[go: up one dir, main page]

CN108913677A - 一种定点突变改造的碱性普鲁兰酶及其应用 - Google Patents

一种定点突变改造的碱性普鲁兰酶及其应用 Download PDF

Info

Publication number
CN108913677A
CN108913677A CN201810813706.8A CN201810813706A CN108913677A CN 108913677 A