72Asn + Leu104Asp + Asn106Gln + Thr113Asp +
Thr217AspSer 70Asp + Gly135Glu + Ala168Glu + Tyr210Ile +
Ala219GlyAsn106Gln + Ser109Glu + Thr113Asp + Thr137Glu +
Trp208AsnAla114Ser + Val138Thr + Pro169Asn + Phe193Asp +
Trp208GluAsn 68Asp + Val138Asp + Ala164Gly + Ala168Glu +
Thr217GlyGly 69Asn + Val138Asp + Ala168Glu + Ser207Asp +
Thr217ProGly110Ser + Thr111Gln + Ala164Glu + Thr215Ser +
Ser222AspLeu104Ser + Gly108Glu + Val115Glu + Val138His +
Ser141GluSer 70Glu + Ala114Ser + Thr137Ser + Ser207Asp +
Ser216AspGly 69Asp + Gly110Asp + Thr137Glu + Pro169Ser +
Tyr171HisSer 70Asp + Thr137Glu + Ala164His + Trp208Ile +
Ser220GluSer 70Glu + Thr137Glu + Thr217Asn + Ser220Glu +
Gly223AsnGly110Gln + Trp112Cys + Val115Glu + Asn140Asp +
Ser216GluAsn 68Asp + Ser109Glu + Asn166Gln + Trp208Pro +
Leu221AspThr113Pro + Thr167Glu + Tyr171Glu + Thr217Gln +
Ser220GluTrp112Ile + Leu134His + Gly139Asp + Gly162Glu +
Trp208ValGly110Asp + Gly162Gln + Trp208Ile + Tyr210Glu +
Gly223AspGly 67Asp + Thr111Asp + Trp208Val + Tyr210Ser +
Ser216GluGly 69Asn + Thr 73Glu + Asn106Asp + Ser141Glu +
Gly162AsnGly 69Glu + Gly135Asn + Pro169Asn + Trp208Ala +
Thr217GluAla203Gly + Trp208Ala + Pro214Glu + Ala219Gln +
Leu221AspThr 73Gly + Gly110Glu + Pro214Asp + Ser220Glu +
Leu221AsnVal103Ala + Thr167Gly + Ser192Glu + Pro214Glu +
Ser220AspAsp105Glu + Tyr171Gln + Pro214Asp + Thr217Ser +
Ser220AspTyr171Val + Ser191Asp + Trp208Pro + Pro214Glu +
Ser220GluGly 67Asp + Gly162Ser + Gly206Ser + Ser207Glu +
Thr217GluGly 67Glu + Trp112Phe + Ser207Asp + Thr217Asp +
Ala219GlnAsn 68Asp + Asn163Glu + Phe193Met + Gly206Ser +
Leu221GluAsn 66Asp + Leu104Ala + Ser109Glu + Ala168Thr +
Ser216GluVal138Met + Gly139Asp + Gly162Ser + Ala168Glu +
Ser220Asp
表36
多环六突变变异体Asn 66Gln + Gly 67Ser + Gly 69Ser + Leu104Gly +Tyr210Pro + Thr217GluSer 70Asp + Gly110Pro + Gly136Gln + Phe193Ser +Trp208Cys + Leu221HisThr113Pro + Gly139Asn + Ser141Glu + Tyr171Pro +Trp208Thr + Leu221GlyGly 69Gln + Leu104Thr + Ser107Asp + Leu134Thr +Thr215Ser + Leu221GlyLeu104Met + Leu134Ala + Thr137Asp + Trp208Gly +Tyr210Val + Pro214GlyGly 69Ser + Thr 73Asp + Gly110Ser + Tyr210Leu +Thr215Gly + Thr217SerAsn 66Ser + Gly 72Gln + Thr 73Pro + Gly110Asp +Trp208Val + Leu221HisThr111Asn + Ala164His + Thr167Glu + Phe193Met +Tyr210Asn + Thr217GlnAsn 66Gln + Thr 73Gly + Thr113Asp + Asn166Ser +Thr167Pro + Tyr210ProLeu104Met + Pro205Gln + Ser207Asp + Trp208Ala +Thr212Gly + Thr217ProLeu134Val + Gly165Gln + Trp208Thr + Pro214Gly +Leu221Ile + Thr224AspAsn 66Ser + Ser109Asp + Ala114Asn + Val115Asn +Trp208Gln + Tyr210SerAsn106Gln + Ala164Pro + Gly165Ser + Tyr171Ala +Ser220Glu + Thr224SerGly110Ser + Gly136Pro + Ile209His + Thr217Gln +Leu221Ile + Thr224AspAsn 66Gln + Val115Pro + Trp208Asp + Thr217Ser +Leu221Ile + Thr224ProGly 67Glu + Thr 73Asn + Asn170Ser + Tyr210Ser +Thr212Gln + Gly223ProVal103Gly + Ala164Thr + Tyr210His + Ala219His +Ser220Glu + Leu221GlyThr 73Pro + Asn140Gln + Thr167Asp + Trp208Met +Thr217Gln + Leu221AsnSer109Asp + Ala164His + Tyr210Pro + Thr212Ser +Gly223Pro + Thr224AsnGly110Gln + Tyr210Pro + Tyr213Thr + Tyr218Met +Ser220Asp + Leu221HisVal138Pro + Ala168Gly + Pro169Gln + Tyr210Ser +Thr217Gln + Ser220AspAla114Thr + Thr137Asn + Gly139Ser + Trp208Pro +Pro214Gln + Ser220GluVal103Met + Thr137Ser + Ala203Ser + Tyr210Thr +Leu221Met + Gly223SerLeu104Glu + Thr111Ser + Val115His + Gly165Asn +Tyr210Met + Ala219HisGly110Pro + Leu134Pro + Asn166Asp + Asn170Ser +Trp208Asn + Thr217AsnThr167Glu + Pro169Gly + Asn170Gln + Trp208Gly +Thr215Ser + Leu221SerThr111Ser + Thr137Ser + Thr195Gln + Ser207Glu +Tyr210Val + Thr217ProAsn106Asp + Gly136Pro + Gly162Ser + Trp208Ile + Tyr210Gly + Pro214GlnSer 70Asp + Gly165Gln + Phe193Asn + Thr195Ser +Trp208Pro + Thr212SerGly 72Ser + Ala114Gln + Asn166Gln + Phe193Glu +Tyr210Gly + Thr224ProAsn 66Ser + Gly136Ser + Gly162Pro + Asn163Ser +Ala164Asp + Ala168GlnGly 67Pro + Asn106Gln + Ser191Asp + Trp208His +Tyr218His + Leu221AsnTrp112Leu + Ala168Pro + Trp208His + Tyr210Ser +Thr217Gly + Ser220AspAsn 66Ser + Thr111Gly + Val115Gly + Ala168Ser +Asn170Gln + Pro214GlyGly110Ser + Trp112Val + Gly162Asn + Thr167Pro +Pro169Gln + Ser216AspGly 69Asn + Asn106Ser + Gly139Glu + Asn170Ser +Tyr210Cys + Leu221GlyGly 69Asn + Ser141Asp + Phe193Asn + Tyr210Ile +Ala219Asn + Leu221SerSer109Asp + Pro169Ser + Trp208His + Pro214Asn +Thr215Asn + Thr217GlyAsn 68Asp + Gly162Gln + Ala164His + Trp208Pro +Tyr210Leu + Leu221ValVal103Thr + Gly139Pro + Ala164Thr + Thr217Ser +Leu221Pro + Thr224ProGly 69Glu + Gly 72Gln + Thr137Pro + Trp208Ile +Tyr218Pro + Gly223GlnGly 67Pro + Leu104Cys + Val138Met + Gly139Ser +Ala203Gln + Ala219AsnTrp112Ser + Phe193Ala + Thr217Gly + Tyr218Glu +Leu221Met + Thr224AsnAsn163Gln + Trp208Leu + Tyr210Ser + Thr217Glu +Ala219Gly + Gly223ProVal103His + Gly110Gln + Gly139Ser + Ser192Asp +Phe193Asn + Thr217GlnGly108Gln + Pro169Asn + Thr195Ser + Trp208Ala +Thr215Pro + Thr217GluSer109Asp + Thr111Asn + Trp112Gln + Thr167Gln +Thr195Gly + Tyr210AsnGly 72Gln + Trp112Leu + Gly136Asp + Tyr171His +Tyr210Thr + Leu221SerGly 69Glu + Ala164Pro + Trp208Val + Tyr210Thr +Tyr218Gln + Thr224SerAla114Pro + Leu134Glu + Asn140Ser + Ala164Gln +Phe193Ile + Trp208AsnAsn 66Ser + Val138Met + Gly139Asn + Asn163Gln +Gly165Gln + Tyr210ThrAsn106Gln + Ser191Asp + Phe193Leu + Trp208Val +Pro214Gln + Thr217SerAla114Thr + Asn166Ser + Tyr171Gly + Ser191Glu +Ala203Pro + Tyr210AlaAsn 68Gln + Tyr171Asp + Phe193Tyr + Gly206Ser +Tyr210Val + Leu221ThrLeu104Ala + Alal14Ser + Asn163Asp + Thr195Gly +Gly206Gln + Leu221AlaGly110Asn + Val115Asn + Trp208Cys + Tyr210His +Ala219Thr + Gly223AspAsn 66Asp + Gly 69Asn + Val115Ala + Ala164Ser +Trp208Gln + Thr217GlnVal115Met + Thr137Ser + Gly162Asn + Asn166Glu +Tyr171His + Pro214SerAsn106Gln + Gly110Gln + Trp208His + Thr217Pro +Ala219Asp + Ser220AspGly 67Asn + Gly 72Asn + Ala114Gly + Asn140Gln +Ser216Asp + Thr217AspGly 69Gln + Thr 73Ser + Pro169Gln + Trp208Ile +Ser220Glu + Leu221AspAsn170Glu + Tyr171Asp + Phe193Ser + Trp208His +Leu221Thr + Thr224GlyGly 67Ser + Gly 69Glu + Ser 70Glu + Val103Gly +Tyr210Gln + Ala219AsnTrp112Gly + Asn166Ser + Trp208Met + Tyr210Glu +Ser220Glu + Thr224AsnLeu134Pro + Thr195Gly + Trp208Glu + Tyr210Asp +Thr217Ser + Ser220GluLeu104Gln + Trp112Gln + Tyr171Pro + Trp208Glu +Thr217Gly + Leu221GluAsp105Glu + Gly108Asp + Gly136Gln + Thr167Gln +Tyr171Gln + Leu221ValSer107Glu + Ser109Glu + Val138Met + Ala168His +Pro169Gln + Phe193SerGly 67Glu + Ser 70Glu + Trp112His + Tyr171Ile +Trp208His + Tyr210AlaVal103Pro + Trp208Glu + Tyr210Ser + Ala219Glu +Ser220Glu + Ser222AspSer191Asp + Ser207Glu + Tyr210Ala + Thr215Gly +Thr217Gln + Gly223SerAsn106Glu + Ser109Asp + Ala168Thr + Tyr171Met +Phe193Ile + Trp208LeuAsn 68Ser + Thr137Gln + Ser191Asp + Ser207Asp +Trp208Asp + Leu221ThrVal103Gln + Val138His + Asn166Asp + Ser192Glu +Trp208His + Leu221GlyVal103Pro + Gly139Pro + Asn163Asp + Gly165Gln +Ala168Glu + Tyr210LeuAsn 68Ser + Thrl13Gln + Leu134Ile + Ser191Glu +Leu221His + Gly223GluGly110Gln + Ser207Glu + Trp208Gln + Thr217Pro +Tyr218Gly + Ser220AspGly162Ser + Phe193Ala + Trp208Glu + Ile209Ser +Leu221Cys + Gly223GluSer 70Asp + Asp105Glu + Gly108Glu + Asn140Gln +Trp208Leu + Thr217GlyGlyl10Asn + Thr137Glu + Pro169Asp + Asn170Ser +Thr195Glu + Tyr210HisAsn 68Gln + Gly162Glu + Ala164Thr + Gly165Asp + Tyr171Ala + Ser192AspVal138Met + Gly139Asn + Asn170Gln + Tyr210Glu +Thr217Asp + Ser220GluGly139Ser + Asn163Asp + Ala164Ser + Ser191Asp +Tyr210Gly + Leu221AlaSer 70Asp + Thr 73Asp + Thr167Ser + Trp208Ser +Thr217Asp + Ala219AspAsn 66Glu + Leu104Glu + Asp105Glu + Ser107Asp +Leu221Ser + Gly223ProAsn 66Glu + Thr 73Asp + Gly110Pro + Ala168Gly +Tyr218Val + Leu221IleGly 67Asn + Asp105Glu + Asn106Glu + Thr111Glu +Gly165Asn + Thr217GlyAsn 66Asp + Tyr210Ser + Pro214Glu + Thr215Asp +Thr217Gln + Tyr218AspThr 73Asn + Ser192Glu + Ala203Thr + Ser207Asp +Trp208Glu + Leu221GluVal138Gly + Pro169Glu + Ser192Glu + Thr195Glu +Trp208Pro + Thr212GlnThr 73Asp + Gly108Asn + Leu134Ser + Tyr210Ala +Thr217Gln + Tyr218GluThr 73Asn + Ala114Thr + Val138Ser + Gly139Asp +Ser141Glu + Asn170GluGly108Gln + Gly135Pro + Gly162Asn + Ser220Glu +Leu221Ala + Gly223GluVal103Gln + Trp112Cys + Ala164Glu + Trp208Gln +Thr217Gly + Gly223AspThr111Gln + Leu134His + Ser191Asp + Phe193Asp +Thr195Gln + Trp208AspVal103Ser + Leu104Ile + Gly110Glu + Gly136Glu +Ala164Asn + Tyr210GlnLeu134Ser + Thr167Gly + Ser191Glu + Ser220Asp +Leu221Pro + Ser222AspGly 69Gln + Trp208Asp + Pro214Glu + Ser220Asp +Ser222Glu + Gly223AsnSer107Asp + Ser109Glu + Trp112Pro + Gly135Asp +Ala168Thr + Thr212GlyAsn140Ser + Thr167Asp + Trp208Glu + Thr217Asn +Ser220Asp + Leu221AspGly135Asp + Gly162Glu + Tyr171Asp + Tyr210Ala +Thr217Ser + Ser222AspVal115Asp + Tyr210Gln + Ser220Glu + Leu221Asp +Ser222Asp + Gly223SerAla114His + Asn166Asp + Tyr171Cys + Tyr210Glu +Ser220Asp + Leu221AspVal115Cys + Gly139Asn + Ala164Gly + Pro205Asn +Thr217Asp + Ser220AspThr167Gln + Tyr210Ile + Thr215Gly + Thr217Asp +Tyr218Asn + Ser220AspThr137Ser + Trp208Leu + Tyr210Leu + Thr217Glu +Ser220Asp + Leu221GlyThr111Asn + Gly135Ser + Trp208Asp + Tyr210Glu +Thr217Glu + Leu221AlaThr113Asn + Ala114Gly + Thr137Asp + Thr167Asp +Trp208Gln + Gly223AsnSer 70Asp + Thr 73Glu + Asn163Asp + Thr167Pro +Pro214Glu + Thr217GlyGly135Glu + Tyr171Asn + Pro205Gln + Ser207Glu +Trp208Glu + Ser220AspGly 69Asp + Asp105Glu + Gly108Asp + Ser207Asp +Trp208Thr + Gly223AsnThr113Gly + Ala114Pro + Tyr210Asp + Pro214Asp +Thr217Gln + Ser220AspAsn 68Glu + Ser 70Asp + Asp105Glu + Ala114Thr +Gly135Ser + Ser220AspThr111Ser + Thr137Glu + Ser207Glu + Trp208Thr +Ser220Glu + Leu221AspGly 69Asp + Leu104Asp + Asp105Glu + Asn140Glu +Asn166Ser + Leu221HisThr137Asp + Gly139Glu + Asn170Glu + Trp208Cys +Tyr210Asp + Leu221ThrGly 67Pro + Trp112Gln + Ser192Asp + Tyr210Ile +Ser220Asp + Ser222AspVal103Gln + Gly108Asp + Gly135Glu + Thr137Glu +Ala168Gln + Tyr210AsnVal115Asp + Leu134Glu + Asn166Gln + Asn170Asp +Tyr210Val + Thr212ProAsn 66Asp +Asn 68Ser + Thr167Gln + Ile209Leu +Ser216Asp + Thr217GluVal103G1u + Gly108Asp + Gly110Glu + Gly165Pro +Thr195Glu + Thr217GlnAsn 66Asp + Ser 70Glu + Thr 73Asp + Ser109Asp +Gly136Pro + Tyr210IleGly 69Asp + Ser 70Glu + Asp105Glu + Asn163Glu +Tyr210Met + Thr217GlnAsn 68Gln + Leu134Glu + Ser207Asp + Trp208Pro +Tyr210Asp + Leu221AspAsn 66Ser + Val115Gly + Gly162Asp + Ser191Glu +Trp208Ile + Thr217SerSer 70Glu + Tyr213Thr + Thr217Asn + Tyr218Gly +Ser222Glu + Gly223AspTrp112Met + Gly165Asp + Pro169Ser + Ser207Glu +Trp208Gly + Ser222AspGly 69Ser + Ser109Asp + Gly110Glu + Ala114Gly +Gly136Asp + Phe193AspAla164Gln + Gly165Pro + Ser192Asp + Phe193Asp +Ser207Glu + Thr217AspAsn 68Glu + Thr 73Asn + Gly162Gln + Trp208Gln +Ala219Glu + Ser220AspSer 70Glu + Leu134Ser + Gly165Asp + Asn166Asp +Ala168Gly + Ile209GlyLeu104Asp + Asp105Glu + Trp208Gly + Tyr210Glu +Tyr218Ala + Gly223ProAsn 68Asp + Gly 69Glu + Thr111Asn + Trp112His +Gly136Asn + Pro169GluGly108Ser + Asn140Asp + Ser141Glu + Ala164Gln + Ser207Glu + Tyr218SerLeu104Cys + Gly162Pro + Ser207Glu + Ser216Glu +Thr217Asp + Ala219ThrAla164Asn + Asn166Asp + Gly206Gln + Tyr210Thr +Ser216Asp + Thr217AspGly108Asp + Ser109Asp + Asn166Ser + Thr217Gln +Ser220Asp + Leu221ThrSer109Asp + Gly110Asp + Val138Ala + Tyr171His +Ala204His + Ser222GluAsn 68Gln + Thr137Gly + Pro169Asp + Ser207Glu +Trp208Glu + Tyr218GlnTrp112Asp + Thr113Glu + Val138Gly + Ser191Glu +Trp208Pro + Leu221SerLeu104Gly + Asp105Glu + Asnl06Glu + Gly206Ser +Trp208His + Tyr218GluGly108Asp + Leu134Asn + Gly136Gln + Trp208Phe +Ser220Asp + Leu221AspGly108Gln + Trp112Tyr + Asn170Glu + Trp208Gln +Ser220Glu + Leu221GluGly 72Asn + Asn166Asp + Phe193Ser + Trp208Tyr +Ser220Glu + Leu221GluAsn 66Glu + Gly 67Glu + Thr111Asn + Thr215Gln +Thr217Asn + Ser220GluAsn106Asp + Ser107Asp + Gly165Asp + Phe193Val +Trp208Val + Leu221ThrLeu104Val + Gly165Gln + Ser192Asp + Phe193Glu +Tyr210Glu + Leu221CysGly136Gln + Pro169Ser + Ser192Asp + Phe193Asp +Trp208Leu + Ser220GluSer107Asp + Val115Thr + Thr137Gln + Gly162Asp +Asn163Asp + Ala203HisVal103Met + Ser107Glu + Trp208Val + Thr215Glu +Ser216Glu + Gly223SerVal103Ser + Asn140Asp + Ser141Glu + Tyr210Glu +Ser220Asp + Gly223AsnAsn163Asp + Asn170Glu + Thr195Asn + Ser207Asp +Tyr210Met + Tyr218ProAsn 66Asp + Gly139Gln + Tyr210Glu + Thr217Ser +Ser220Asp + Thr224AsnGly 69Asp + Asn106Asp + Gly110Asp + Gly165Asn +Pro169Ser + Thr217AspPhe193Ile + Thr215Ser + Thr217Glu + Ser220Asp +Leu221Pro + Gly223GluVal103Gly + Asp105Glu + Thr195Asn + Ser207Asp +Ser222Asp + Gly223AsnGly 69Asp + Asn140Gln + Ala164His + Ala168Ser +Ser207Asp + Ser222AspAsn 68Asp + Thr111Asn + Ala114Gly + Gly135Gln +Ser207Glu + Ser222GluAsn106Asp + Gly139Asn + Gly162Glu + Pro169Ser +Ser191Asp + Thr224AspThr113Asp + Ser192Asp + Ser207Glu + Tyr213Thr +Thr215Pro + Ser222AspGly 69Pro + Asn163Asp + Thr195Glu + Trp208Ser +Tyr210Glu + Leu221CysGly 69Pro + Ser 70Asp + Gly110Ser + Gly135Glu +Asn170Glu + Leu221IleGly 69Glu + Ala164His + Thr195Gln + Ser220Glu +Leu221Glu + Thr224GluGly 69Gln + Gly136Asp + Val138Asp + Pro169Gly +Asn170Gln + Ser220GluGly136Glu + Val138Asp + Thr195Asn + Ser220Glu +Leu221Gly + Gly223ProAsn 68Gln + Ser107Glu + Ser109Glu + Gly139Gln +Ala164Gln + Ser207AspSer107Glu + Ser109Glu + Pro169Gln + Tyr210Met +Thr217Pro + Ser222AspGly 67Asp + Ser 70Glu + Val103Ala + Thr111Glu +Tyr210Asn + Pro214SerAsn 66Glu + Gly 67Asp + Leu134His + Ser191Asp +Ser207Glu + Trp208ThrAsn 66Glu + Val103Asp + Gly108Asn + Asn170Ser +Thr217Asn + Leu221MetThr 73Glu + Trp112Pro + Asn166Ser + Phe193His +Tyr218Ile + Ser220GluVal115His + Ser207Glu + Trp208Tyr + Tyr210Leu +Pro214Glu + Leu221GluAsn 66Gln + Ser107Glu + Ser109Glu + Ser207Glu +Gly223Asp + Thr224GlyAsn 66Glu +Asn 68Asp + Thr 73Gln + Thr195Ser +Ser207Asp + Tyr218ProSer 70Asp + Leu104Ile + Asn163Asp + Thr217Gln +Leu221Val + Thr224GluGly 67Asp + Val103Glu + Asn106Asp + Ala168Asn +Thr195Glu + Trp208LeuThr137Gly + Gly162Glu + Phe193Asp + Trp208Asp +Thr217Asn + Tyr218ValGly135Pro + Gly139Glu + Ser207Glu + Trp208Ala +Tyr210Cys + Gly223GluAsn106Ser + Thr137Asp + Gly139Glu + Ser141Asp +Ala204Gln + Trp208GluTrp112Ala + Asn163Glu + Ser192Glu + Trp208Val +Tyr210Glu + Ser220GluGly110Asp + Trp112Asp + Tyr210Thr + Ser220Glu +Leu221Met + Ser222GluGly 67Asp + Gly 69Asp + Thr 73Pro + Ser109Asp +Trp208Gln + Ser222AspAsn 66Ser + Val103Glu + Asp105Glu + Trp112His +Ser191Glu + Tyr210AlaGly 67Glu + Gly 69Asn + Gly162Glu + Ser207Glu +Thr217Asn + Gly223GluSer 70Asp + Gly165Asp + Ala168Asp + Trp208Pro +Thr217Gly + Leu221IleGly 67Glu + Val103Asn + Gly136Gln + Trp208Pro +Ser216Glu + Leu221MetGly 67Asn + Gly139Glu + Ser141Glu + Tyr213Gly + Ser220Asp + Leu221AsnGly 67Glu + Leu104Ser + Tyr210Ser + Ser220Asp +Ser222Glu + Gly223GlnGly 67Asp +Asn 68Glu + Gly136Asp + Ala168Gln +Pro169Glu + Phe193AsnAsn 66Glu + Gly 69Asp + Asn140Gln + Ser192Asp +Phe193Asp + Tyr210LeuLeu104Asp + Asn106Glu + Asn170Glu + Tyr210Ile +Thr215Ser + Thr224ProLeu104Asp + Ser107Glu + Gly135Asp + Gly139Asn +Ala164Glu + Thr217GlyTrp112Glu + Gly139Glu + Ala164Glu + Thr195Glu +Thr212Ser + Tyr213SerAsn 68Asp + Thr111Gly + Thr113Ser + Val138Asp +Asn170Asp + Phe193SerAsn163Gln + Ala168Asp + Asn170Asp + Ser207Glu +Thr217Gly + Ser220AspGly 67Asp + Asn106Glu + Thr113Ser + Gly136Gln +Asn166Glu + Leu221CysAsn 66Gln + Gly110Asp + Ala114His + Gly136Glu +Thr137Asp + Ser222GluGly 67Asp +Asn 68Ser + Gly 72Pro + Gly139Ser +Thr167Glu + Thr195AspAsn 66Asp + Asn 68Gln + Ser191Glu + Ser207Asp +Trp208Val + Ser220AspSer109Glu + Ala114Glu + Leu134Ile + Gly136Ser +Ala219Glu + Ser220AspVal115Glu + Ala168His + Pro169Asn + Tyr210Asp +Ser216Glu + Ala219AspAsn 66Glu + Asn106Asp + Asnl70Ser + Trp208Thr +Ser216Asp + Tyr218GlyGly162Asn + Asn170Asp + Phe193Glu + Tyr210Gln +Ser220Glu + Thr224AspGly108Ser + Leu134Pro + Thr137Asp + Pro169Glu +Ser207Glu + Thr217GlyThr137Asp + Val138Glu + Asn163Glu + Ala164Asn +Ala168Ser + Thr217GluGly162Glu + Trp208Glu + Tyr210Val + Thr217Glu +Ser220Glu + Gly223ProGly135Asp + Val138Thr + Ser207Asp + Thr217Pro +Tyr218Ile + Ser220GluLeu104Gly + Ser192Asp + Thr195Asp + Thr217Pro +Ser220Glu + Leu221AsnAsn 66Ser + Gly136Glu + Val138Glu + Trp208Leu +Thr217Asp + Ser220GluGly 69Ser + Val103Asp + Ser109Asp + Ser191Asp +Gly206Gln + Tyr210ProAla114Gly + Gly162Asp + Pro214Gly + Thr215Asp +Thr217Glu + Ser220GluThr137Gln + Gly139Asp + Gly165Pro + Tyr171Glu +Thr217Glu + Ser220GluGly 67Glu + Asp105Glu + Asn140Glu + Phe193Gly +Trp208Leu + Leu221CysLeu104Glu + Trp112Leu + Phe193Leu + Ser207Asp +Trp208Ala + Thr224AspSer 70Glu + Thr113Gly + Gly162Ser + Asn163Asp +Ala164Glu + Ser220AspGly 69Glu + Ser 70Asp + Thr111Glu + Ser220Asp +Leu221Pro + Gly223SerAsn 66Gln + Leu104Pro + Gly136Glu + Asn163Asp +Tyr171Ile + Leu221GluSer 70Asp + Leu104Glu + Ala168Asn + Pro169Glu +Thr195Asp + Trp208PheSer 70Glu + Gly162Glu + Trp208Glu + Thr217Gly +Leu221Thr + Ser222AspGly 69Asn + Thr137Glu + Gly139Pro + Asn166Asp +Pro169Asp + Trp208AspGly 69Glu + Leu104Glu + Val115Ser + Asn140Ser +Ser192Glu + Pro214AspLeu104His + Asp105Glu + Val138His + Asn163Asp +Ser192Glu + Thr217AsnAla114Asn + Asn163Glu + Asn170Ser + Ser192Asp +Ala204Asn + Thr217AspAsn 68Gln + Thr 73Asp + Ser109Glu + Trp208Phe +Pro214Glu + Ser220AspVal115Cys + Leu134Asp + Gly135Pro + Ala164Thr +Asn170Asp + Trp208AspAsn 66Glu + Gly 69Glu + Trp112Gln + Gly135Asn +Gly139Asp + Asn170GluThr 73Gly + Ser107Glu + Gly135Asp + Pro169Asp +Trp208Thr + Leu221AsnGly 67Ser + Ser 70Glu + Gly136Asp + Pro169Asn +Leu221Asp + Gly223AspSer 70Asp + Gly110Glu + Val138Ser + Tyr210Val +Leu221Asp + Gly223AspAsn 66Asp + Leu104Glu + Ser109Glu + Tyr210Asn +Thr217Asn + Ser220AspAsn 66Glu + Gly 67Asp + Asn163Glu + Ser207Glu +Thr215Gly + Leu221ThrGly 69Glu + Tyr210Glu + Thr215Asp + Thr217Gln +Tyr218Gln + Ser220GluSer 70Asp + Thr137Glu + Ala164His + Thr167Gly +Trp208Asp + Ser220GluVal138Met + Gly139Gln + Asn140Glu + Ala168Pro +Tyr171Glu + Thr215GluVal103Gly + Asn106Ser + Ser107Asp + Thr111Asp +Ser222Glu + Gly223GluGly 72Gln + Asn106Glu + Gly110Asp + Thr113Glu +Gly135Asn + Ala219GluSer109Glu + Thr113Asp + Trp208Ile + Ser220Glu +Leu221Asp + Thr224SerGly 67Asn + Ser 70Asp + Gly108Asp + Asn170Glu +Thr215Pro + Leu221ThrGly 67Asp + Ser107Asp + Thr167Gly + Tyr210Asn +Ser220Asp + Ser222GluGly 69Asp + Thr 73Glu + Asn106Gln + Thr111Asn + Val138Glu + Thr217AspThr111Asn + Ser191Asp + Thr195Asp + Ala204Thr +Ala219Ser + Ser220GluSer109Asp + Ser191Glu + Tyr210Val + Thr215Asn +Leu221Gly + Ser222GluThr 73Glu + Leu104Asp + Asn106Gln + Thr195Ser +Ser207Glu + Ser220AspAsn 66Asp + Thr 73Asp + Leu104Glu + Gly136Gln +Gly162Asp + Leu221GlyGly 72Asn + Leu104Asp + Asn163Asp + Tyr171Ser +Ser191Glu + Thr217AsnVal115Asp + Gly136Gln + Tyr210Glu + Tyr218Cys +Leu221Asn + Ser222AspThr 73Asp + Asn140Ser + Asn163Glu + Thr195Asp +Ser220Asp + Leu221ProAsn 68Glu + Leu104Asp + Pro169Asn + Trp208Val +Thr217Asn + Gly223GluGly110Glu + Thr167Gln + Ser192Glu + Ser207Glu +Trp208Cys + Ser220AspVal103Gly + Gly136Glu + Asn140Asp + Ser207Asp +Tyr213Asn + Ser220AspThr113Asp + Gly136Asp + Thr137Gln + Asn163Glu +Asn166Glu + Trp208PheAsn 66Glu + Gly 69Glu + Ser109Asp + Ser192Asp +Phe193Val + Tyr218AlaSer 70Glu + Ser141Asp + Asn163Ser + Trp208Met +Tyr210Pro + Thr215AspAsp105Glu + Gly110Asn + Thr111Glu + Leu134Ala +Ser141Glu + Phe193TyrAsn 66Glu + Gly 69Ser + Gly108Asp + Thr111Asp +Thr195Gly + Tyr210LeuGly165Glu + Tyr171Asn + Ser207Glu + Thr215Glu +Thr217Gly + Thr224GluAsn 66Gln + Thr 73Ser + Gly139Asn + Pro214Asp +Ser220Asp + Leu221IleAsn166Glu + Ser192Asp + Trp208Thr + Tyr210Gly +Thr215Asp + Ser222GluAsn 66Asp + Ser191Asp + Thr215Asn + Ala219Asp +Ser222Asp + Gly223GlnAsp105Glu + Gly108Glu + Thr195Asn + Ile209Leu +Pro214Asp + Ser220AspThr111Glu + Val138Asp + Asn163Ser + Tyr218Met +Leu221Glu + Gly223AspGly 67Glu + Val138Glu + Ser141Glu + Trp208Ile +Pro214Asn + Thr215AspTrpl12Leu + Thr113Gln + Gly165Glu + Thr167Asp +Thr217Glu + Ser220AspGly 67Gln + Asn 68Asp + Gly 69Asp + Phe193Ala +Ser216Glu + Ser222GluSer 70Asp + Val115Glu + Gly162Ser + Ala168Thr +Tyr210Gly + Thr217AspSer 70Asp + Gly136Asn + Gly165Glu + Tyr210Asn +Thr217Asp + Leu221HisThr 73Ser + Val103Asn + Ser109Asp + Ser192Asp +Ser207Asp + Trp208IleAsn106Asp + Ser192Glu + Phe193Pro + Ser207Glu +Thr217Gln + Tyr218IleSer141Asp + Thr167Asp + Ala168Asp + Ser191Asp +Ala219Asn + Leu221ThrAsn 66Gln + Ser141Asp + Asn170Ser + Thr217Gln +Ser220Asp + Gly223AspThr 73Gly + Asp105Glu + Ala114His + Ser220Glu +Leu221Asn + Gly223GluAsn106Asp + Asn140Gln + Trp208Ser + Tyr210Cys +Ser220Asp + Gly223GluGly 67Gln + Asp105Glu + Ala164Asp + Pro169Asn +Tyr218Leu + Gly223AspTrp112Val + Asn166Asp + Phe193Gly + Pro214Glu +Ser220Asp + Ser222GluGly108Asp + Thr111Asn + Ser192Glu + Thr217Asn +Ala219Glu + Ser220AspVal103Ser + Val115Asp + Gly139Glu + Asn166Ser +Ser207Glu + Ser220GluLeu104Asp + Asp105Glu + Asn140Glu + Thr167Asp +Pro214Gly + Ala219GlyGly 67Asp + Asn 68Asp + Thr111Asp + Ala164Gln +Phe193Ser + Ser207AspSer109Glu + Trp208Asp + Ser216Glu + Thr217Asp +Tyr218Asn + Leu221HisSer141Asp + Ala168Glu + Tyr210Met + Ser216Glu +Thr217Asp + Tyr218AlaSer109Asp + Gly110Asp + Asn166Asp + Asn170Ser +Thr215Ser + Leu221AspGly110Glu + Thr167Glu + Ala168Asp + Trp208Met +Ser220Asp + Thr224GlnAsp105Glu + Asn106Asp + Gly165Asp + Thr195Ser +Tyr218Ile + Ser220AspAsn106Glu + Ser141Asp + Tyr218Ser + Ala219Thr +Ser220Asp + Leu221GluThr 73Gln + Thr111Glu + Asn166Asp + Trp208Ser +Ser220Asp + Leu221AspSer107Glu + Ala168Asn + Pro169Asn + Ser191Asp +Ser192Asp + Thr217AspAla114Glu + Asn166Ser + Thr212Pro + Thr217Glu +Tyr218Asp + Thr224AspAsn106Glu + Ser107Asp + Gly139Asp + Ala203Asn +Thr217Ser + Ser220AspThr113Asn + Gly139Ser + Ser141Glu + Thr215Glu +Ser216Asp + Ser220AspGly 69Gln + Asn140Glu + Gly162Glu + Asn163Asp +Tyr210Gln + Ser220AspAsn 68Asp + Ala114Pro + Gly135Glu + Trp208Asp +Leu221Ile + Gly223AspAsn 68Asp + Ala164Glu + Trp208Cys + Tyr210Glu +Ala219Thr + Ser220AspThr111Asp + Asn166Asp + Asn170Gln + Tyr210Asp + Thr212Ser + Ser220GluSer141Glu + Gly165Asp + Trp208Ile + Tyr210Asp +Thr217Gln + Ser220AspAsn 66Gln + Val103Asp + Gly108Pro + Thr137Glu +Tyr210Glu + Ser220GluGly108Glu + Thr137Gln + Asn163Glu + Tyr210Glu +Ser220Asp + Leu221CysGly 69Gln + Ser 70Glu + Gly135Glu + Asn163Asp +Thr217Gly + Ser220AspAsn106Glu + Asn140Glu + Asn163Glu + Asn170Glu +Thr195Asn + Leu221HisAsn106Gln + Asn166Asp + Ala168Asn + Asn170Glu +Ser207Glu + Ser220AspAsn 66Asp + Ser 70Asp + Gly110Gln + Gly136Asp +Pro214Gln + Ser220AspAsn 68Ser + Pro169Asp + Ser192Glu + Ala219Asp +Gly223Ser + Thr224AspThr 73Pro + Gly135Ser + Phe193Glu + Gly206Gln +Tyr210Asp + Thr217GluTrp112Glu + Val115Gln + Asn170Gln + Thr217Asp +Tyr218Met + Ser220AspGly110Asn + Val115Glu + Thr137Gln + Ile209Gly +Thr217Asp + Ser220GluGly108Glu + Thr113Gln + Pro214Asn + Thr217Glu +Tyr218Gln + Ser220AspGly110Ser + Thr111Gly + Thr113Glu + Phe193Pro +Thr217Glu + Ser220AspAsn166Glu + Trp208Ile + Tyr210Met + Thr217Glu +Tyr218Val + Ser220AspSer 70Glu + Val103Glu + Val115Gln + Pro169Ser +Tyr210Ala + Ser220AspAsn 66Asp + Ser107Asp + Trp208Met + Tyr210Pro +Ser220Asp + Ser222GluGly110Ser + Thr111Asp + Gly135Gln + Gly136Asp +Thr137Asn + Trp208GluThr167Asp + Ala168Ser + Pro169Glu + Trp208Thr +Thr215Asp + Leu221AspVal115Gln + Ala168Gln + Pro169Gln + Phe193Asp +Tyr210Ser + Leu221AspAsn 66Glu + Gly136Pro + Asn140Gln + Ser191Asp +Trp208Asp + Leu221HisGly136Glu + Gly139Glu + Ser192Asp + Ala203Asn +Trp208Asp + Leu221HisGly 67Asn + Thr113Gln + Thr137Glu + Thr167Asp +Ala219Gln + Leu221GluGly 69Asn + Thr 73Asp + Thr111Pro + Asn166Asp +Ser191Asp + Leu221ValVal138Pro + Asn166Glu + Ser191Asp + Tyr218Asn +Ser220Asp + Leu221GlnAsp105Glu + Gly139Asn + Gly165Glu + Trp208Met +Tyr218Thr + Gly223AspGly110Asn + Thr111Pro + Ala114Asp + Asn170Asp +Trp208Asp + Tyr210AspGly 67Gln + Gly 69Glu + Ser107Asp + Thr167Glu +Ala168Asn + Ser207AspGly 69Glu + Gly165Glu + Trp208Cys + Tyr210Cys +Pro214Asp + Ser220AspSer 70Glu + Gly 72Gln + Val103Thr + Ser109Glu +Thr113Glu + Ser220AspGly 69Pro + Val115Glu + Tyr210Asn + Ser216Asp +Ser220Glu + Ser222GluGly136Glu + Trp208Ser + Ser216Asp + Ser220Asp +Leu221Asn + Ser222AspLeu104Glu + Val115Pro + Gly162Ser + Asn166Asp +Ser220Asp + Ser222AspGly 67Glu + Val138Asp + Thr217Ser + Ser220Asp +Leu221His + Ser222AspAsn 68Asp + Asn170Glu + Gly206Asn + Ser207Asp +Trp208Ile + Leu221AsPAsn106Gln + Ser109Glu + Thr113Asp + Leu134Gln +Thr137Glu + Trp208AsnAsn106Glu + Gly139Gln + Thr167Glu + Pro169Gln +Ser191Asp + Ser207GluSer107Glu + Val115Asp + Gly165Asn + Trp208Thr +Tyr218Asp + Ser220GluGly72Asn + Asn106Gln + Gly139Asp + Pro169Glu +Trp208Pro + Ser220GluVal138Asp + Ser141Glu + Pro169Ser + Ser207Glu +Ala219Glu + Leu221ThrAsn106Gln + Ala114Asp + Gly135Asp + Thr195Asn +Ser207Glu + Ser220AspSer 70Asp + Asn166Glu + Asn170Gln + Ser216Glu +Thr217Gln + Thr224GlyGly 69Asp + Asn140Ser + Gly162Pro + Trp208Gly +Thr217Glu + Leu221AspAsn 68Asp + Ser107Asp + Ala114Gln + Gly162Asp +Thr217Pro + Ser220GluSer 70Asp + Thr111Asp + Ile209Met + Thr217Gly +Ser220Glu + Leu221AsnSer 70Asp + Leu104His + Thr111Gln + Tyr171Ser +Phe193Asp + Ser220GluThr113Gly + Gly139Pro + Ser141Glu + Tyr171Glu +Ser192Asp + Ile209SerTrp112Ala + Thr113Glu + Val138Gln + Ser141Asp +Ser216Asp + Leu221SerGly 69Asn + Asn106Asp + Trp112Gly + Gly162Asp +Phe193Glu + Ser220GluAsn 68Ser + Val103Glu + Asn106Asp + Ala168Pro +Pro169Asp + Pro214GluLeu104Glu + Ala114Ser + Asn140Ser + Gly165Asp +Ser207Asp + Ser220GluAsn 68Asp + Ser109Glu + Gly139Glu + Ala204Ser +Leu221His + Gly223AsnThr111Gly + Val138Pro + Gly139Asp + Ser207Glu +Ser216Glu + Ser220AspAsn106Asp + Asn170Ser + Ser192Glu + Thr195Asp + Trp208His + Ser220GluAsn106Asp + Gly162Asp + Asn166Gln + Thr167Asp +Pro214Asp + Ala219ThrVal115Glu + Gly136Glu + Ser207Glu + Tyr218Gly +Ala219Pro + Leu221ProGly136Ser + Gly162Asp + Asn163Gln + Ala164His +Ser216Asp + Ser222GluAsn 68Ser + Thr 73Glu + Asn166Glu + Asn170Gln +Tyr171Gly + Leu221AspThr113Glu + Val115Cys + Thr137Glu + Ser191Glu +Thr217Ser + Thr224GluGly 67Asp + Ser107Asp + Gly139Asp + Gly165Pro +Thr167Ser + Thr217GluThr73Gly + Gly108Asp + Thr137Glu + Gly162Glu +Tyr210Gln + Ser220AspLeu104Asp + Gly139Pro + Tyr171Asp + Ser191Asp +Ser222Glu + Thr224SerThr113Asp + Pro169Glu + Tyr210Gln + Pro214Ser +Ser220Glu + Gly223AspGly108Asp + Gly136Glu + Ser141Asp + Thr195Asp +Thr215Ser + Thr217GlnSer107Asp + Asn163Glu + Pro169Gly + Ser207Asp +Trp208Asn + Gly223AsnGly110Asp + Asn163Asp + Ser191Asp + Thr195Gln +Tyr210Glu + Thr217ProVal103Glu + Gly108Asp + Ala203Gly + Trp208Met +Ala219Asp + Thr224GluThr73Gln + Thr111Asp + Leu134Asp + Gly136Ser +Asn163Gln + Ser207AspII.洗涤组合物
在本发明的另一实施方案中，有效量的一种或多种酶变异体包括在用于洗涤各种需要除去蛋白质污垢的表面的组合物中。这种洗涤组合物包括用于洗涤硬表面的非限制形式洗涤组合物(例如，液体或颗粒)；用于洗涤织物的非限制形式的洗涤组合物(颗粒、液体或条状制剂)；餐具洗涤组合物(非限制形式)；非限制形式的口腔用洗涤组合物(例如，牙粉、牙膏和漱口剂制剂)；非限制形式的假牙洗涤组合物(例如，液体、片剂)；和非限制形式的接触透镜洗涤组合物(例如，液体、片剂)。
除了本文上述的热酶变异体之外，洗涤组合物还包括与蛋白酶相容的一种或多种洗涤组合物材料。本文使用的术语“洗涤组合物材料”指的是选择用于所需的洗涤组合物的具体类型和产物形式(例如，液体、颗粒、条状、喷雾剂、棒状、膏状、凝胶)的任何液体、固体或气态材料，该材料还应与组合物中使用的热酶变异体相容。通过考虑所洗涤的表面材料、在使用过程中用于洗涤条件下所需的组合物的形式(例如通过洗涤剂使用)很容易地具体选择洗涤组合物材料。本文中使用的术语“相容”指的是洗涤组合物材料不降低热酶变异体的蛋白水解活性至在正常使用场合根据需要蛋白酶失效的程度。具体的洗涤组合物材料在下文详细举例说明。
本文中使用的“有效量的酶变异体”指的是为获得在具体洗涤组合物中所需的酶活性而必需的酶变异体的量。这种有效量易于被本领域的技术人员确定，其基于许多因素，例如所用的具体的酶变异体、洗涤应用、洗涤组合物的具体组成，和是否需要液体或无水(例如颗粒、条状)组合物等等。洗涤组合物优选含有约0.0001％-约10％一种或多种本发明的酶变异体，更优选约0.001％-约1％，还优选约0.01％-约0.1％。其中使用酶变异体的各种洗涤组合物的若干实例在下文中进一步地讨论。本文使用的所有份数、百分数和比率均为按重量计，除非另有说明。
本文中使用的“非织物洗涤组合物”包括硬表面洗涤组合物、餐具洗涤组合物、口腔洗涤组合物、假牙洗涤组合物和接触透镜洗涤组合物。A.用于硬表面、餐具和织物的洗涤组合物
本发明的酶变异体可用于其中需要高泡和高效不溶底物去除的各种洗涤组合物中。因此，酶变异体可与各种常规成分一起使用以提供全配制的硬表面洗涤剂，餐具洗涤组合物、织物洗涤组合物等等。这种组合物可以是液体、颗粒、条状形式等等。这种组合物可配制成含有多达按表面活性剂重量计30％-60％的现代“浓缩”洗涤剂。
本发明的洗涤组合物可选择性地，并优选地含有各种阴离子、非离子、两性离子等等的表面活性剂。这些表面活性剂通常以组合物的约5％-约35％的含量存在。
用于本发明的表面活性剂的非限制性实例包括常规的C11-C18烷基苯磺酸盐和伯和无规烷基硫酸盐、式为CH3(CH2)x(CHOSO3 -M+)CH3和CH3(CH2)y(CHOSO3 -M+)CH2CH3的C10-C18仲(2，3)烷基硫酸盐，其中x和(y+1)是至少约7，优选至少约9的整数，M是水溶性阳离子，尤其是钠、C10-C18烷基烷氧基硫酸盐(尤其是EO 1-5乙氧基硫酸盐)、C10-C18烷基烷氧基羧酸盐(尤其是EO 1-5乙氧基羧酸盐)、C10-C18烷基聚糖苷和它们相应的硫酸化聚糖苷，和C12-C18α-磺化脂肪酸酯、C12-C18烷基和烷基苯酚烷氧基化物(尤其是乙氧基化物和混合的乙氧基化物/丙氧基化物)、C12-C18甜菜碱和磺基甜菜碱(“sultaines”)、C10-C18胺氧化物等等。本发明优选烷基烷氧基硫酸盐(AES)和烷基烷氧基羧酸盐(AEC)。(根据配制者的需要，使用这种表面活性剂与上述的胺氧化物和/或甜菜碱或磺基甜菜碱表面活性剂的混合物也是优选的)。其它常规使用的表面活性剂在标准文献中列出。尤其有用的表面活性剂包括在1993年3月16日颁布的Connor等人的US5194639中的C10-C18 N-甲基葡糖酰胺(glucamides)，列为本文参考文献。
用于洗涤组合物中的各种各样的其它成分可包括在本发明的组合物中，其包括其它活性成分、载体、增溶剂、加工助剂、染料或颜料、用于液体配方的溶剂等等。如果需要附加地增加泡沫，在组合物中，通常以约1％-约10％的含量加入发泡剂，例如C10-C16烷醇酰胺。C10-C14单乙醇和二乙醇酰胺说明了这种发泡剂的典型种类。与上述的高泡辅助表面活性剂，例如胺氧化物、甜菜碱和磺基甜菜碱一起使用这类发泡剂也是有利的。如果需要，溶解的镁盐，例如氯化镁、硫酸镁等等可以通常约0.1％-约2％的含量加入以提供附加的起泡。
本发明的液体洗涤组合物可含有作为载体的水和其它溶剂。低分子量的伯或仲醇，例如甲醇、乙醇、丙醇和异丙醇是适宜的。单羟基醇对于表面活性剂的增溶是优选的，但多元醇，例如含有约2-约6个碳原子和约2-约6个羟基的多元醇(例如，1，3-丙二醇、乙二醇、甘油和1，2-丙二醇)也可以使用。组合物可含有约5％-约90％，典型地约10％-约50％的这种载体。
本发明的洗涤组合物将优选配制成在使用过程以含水洗涤操作，洗涤水将具有约6.8-约11.0的pH，最终的产物因此通常在该范围内配制.控制pH在所需要的范围内的技术包括使用缓冲剂、碱、酸等等，是本领域技术人员已知的。
在配制本发明的硬表面洗涤组合物和织物洗涤组合物时，配制者可以按重量计约5％-约50％的含量使用各种助剂。典型的助剂包括1-10微米沸石、多羧酸盐，例如柠檬酸盐和氧联二琥珀酸盐、层状硅酸盐、磷酸盐等等。其它常规的助剂在标准配方中列出。
同样，配制者可在组合物中，通常以按重量计约0.001％-1％的含量使用各种其它的酶，例如纤维素酶、脂酶、淀粉酶和蛋白酶。在洗衣洗涤剂领域中已知各种洗涤和织物护理酶。
各种漂白化合物，例如过碳酸盐、过硼酸盐等等可通常以按重量计约1％-约15％的含量用于该组合物中。如果需要，该组合物还可以含有漂白活性剂，例如四乙酰基乙二胺、壬酰氧基苯磺酸盐等等，它们也是现有技术中已知的。使用含量通常为按重量计约1％-约10％。
各种污垢解脱剂，尤其是阴离子低聚酯类型、各种螯合剂，尤其是氨基膦酸盐和乙二胺二琥珀酸盐、各种粘土去污剂，尤其是乙氧基化四亚乙五胺、各种分散剂，尤其是聚丙烯酸酯和聚天冬氨酸酯、各种增白剂，尤其是阴离子增白剂、各种泡沫抑制剂，尤其硅烷和仲醇、各种织物柔软剂，尤其是绿土等等都可以按重量计约1％-约35％的含量用于组合物。在标准配方和公开的专利中有此类常规物质的详细描述。
在洗涤组合物中也可使用酶稳定剂。酶稳定剂包括丙二醇(优选约1％-约10％)、甲酸钠(优选约0.1％-约1％)和甲酸钙(优选约0.1％-约1％)。l.硬表面洗涤组合物
本文使用的“硬表面洗涤组合物”指的是用于洗涤硬表面，例如地板、墙壁、浴室瓷砖等等的液体和颗粒洗涤组合物。本发明的硬表面洗涤组合物含有有效量的一种或多种本发明的酶变异体，按组合物活性酶重量计优选为约0.001％-约10％，更优选约0.01％-约5％，还优选约0.05％-约1％。除了含有一种或多种酶变异体之外，硬表面洗涤组合物通常含有表面活性剂和水溶性螯合剂。然而，在某些特殊的产物，例如喷雾玻璃清洁剂中，有时是不使用表面活性剂的，因为它们会产生残留在玻璃表面的膜状/条纹残余物。
当存在时，表面活性剂组分可含有少至组合物的0.1％，但通常组合物将含有约0.25％-约10％，更优选约1％-约5％的表面活性剂。
组合物通常含有约0.5％-约50％的洗涤剂助剂，优选为约1％-约10％。
pH值优选在约8-12的范围内。如果需要进行调节，可使用常规的pH调节剂，例如氢氧化钠、碳酸钠或盐酸。
在组合物中可包括溶剂。有用的溶剂包括，但不限制于，乙二醇醚，例如二乙二醇单己基醚、二乙二醇单丁基醚、乙二醇单丁基醚、乙二醇单己基醚、丙二醇单丁基醚、二丙二醇单丁基醚，和二醇，例如2，2.4-三甲基-1，3-戊二醇和2-乙基-1，3-己二醇。当使用时，溶剂通常以约0.5％-约15％，优选约3％-约11％的含量存在。
此外，当组合物以“完全强度”应用于表面后表面不进行漂洗时在本发明的组合物中可使用高挥发性溶剂，例如异丙醇或乙醇，以有助于组合物从表面快速蒸发。当使用时，挥发性溶剂通常以约2％-约12％的含量存在于组合物中。
本发明的硬表面洗涤组合物的实施方案通过如下实施例说明。
实施例7-12
液体硬表面洗涤组合物
实施例组分 7 8 9 10 11 12Gln66Asn 0.05 0.50 0.02 0.03 0.10 0.03Gly206Asn - - - - 0.20 0.02Na2DIDA*EDTA** - - 2.90 2.90 - -柠檬酸钠 - - - - 2.90 2.90C12烷基苯磺酸钠 1.95 - 1.95 - 1.95 -C12烷基硫酸钠 - 2.20 - 2.20 - 2.20C12(乙氧基)***硫酸钠 - 2.20 - 2.20 - 2.20C12二甲基胺氧化物 - 0.50 - 0.50 - 0.50枯烯磺酸钠 1.30 - 1.30 - 1.30 -己基卡必醇*** 6.30 6.30 6.30 6.30 6.30 6.30水**** 平衡至100％*N-二乙二醇-N，N-亚氨基二乙酸二钠**乙二胺二乙酸四钠***二乙二醇单己基醚****所有配方调节到pH7
在实施例7-10中，用表2-36中所列的热酶变异体代替Gln66Asn，得到基本上类似的结果。
在实施例11-12中，用表2-36中所列的热酶变异体的任何组合代替Gln66Asn和Gly206Asn，得到基本上类似的结果。
实施例13-18
用于清洁硬表面和除去家庭霉变的喷雾组合物
实施例组分 13 14 15 16 17 18Thr111Asn 0.50 0.05 0.60 0.30 0.20 0.30Thr217Gly+Ser222Glu - - - - 0.30 0.10辛基硫酸钠 2.00 2.00 2.00 2.00 2.00 2.00十二烷基硫酸钠 4.00 4.00 4.00 4.00 4.00 4.00氢氧化钠 0.80 0.80 0.80 0.80 0.80 0.80硅酸盐(钠) 0.04 0.04 0.04 0.04 0.04 0.04香料 0.35 0.35 0.35 0.35 0.35 0.35水 平衡至100％产物pH为约7。
在实施例13-16中，用表2-36中所列的热酶变异体代替Thr111Asn，得到基本上类似的结果。
在实施例17-18中，用表2-36中所列的热酶变异体的任何组合代替Thr111Asn和Thr217Gly+Ser222Glu，得到基本上类似的结果。
2.餐具洗涤组合物
在本发明的另一实施方案中，餐具洗涤组合物含有一种或多种本发明的酶变异体。本文使用的“餐具洗涤组合物”指的是用于洗涤餐具的组合物的所有形式，其包括，但不限制于，颗粒和液体形式。本发明的餐具洗涤组合物的实施方案由如下实施例说明。
实施例19-24
餐具洗涤组合物
实施例组分 19 20 21 22 23 24Gly139Asn 0.05 0.50 0.02 0.40 0.10 0.03Ser207Glu+Tyr210Ser - - - - 0.40 0.02+Gly223AsnC12-C14N-甲基葡糖酰 0.90 0.90 0.90 0.90 0.90 0.90胺C12乙氧基(1)硫酸盐 12.00 12.00 12.00 12.00 12.00 12.002-甲基十一烷酸 4.50 4.50 4.50 4.50 4.50 4.50C12乙氧基(2)羧酸盐 4.50 4.50 4.50 4.50 4.50 4.50C12醇乙氧基化物(4) 3.00 3.00 3.00 3.00 3.00 3.00C12胺氧化物 3.00 3.00 3.00 3.00 3.00 3.00枯烯磺酸钠 2.00 2.00 2.00 2.00 2.00 2.00乙醇 4.00 4.00 4.00 4.00 4.00 4.00Mg++(作为氯化镁) 0.20 0.20 0.20 0.20 0.20 0.20Ca++(作为氯化钙) 0.40 0.40 0.40 0.40 0.40 0.40水 平衡至100％产物pH调节至7。
在实施例19-22中，用表2-36中所列的热酶变异体代替Gly139Asn，得到基本上类似的结果。
在实施例23-24中，用表2-36中所列的热酶变异体的任何组合代替Gly139Asn和Ser207Glu+Tyr210Ser+Gly223Asn，得到基本上类似的结果。
3.织物洗涤组合物
在本发明的另一实施方案中，织物洗涤组合物含有一种或多种本发明的酶变异体。本文使用的“织物洗涤组合物”指的是用于洗涤织物的组合物的所有形式，其包括，但不限制于，颗粒、液体和条状形式。优选的织物洗涤组合物是液体形式。a.颗粒织物洗涤组合物
本发明的颗粒织物洗涤组合物含有有效量的一种或多种本发明的酶变异体，按组合物活性酶重量计优选为约0.001％-约10％，更优选约0.005％-约5％，还优选约0.01％-约1％。除了含有一种或多种酶变异体之外，颗粒织物洗涤组合物通常含有至少一种表面活性剂、一种或多种助剂和，在某些情况下，漂白剂。
本发明的颗粒织物洗涤组合物的实施方案由如下实施例说明。
实施例25-28
颗粒织物洗涤组合物
实施例组分 25 26 27 28Ala168Asn 0.10 0.20 0.03 0.05Tyr210Gly+Thr212Pro+Pro - - 0.02 0.05214Gly+Ser222AspC13直链烷基苯磺酸盐 22.00 22.00 22.00 22.00磷酸盐(作为三聚磷酸钠) 23.00 23.00 23.00 23.00碳酸钠 23.00 23.00 23.00 23.00硅酸钠 14.00 14.00 14.00 14.00沸石 8.20 8.20 8.20 8.20螯合剂(二亚乙三胺五乙酸) 0.40 0.40 0.40 0.40硫酸钠 5.50 5.50 5.50 5.50水 平衡至100％
在实施例25-26中，用表2-36中所列的热酶变异体代替Ala168Asn，得到基本上类似的结果。
在实施例27-28中，用表2-36中所列的热酶变异体的任何组合代替Ala168Asn和Tyr210Gly+Thr212Pro+Pro214Gly+Ser222Asp，得到基本上类似的结果。
实施例29-32
颗粒织物洗涤组合物
实施例组分 29 30 31 32Thr195Pro 0.10 0.20 0.03 0.05Trp208Phe+Tyr213Met+Thr215 - - 0.02 0.05Pro+Ser220Glu+Thr224GlyC12烷基苯磺酸盐 12.00 12.00 12.00 12.00沸石A(1-10微米) 26.00 26.00 26.00 26.002-丁基辛酸 4.00 4.00 4.00 4.00C12-C14仲(2，3)烷基硫酸钠 5.00 5.00 5.00 5.00柠檬酸钠 5.00 5.00 5.00 5.00荧光增白剂 0.10 0.10 0.10 0.10硫酸钠 17.00 17.00 17.00 17.00水和少量组分 平衡至100％
在实施例29-30中，用表2-36中所列的热酶变异体代替Thr195Pro，得到基本上类似的结果。
在实施例31-32中，用表2-36中所列的热酶变异体的任何组合代替Thr195Pro和Trp208Phe+Tyr213Met+Thr215Pro+Ser220Glu+Thr224Gly，得到基本上类似的结果。
实施例33-36
颗粒织物洗涤组合物
实施例组分 33 34 35 36Gly67Ser+Gly72Ser 0.10 0.20 0.03 0.05Tyr171Thr - - 0.02 0.05C13直链烷基苯磺酸盐 22.00 22.00 22.00 22.00磷酸盐(作为三聚磷酸钠) 23.00 23.00 23.00 23.00碳酸钠 23.00 23.00 23.00 23.00硅酸钠 14.00 14.00 14.00 14.00沸石 8.20 8 20 8.20 8.20螯合剂(二亚乙三胺五乙酸) 0.40 0.40 0.40 0.40硫酸钠 5.50 5.50 5.50 5.50水 平衡至100％
在实施例33-34中，用表2-36中所列的热酶变异体代替Gly67Ser+Gly72Ser，得到基本上类似的结果。
在实施例35-36中，用表2-36中所列的热酶变异体的任何组合代替Gly67Ser+Gly72Ser和Tyr171Thr，得到基本上类似的结果。
实施例37-40
颗粒织物洗涤组合物
实施例组分 37 38 39 40Ser192Asp+Phe193Leu+Thr195As 0.10 0.20 0.03 0.05nTyr213Ser+Thr217Gly+Gly223Glu - - 0.02 0.05C12烷基苯磺酸盐 12.00 12.00 12.00 12.00沸石A(1-10微米) 26.00 26.00 26.00 26002-丁基辛酸 4.00 4.00 4.00 4.00C12-C14仲(2，3)烷基硫酸钠 5.00 5.00 5.00 5.00柠檬酸钠 5.00 5.00 5.00 5.00荧光增白剂 0.10 0.10 0.10 0.10硫酸钠 17.00 17.00 17.00 17.00水和少量组分 平衡至100％
在实施例37-38中，用表2-36中所列的热酶变异体代替Ser192Asp+Phe193Leu+Thr195Asn，得到基本上类似的结果。
在实施例39-40中，用表2-36中所列的热酶变异体的任何组合代替Ser192Asp+Phe193Leu+Thr195Asn和Tyr213Ser+Thr217Gly+Gly223Glu，得到基本上类似的结果。
实施例41-42
颗粒织物洗涤组合物
实施例组分 41 42直链烷基苯磺酸盐 11.4 10.70动物脂烷基硫酸盐 1.80 2.40C14-15烷基硫酸盐 3.00 3.10C14-15醇7倍乙氧基化 4.00 4.00动物脂醇11倍乙氧基化 1.80 1.80分散剂 0.07 0.1硅氧烷液体 0.80 0.80柠檬酸三钠 14.00 15.00柠檬酸 3.00 2.50沸 32.50 32.10马来酸丙烯酸共聚物 5.00 5.00二亚乙三胺五亚甲基膦酸 1.00 0.20Leu104Asp+Gly139Pro+Tyr171Asp 0.30 0.30+Ser191Asp+Ser222Glu+Thr224Ser脂酶 0.36 0.40淀粉酶 0.30 0.30硅酸钠 2.00 2.50硫酸钠 3.50 5.20聚乙烯吡咯烷酮 0.30 0.50过硼酸盐 0.5 1苯酚磺酸盐 0.1 0.2过氧化酶 0.1 0.1少量组分 至100 至100
实施例43-44
颗粒织物洗涤组合物
实施例组分 43 44直链C12烷基苯磺酸钠 6.5 8.0硫酸钠 15.0 18.0沸石A 26.0 22.0次氮基三乙酸钠 5.0 5.0聚乙烯吡咯烷酮 0.5 0.7四乙酰基乙二胺 3.0 3.0硼酸 4.0 -过硼酸盐 0.5 1苯酚磺酸盐 0.1 0.2Pro205Gly+Trp208Leu+Thr217Gln+Leu 0.4 0.4221Gln+Gly223Gln+Thr224Glu填料(例如，硅酸盐；碳酸盐；香料；水) 至100 至100
实施例45
压型颗粒织物洗涤组合物组分 重量％烷基硫酸盐 8.0烷基乙氧基硫酸盐 2.0C25和C45醇3和7倍乙氧基化的混合物 6.0多羟基脂肪酸酰胺 2.5沸石 17.0层状硅酸盐/柠檬酸盐 16.0碳酸盐 7.0马来酸丙烯酸共聚物 5.0污垢解脱聚合物 0.4羧甲基纤维素 0.4聚(4-乙烯基吡啶)-N-氧化物 0.1乙烯基咪唑和乙烯基吡咯烷酮共聚物 0.1PEG2000 0.2Asn66Ser+Ser70Glu+Gly72Asn 0.5脂酶 0.2纤维素酶 0.2四乙酰基乙二胺 6.0过碳酸盐 22.0乙二胺二琥珀酸 0.3泡沫抑制剂 3.54，4’-双(2-吗啉基-4-苯胺基-s-三嗪-6-基氨基) 0.25芪-2，2’-二磺酸二钠4，4’-双(2-磺基苯乙烯基)联苯二钠 0.05水、香料和少量组分 至100
实施例46
颗粒织物洗涤组合物组分 重量％直链烷基苯磺酸盐 7.6C16-C18烷基硫酸盐 1.3C14-15醇7倍乙氧基化物 4.0椰子烷基二甲基羟基乙基铵氯化物 1.4分散剂 0.07硅氧烷液体 0.8柠檬酸三钠 5.0沸石4A 15.0马来酸丙烯酸共聚物 4.0二亚乙三胺五亚甲基膦酸 0.4过硼酸盐 15.0四乙酰基乙二胺 5.0绿土 10.0聚(环氧乙烷)(MW300000) 0.3Ser109Glu+Thr113Gly 0.4脂酶 0.2淀粉酶 0.3纤维素酶 0.2硅酸钠 3.0碳酸钠 10.0羧甲基纤维素 0.2增白剂 0.2水、香料和少量组分 至100
实施例47颗粒织物洗涤组合物组分 重量％直链烷基苯磺酸盐 6.92动物脂烷基硫酸盐 2.05C14-15醇7倍乙氧基化物 4.4C12-15烷基乙氧基硫酸盐-3倍乙氧基化 0.16沸石 20.2柠檬酸盐 5.5碳酸盐 15.4硅酸盐 3.4马来酸丙烯酸共聚物 4.0羧甲基纤维素酶 0.31污垢解脱聚合物 0.30Gly135Gln+Val138Asp+Gly139Ser 0.20脂酶 0.36纤维素酶 0.13过硼酸盐四水合物 11.64过硼酸盐单水合物 8.7四乙酰基乙二胺 5.0二亚乙三胺五甲基膦酸 0.38硫酸镁 0.40增白剂 0.19香料、硅氧烷、泡沫抑制剂 0.85少量组分 至100b.液体织物洗涤组合物
本发明的液体织物洗涤组合物含有有效量的一种或多种本发明的酶变异体，按组合物活性酶重量计优选为约0.005％-约5％，更优选为约0.01％-约1％。该液体织物洗涤组合物通常还含有一种阴离子表面活性剂、脂肪酸、水溶性洗涤剂助剂和水。
本发明的液体织物洗涤组合物的实施方案由如下实施例说明。
实施例48-52
液体织物洗涤组合物
实施例组分 48 49 50 51 52Asn166Asp+Thr167Asn+ 0.05 0.03 0.30 0.03 0.10Asn170Ser+Tyr171ThrSer191Glu+Ser192Asp+ - - - 0.01 0.20Phe193Asp+Thr195ProC12-C14烷基硫酸钠 20.00 20.00 20.00 20.00 20.002-丁基辛酸 5.00 5.00 5.00 5.00 5.00柠檬酸钠 1.00 1.00 1.00 1.00 1.00C10醇乙氧基化物(3) 13.00 13.00 13.00 13.00 13.00单乙醇胺 2.50 2.50 2.50 2.50 2.50水/丙二醇/乙醇(100∶1∶1) 平衡至100％
在实施例48-50中，用表2-36所列的热酶变异体代替Asn166Asp+Thr167Asn+Asn170Ser+Tyr171Thr，得到基本上类似的结果。
在实施例51-52中，用表2-36中所列的热酶变异体的任何组合代替Asn166Asp+Thr167Asn+Asn170Ser+Tyr171Thr和Ser191Glu+Ser192Asp+Phe193Asp+Thr195Pro，得到基本上类似的结果。
实施例53-57
液体织物洗涤组合物
实施例组分 53 54 55 56 57Pro205Gln+Gly206Ser+Trp208V 0.05 0.03 0.30 0.03 0.10al+Tyr210Leu+Thr212Asn+Tyr218Met+Ser220GluAsn68Asp+Val138Asp+Ala164 - - - 0.01 0.20Gly+Ala168Glu+Thr217GlyC12-C14烷基硫酸钠 20.00 20.00 20.00 20.00 20.002-丁基辛酸 5.00 5.00 5.00 5.00 5.00柠檬酸钠 1.00 1.00 1.00 1.00 1.00C10醇乙氧基化物(3) 13.00 13.00 13.00 13.00 13.00单乙醇胺 2.50 2.50 2.50 2.50 2.50水/丙二醇/乙醇(100∶1∶1) 平衡至100％
在实施例53-55中，用表2-36中所列的热酶变异体代替Pro205Gln+Gly206Ser+Trp208Val+Tyr210Leu+Thr212Asn+Tyr218Met+Ser220Glu，得到基本上类似的结果。
在实施例56-57中，用表2-36中所列的热酶变异体的任何组合代替Pro205Gln+Gly206Ser+Trp208Val+Tyr210Leu+Thr212Asn+Tyr218Met+Ser220Glu和Asn68Asp+Val138Asp+Ala164Gly+Ala168Glu+Thr217Gly，得到基本上类似的结果。
实施例58-59
液体织物洗涤组合物
实施例组分 58 59C12-14烯基琥珀酸 3.0 8.0柠檬酸单水合物 10.0 15.0C12-15烷基硫酸钠 8.0 8.0C12-15醇2倍乙氧基化硫酸钠 - 3.0C12-15醇7倍乙氧基化物 - 8.0C12-15醇5倍乙氧基化物 8.0 -二亚乙三胺五(亚甲基膦酸) 0.2 -油酸 1.8 -乙醇 4.0 4.0丙二醇 2.0 2.0Leu134Cys+Thr137Glu+Val138Gly 0.2 0 2聚乙烯吡咯烷酮 1.0 2.0泡沫抑制剂 0.15 0.15氢氧化钠 至pH7.5过硼酸盐 0.5 1苯酚磺酸盐 0.1 0.2过氧化酶 0.4 0.1水和少量组分 至100份
在实施例58和59中，用表2-36中所列的热酶变异体代替Leu134Cys+Thr137Glu+Val138Gly，得到基本上类似的结果。
实施例60-62
液体织物洗涤组合物
实施例组分 60 61 62柠檬酸 7.10 3.00 3.00脂肪酸 2.00 - 2.00乙醇 1.93 3.20 3.20硼酸 2.22 3.50 3.50单乙醇胺 0.71 1.09 1.091，2-丙二醇 7.89 8.00 8.00枯烯磺酸钠 1.80 3.00 3.00甲酸钠 0.08 0.08 0.08氢氧化钠 6.70 3.80 3.80含硅消泡剂 1.16 1.18 1.18Gly67Glu+Ser70Asp+Gly72Ser+Thr73Ser 0.0145 - -Ile209Ala+Ala219Pro - 0.0145 -Leu216Asn - - 0.0145脂酶 0.200 0.200 0.200纤维素酶 - 7.50 7.50污垢解脱聚合物 0.29 0.15 0.15消泡剂 0.06 0.085 0.085增白剂36 0.095 - -增白剂3 - 0.05 0.05C12烷基苯磺酸 9.86 - -C12-15烷基多乙氧基化(2.5)硫酸盐 13.80 18.00 18.00C12葡糖酰胺 - 5.00 5.00C12-13烷基聚乙氧基化物(9) 2.00 2.00 2.00水、香料和少量组分 平衡至100％c.条状织物洗涤组合物
本发明的适合于手洗脏织物的条状织物洗涤组合物含有有效量的一种或多种本发明的酶变异体，按组合物活性酶重量计优选为约0.001％-约10％，更优选为约0.01％-约1％。
本发明的条状织物洗涤组合物的实施方案由如下实施例说明。
实施例63-66
条状织物洗涤组合物
实施例组分 63 64 65 66Trp112Cys+Thr217Gly 0.3 - 0.1 0.02Val103Ala+Thr212Ser - - 0.4 0.03C12-C16烷基硫酸钠 20.0 20.0 20.0 20.00C12-C14N-甲基葡糖酰胺 5.0 5.0 5.0 5.00C11-C13烷基苯磺酸钠 10.0 10.0 10.0 10.00碳酸钠 25.0 25.0 25.0 25.00焦磷酸钠 7.0 7.0 7.0 7.00三聚磷酸钠 7.0 7.0 7.0 7.00沸石A(0.1-10μ) 5.0 5.0 5.0 5.00羧甲基纤维素 0.2 0.2 0.2 0.20聚丙烯酸酯(MW1400) 0.2 0.2 0.2 0.20椰子烷基单乙醇胺 5.0 5.0 5.0 5.00增白剂、香料 0.2 0.2 0.2 0.20硫酸钙 1.0 1.0 1.0 1.00硫酸镁 1.0 1.0 1.0 1.00水 4.0 4.0 4.0 4.00填料* 平衡至100％*可以选自常规材料，例如碳酸钙、滑石、粘土、硅酸盐等等。
在实施例63-64中，用表2-36中所列的热酶变异体代替Trp112Cys+Thr217Gly，得到基本上类似的结果。
在实施例65-66中，用表2-36中所列的热酶变异体的任何组合代替Trp112Cys+Thr217Gly和Val103Ala+Thr212Ser，得到基本上类似的结果。
实施例67-70
条状织物洗涤组合物
实施例组分 67 68 69 70Ser109Glu 0.3 - 0.1 0.02Pro169Glu - 0.3 0.4 0.03C12-C16烷基硫酸钠 20.0 20.0 20.0 20.0C12-C14N-甲基葡糖酰胺 5.0 5.0 5.0 5.00C11-C13烷基苯磺酸钠 10.0 10.0 10.0 10.00碳酸钠 25.0 25.0 25.0 25.00焦磷酸钠 7.0 7.0 7.0 7.00三聚磷酸钠 7.0 7.0 7.0 7.00沸石A(0.1-10μ) 5.0 5.0 5.0 5.00羧甲基纤维素 0.2 0.2 0.2 0.20聚丙烯酸酯(MW1400) 0.2 0.2 0.2 0.20椰子烷基单乙醇胺 5.0 5.0 5.0 5.00增白剂、香料 0.2 0.2 0.2 0.20硫酸钙 1.0 1.0 1.0 1.00硫酸镁 1.0 1.0 1.0 1.00水 4.0 4.0 4.0 4.00填料* 平衡至100％*可以选自常规材料，例如碳酸钙、滑石、粘土、硅酸盐等等。
在实施例67中，用表2-36中所列的热酶变异体代替Ser109Glu，得到基本上类似的结果。
在实施例68中，用表2-36中所列的热酶变异体代替Pro169Glu，得到基本上类似的结果。
在实施例69-70中，用表2-36中所列的热酶变异体的任何组合代替Ser109Glu和Pro169Glu，得到基本上类似的结果。B.其它洗涤组合物
除了上述硬表面洗涤、餐具洗涤和织物洗涤组合物之外，本发明的一种或多种酶变异体可加入各种需要水解不溶解底物的其它洗涤组合物中。其它洗涤组合物包括，但不限制于，口腔洗涤组合物、假牙洗涤组合物和接触透镜洗涤组合物。
1.口腔洗涤组合物
在本发明的另一实施方案中，在组合物中包含一种或多种可药用量的本发明的酶变异体以从牙齿或假牙中除去蛋白质污垢。本文使用的“口腔洗涤组合物”指的是洁齿粉、牙膏、牙凝胶、牙粉、漱口剂、口腔喷雾剂、口腔凝胶、口香糖、锭剂、香囊、片剂、生物凝胶、预防膏、假牙处理溶液等等。按组合物的重量计，洗涤组合物优选含有约0.0001-约20％，更优选约0.001％-约10％，还优选约0.01％-约5％的一种或多种本发明的酶变异体和可药用的载体。本文使用的“可药用的”指的是该术语描述的药物、药剂或惰性成分适合于与人体和低级动物的组织接触，而没有不适宜的毒性、不适性、不稳定性、刺激性、过敏反应等等，与有益效果/风险比率相当。
口腔洗涤组合物的可药用的口腔洗涤载体组分按组合物重量计通常占约50％-约99.99％，优选约65％-约99.99％，更优选约65％-约99％。
可包括在本发明的口腔洗涤组合物中的可药用的载体组分和选择性的组分是本领域技术人员已知的。用于口腔洗涤组合物的各种组合物类型、载体组分和选择性组分在1992年3月17日授权的Seibel的US5096700；1991年7月2日授权的Sampathkumar的US5028414和1991年7月2日授权的Benedict、Bush和Sunberg的US5028415中，列为本文参考文献。
本发明的口腔洗涤组合物的实施方案在如下实施例中说明。
实施例71-74
洁牙粉组合物
实施例组分 71 72 73 74Tyr218Ala 2.000 3.500 1.500 2.000山梨糖醇(70％水溶液) 35.000 35.000 35.000 35.000PEG-6* 1.000 1.000 1.000 1.000氧化硅牙用磨料** 20.000 20.000 20.000 20.000氟化钠 0.243 0.243 0.243 0.243二氧化钛 0.500 0.500 0.500 0.500糖精钠 0.286 0.286 0.286 0.286烷基硫酸钠(27.9％水溶液) 4.000 4.000 4.000 4.000香料 1.040 1.040 1.040 1.040羧乙烯基聚合物*** 0.300 0.300 0.300 0.300Carrageenan**** 0.800 0.800 0.800 0.800水 平衡至100％*PEG-6＝分子量600的聚乙二醇。**由J.M.Huber提供的沉淀的氧化硅Zeodent119***由B.F.Goodrich Chemical Company提供的Carbopol****由Hercules Chemical Company提供的lota Carrageenan。
在实施例71-74中，用表2-36中所列的热酶变异体代替Tyr218Ala，得到基本上类似的结果。
实施例75-78
漱口剂组合物
实施例组分 75 76 77 78Ala164His+Ser220Glu 3.00 7.50 1.00 5.00SDA40醇 8.00 8.00 8.00 8.00香料 0.08 0.08 0.08 0.08乳化利 0.08 0.08 0.08 0.08氟化钠 0.05 0.05 0.05 0.05甘油 10.00 10.00 10.00 10.00增甜剂 0.02 0.02 0.02 0.02苯甲酸 0.05 0.05 0.05 0.05氢氧化钠 0.20 0.20 0.20 0.20染料 0.04 0.04 0.04 0.04水 平衡至100％在实施例75-78中，用表2-36中所列的热酶变异体代替Ala164His+Ser220Glu，得到基本上类似的结果。
实施例79-82
锭剂组合物
实施例组分 79 80 81 82Leu221Val+Thr224Gln 0.01 0.03 0.10 0.02山梨糖醇 17.50 17.50 17.50 17.50甘露糖醇 17.50 17.50 17.50 17.50淀粉 13.60 13.60 13.60 13.60增甜剂 1.20 1.20 1.20 1.20香料 11.70 11.70 11.70 11.70色素 0.10 0.10 0.10 0.10玉米糖浆 平衡至100％
在实施例79-82中，用表2-36中所列的热酶变异体代替Leu221Val+Thr224Gln，得到基本上类似的结果。
实施例83-86
咀嚼胶组合物
实施例组分 83 84 85 86Ser191Asp+Phe193Ile+Thr195Gly 0.03 0.02 0.10 0.05山梨糖醇结晶 38.44 38.40 38.40 38.40Palija-T胶基料* 20.00 20.00 20.00 20.00山梨糖醇(70％水溶液) 22.00 22.00 22.00 22.00甘露糖醇 10.00 10.00 10.00 10.00甘油 7.56 7.56 7.56 7.56香料 1.00 1.00 1.00 1.00*L.A.Dreyfus Company提供。
在实施例83-86中，用表2-36中所列的热酶变异体代替Ser191Asp+Phe193Ile+Thr195Gly，得到基本上类似的结果。
2.假牙洗涤组合物在本发明的另一实施方案中，用于在口腔外洗涤假牙的假牙洗涤组合物含有一种或多种本发明的酶变异体。该假牙洗涤组合物含有有效量的一种或多种酶变异体，优选按组合物重量计，约0.0001-约50％，更优选约0.001％-约35％，还优选约0.01％-约20％的一种或多种本发明的酶变异体，和假牙洗涤载体。各种假牙洗涤组合物配方，如泡腾片等等是现有技术中已知的(参见例如Young的US5055305，列为本文参考文献)，通常适合于加入一种或多种酶变异体以便从假牙除去蛋白质污垢。
本发明的假牙洗涤组合物的实施方案由如下实施例说明。
实施例87-90
双层泡腾假牙洗涤片
实施例组分 87 88 89 90酸性层Gly135Gln+Gly139Asn+ 1.0 1.5 0.01 0.05Asn140Gln+Ser141Asp酒石酸 24.0 24.0 24.00 24.00碳酸钠 4.0 4.0 4.00 4.00氨基磺酸 10.0 10.0 10.00 10.00PEG20000 4.0 4.0 4.00 4.00碳酸氢钠 24.5 24.5 24.50 24.50过硫酸钾 15.0 15.0 15.00 15.00酸性焦磷酸钠 7.0 7.0 7.00 7.00热解氧化硅 2.0 2.0 2.00 2.00TAED* 7.0 7.0 7.00 7.00蓖麻酰基磺基琥珀酸盐 0.5 0.5 0.50 0.50香料 1.0 1.0 1.00 1.00碱性层过硼酸钠单水合物 32.0 32.0 32.00 32.00碳酸氢钠 19.0 19.0 19.00 19.00EDTA 3.0 3.0 3.00 3.00三聚磷酸钠 12.0 12.0 12.00 12.00PEG20000 2.0 2.0 2.00 2.0过硫酸钾 26.0 26.0 26.00 26.00碳酸钠 2.0 2.0 2.00 2.00热解氧化硅 2.0 2.0 2.00 2.00染料/香料 2.0 2.0 2.00 2.00*四乙酰基乙二胺
在实施例87-90中，用表2-36中所列的热酶变异体代替Gly135Gln+Gly139Asn+Asn140Gln+Ser141Asp，得到基本上类似的结果。
2.接触透镜洗涤组合物
在本发明的另一实施方案中，接触透镜洗涤组合物含有一种或多种本发明的酶变异体。该接触透镜洗涤组合物含有有效量的一种或多种酶变异体，优选按组合物重量计，约0.01-约50％，更优选约0.01％-约20％，还优选约1％-约5％的一种或多种本发明的酶变异体，和接触透镜洗涤载体。各种接触透镜洗涤组合物配方，例如片剂、液体等等在现有技术中是已知的(参见例如1989年9月5日授权的Davies，Meaken和Rees，US4863627；1988年5月24日再次授权的Huth，Lam和Kirai，USRe32672；有1986年9月2日授权的Schafer，US4609493；1987年9月1日授权的Ogunbiyi和Smith，US4690793；1986年9月30日授权的Ogunbiyi，Riedhammer和Smiht，US4614549；和1981年8月25日授权的Ogata，US4285738；均列为本文参考文献)，通常适合于加入本发明的一种或多种酶变异体以便从接触透镜除去蛋白质污垢。
本发明的接触透镜洗涤组合物的实施方案由如下实施例说明。
实施例91-94
酶解接触透镜洗涤溶液
实施例组分 91 92 93 94Leu221Gln 0.01 0.5 0.1 2.0葡萄糖 50.00 50.0 50.0 50.0非离子表面活性剂(聚氧乙烯- 2.00 2.0 2.0 2.0聚氧丙烯共聚物)阴离子表面活性剂(聚氧乙烯- 1.00 1.0 1.0 1.0烷基苯基醚硫酸酯钠)氯化钠 1.00 1.0 1.0 1.0硼砂 0.30 0.3 0.3 0.3水 平衡至100％
在实施例91-94中，用表2-36中所列的热酶变异体代替Leu221Gln，得到基本上类似的结果。
尽管描述了本发明的具体实施方案，但显然本领域的技术人员可作出本发明的各种改变和改进而不会违背本发明的精神和范围。在所附的权利要求中，本发明的范围覆盖了所有这些改进。
Claims (9)
1.具有热酶(thermitase)野生型氨基酸序列的修饰氨基酸序列的热酶变异体，野生型氨基酸序列包括第一环区域、第二环区域、第三环区域、第四环区域、第五环区域和第六环区域；其中修饰的氨基酸序列在一个或多个环区域内的一个或多个位点上含有取代基；其中
A.当取代发生在第一环区域中时，取代发生在位点66、69、70、72或73的一个或多个上；其中
a.当取代发生在位点66时，取代氨基酸是Asn；
b.当取代发生在位点69时，取代氨基酸是Gln；
c.当取代发生在位点70时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser；
d.当取代发生在位点72时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser；
e.当取代发生在位点73时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser；
B.当取代发生在第二环区域中时，取代发生在位点103、104、105、106、108、110、112、113或114的一个或多个上；其中
a.当取代发生在位点103时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Met、Pro、Ser或Thr；
b.当取代发生在位点104时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Gly、His、Ile、Met、Pro或Ser；
c.当取代发生在位点105时，取代氨基酸是Glu；
d.当取代发生在位点106时，取代氨基酸是Gln；
e.当取代发生在位点108时，取代氨基酸是Asn、Gln、Pro或Ser；
f.当取代发生在位点110时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser；
g.当取代发生在位点112时，取代氨基酸是Asn、Asp、Cys、Gln、Glu、His、Ile、Met、Phe、Pro、Thr或Tyr；
h.当取代发生在位点113时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser；和
i.当取代发生在位点114时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、His、Pro、Ser或Thr；
C.当取代发生在第三环区域中时，取代发生位点134、135、136、137、138、139或141的一个或多个上；其中
a.当取代发生在位点134时，取代氨基酸是Ala、Asn、Cys、Gln、Gly、His、Met、Pro、Ser、Thr或Val；
b.当取代发生在位点135时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser；
c.当取代发生在位点136时，取代氨基酸是Pro；
d.当取代发生在位点137时，取代氨基酸是Asn、Asp、Gln、Glu或Ser；
e.当取代发生在位点138时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Met、Pro、Ser或Thr；
f.当取代发生在位点139时，取代氨基酸是Asn、Gln、Pro或Ser；和
g.当取代发生在位点141时，取代氨基酸是Asp或Glu；
D.当取代发生在第四环区域中时，取代发生在位点167、169或171一个或多个上；其中
a.当取代发生在位点167时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser；
b当取代发生在位点169时，取代氨基酸是Asn、Asp、Gln、Glu、Gly或Ser；和
c.当取代发生在位点171时，取代氨基酸是His、Ile、Leu、Met或Pro；
E.当取代发生在第五环区域中时，取代发生在位点193一个或多个上；其中
a.当取代发生在位点193时，取代氨基酸是Asn、Cys、Gln、His、Ile、Met、Thr或Tyr；
F.当取代发生在第六环区域中时，取代发生在位点204、205、206、207、208、209、210、211、212、213、214、215、216、217、220、221、223或224的一个或多个上，其中
a.当取代发生在位点204时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、His、Pro、Ser或Thr；
b.当取代发生在位点205时，取代氨基酸是Asn、Asp、Gln、Glu、Gly或Ser；
c.当取代发生在位点206时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser；
d.当取代发生在位点207时，取代氨基酸是Asp或Glu；
e.当取代发生在位点208时，取代氨基酸是Asp或Glu；
f.当取代发生在位点209时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Leu、Met、Pro、Ser、Thr或Val；
g.当取代发生在位点210时，取代氨基酸是Asp、His、Ile、Leu、Met或Pro；
h.当取代发生在位点211时，取代氨基酸是Asp或Glu；
i.当取代发生在位点212时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser；
j.当取代发生在位点213时，取代氨基酸是Asp、Glu、His、Ile、Leu、Met、Phe、Pro或Val；
i.当取代发生在位点214时，取代氨基酸是Asn、Gln、Gly或Ser；
j.当取代发生在位点215时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser；
m.当取代发生在位点216时，取代氨基酸是Asp或Glu；
n.当取代发生在位点217时，取代氨基酸是Asp、Glu或Pro；
o.当取代发生在位点220时，取代氨基酸是Asp或Glu；
p.当取代发生在位点221时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Met、Pro、Ser、Thr或Val；
q.当取代发生在位点222时，取代氨基酸是Asp或Glu；
r.当取代发生在位点223时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser；
s.当取代发生在位点224时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser；
从而，与野生型热酶相比较，热酶变异体对不溶解底物具有降低的吸附作用和增加的水解作用。
2.权利要求1的热酶变异体，其中一个或多个取代基发生在第一环区域。
3.权利要求1的热酶变异体，其中一个或多个取代基发生在第二环区域。
4.权利要求1的热酶变异体，其中一个或多个取代基发生在第三环区域。
5.权利要求1的热酶变异体，其中一个或多个取代基发生在第四环区域。
6.权利要求1的热酶变异体，其中一个或多个取代基发生在第五环区域。
7.权利要求1的热酶变异体，其中一个或多个取代基发生在第六环区域。
8.一种选自硬表面洗涤组合物、餐具洗涤组合物、口腔洗涤组合物、假牙洗涤组合物、接触透镜洗涤组合物和织物洗涤组合物的洗涤组合物，其特征在于其中洗涤组合物含有权利要求1-7的任何一项的热酶变异体和洗涤组合物载体；洗涤组合物优选是硬表面洗涤组合物或洗涤组合物是织物洗涤组合物；组合物优选含有按组合物重量计至少约5％的表面活性剂和至少约5％助剂；组合物还优选含有选自溶剂、缓冲剂、酶、污垢解脱剂、粘土去污剂、分散剂、增白剂、泡沫抑制剂、织物柔软剂、增泡剂、酶稳定剂、漂白剂、染料、香料和它们的混合物的洗涤组合物材料。
9.编码权利要求1-7的任何一项的热酶变异体的DNA序列。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US40157395A | 1995-03-09 | 1995-03-09 | |
| US08/401,573 | 1995-03-09 |
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| Publication Number | Publication Date |
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| CN1183118A true CN1183118A (zh) | 1998-05-27 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN96193608A Pending CN1183118A (zh) | 1995-03-09 | 1996-03-06 | 具有降低的吸附作用和增加的水解作用的热酶变异体 |
Country Status (14)
| Country | Link |
|---|---|
| EP (1) | EP0815244A1 (zh) |
| JP (1) | JPH11501816A (zh) |
| CN (1) | CN1183118A (zh) |
| AU (1) | AU5182996A (zh) |
| BR (1) | BR9607756A (zh) |
| CA (1) | CA2214578A1 (zh) |
| CZ (1) | CZ280097A3 (zh) |
| HU (1) | HUP9802069A3 (zh) |
| IL (1) | IL117352A0 (zh) |
| IN (1) | IN187236B (zh) |
| MA (1) | MA23819A1 (zh) |
| MX (1) | MX9706846A (zh) |
| WO (1) | WO1996028558A1 (zh) |
| ZA (1) | ZA961822B (zh) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| AU1200997A (en) * | 1995-12-22 | 1997-07-17 | Helix Biotechnology Ltd | Thermostable proteolytic enzyme from thermoactinomyces thalpophilus thm1 |
| ATE409743T1 (de) * | 1996-11-04 | 2008-10-15 | Novozymes As | Subtilase varianten und verbindungen |
| JPH10204500A (ja) * | 1997-01-20 | 1998-08-04 | Soft Kyukyu Corp:Kk | 発泡錠剤洗浄剤 |
| AR015977A1 (es) * | 1997-10-23 | 2001-05-30 | Genencor Int | Variantes de proteasa multiplemente substituida con carga neta alterada para su empleo en detergentes |
| ES2368718T3 (es) | 1997-10-23 | 2011-11-21 | Danisco Us Inc. | Variantes de subtilisina con múltiples sustituciones. |
| AU773066B2 (en) * | 1998-12-18 | 2004-05-13 | Novozymes A/S | Subtilase enzymes of the I-S1 and I-S2 sub-groups having an additional amino acid residue in an active site loop region |
| CN1198913C (zh) * | 1998-12-18 | 2005-04-27 | 诺沃奇梅兹有限公司 | 在活性位点环区中具有额外氨基酸残基的i－s1和i－s2亚组枯草杆菌酶 |
| US7319112B2 (en) | 2000-07-14 | 2008-01-15 | The Procter & Gamble Co. | Non-halogenated antibacterial agents and processes for making same |
| DE10153792A1 (de) | 2001-10-31 | 2003-05-22 | Henkel Kgaa | Neue Alkalische Protease-Varianten und Wasch- und Reinigungsmittel enthaltend diese neuen Alkalischen Protease-Varianten |
| DE10162727A1 (de) | 2001-12-20 | 2003-07-10 | Henkel Kgaa | Neue Alkalische Protease aus Bacillus gibsonii (DSM 14391) und Wasch-und Reinigungsmittel enthaltend diese neue Alkalische Protease |
| DE10162728A1 (de) | 2001-12-20 | 2003-07-10 | Henkel Kgaa | Neue Alkalische Protease aus Bacillus gibsonii (DSM 14393) und Wasch-und Reinigungsmittel enthaltend diese neue Alkalische Protease |
| JP5339672B2 (ja) * | 2006-07-03 | 2013-11-13 | 小林製薬株式会社 | 漂白洗浄剤組成物 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US5116741A (en) * | 1988-04-12 | 1992-05-26 | Genex Corporation | Biosynthetic uses of thermostable proteases |
| JPH04500384A (ja) * | 1989-06-26 | 1992-01-23 | ユニリーバー・ナームローゼ・ベンノートシヤープ | 酵素洗剤組成物 |
| BR9006827A (pt) * | 1989-06-26 | 1991-08-06 | Unilever Nv | Composicoes detergentes enzimaticas |
| DK316989D0 (da) * | 1989-06-26 | 1989-06-26 | Novo Nordisk As | Enzymer |
| EP0719339B1 (en) * | 1993-09-15 | 2006-10-25 | The Procter & Gamble Company | Subtilisin bpn' variants with decreased adsorption and increased hydrolysis |
-
1996
- 1996-03-04 IL IL11735296A patent/IL117352A0/xx unknown
- 1996-03-06 MX MX9706846A patent/MX9706846A/es unknown
- 1996-03-06 HU HU9802069A patent/HUP9802069A3/hu unknown
- 1996-03-06 CA CA002214578A patent/CA2214578A1/en not_active Abandoned
- 1996-03-06 BR BR9607756A patent/BR9607756A/pt not_active Application Discontinuation
- 1996-03-06 IN IN457DE1996 patent/IN187236B/en unknown
- 1996-03-06 CN CN96193608A patent/CN1183118A/zh active Pending
- 1996-03-06 AU AU51829/96A patent/AU5182996A/en not_active Abandoned
- 1996-03-06 WO PCT/US1996/003009 patent/WO1996028558A1/en not_active Application Discontinuation
- 1996-03-06 JP JP8527693A patent/JPH11501816A/ja active Pending
- 1996-03-06 CZ CZ972800A patent/CZ280097A3/cs unknown
- 1996-03-06 ZA ZA961822A patent/ZA961822B/xx unknown
- 1996-03-06 EP EP96908663A patent/EP0815244A1/en not_active Withdrawn
- 1996-03-07 MA MA24176A patent/MA23819A1/fr unknown
Also Published As
| Publication number | Publication date |
|---|---|
| WO1996028558A1 (en) | 1996-09-19 |
| CA2214578A1 (en) | 1996-09-19 |
| CZ280097A3 (cs) | 1998-01-14 |
| BR9607756A (pt) | 1999-03-30 |
| EP0815244A1 (en) | 1998-01-07 |
| AU5182996A (en) | 1996-10-02 |
| MX9706846A (es) | 1997-11-29 |
| HUP9802069A2 (hu) | 1998-12-28 |
| ZA961822B (en) | 1996-09-11 |
| HUP9802069A3 (en) | 2000-11-28 |
| JPH11501816A (ja) | 1999-02-16 |
| IL117352A0 (en) | 1996-07-23 |
| IN187236B (zh) | 2002-03-09 |
| MA23819A1 (fr) | 1996-10-01 |
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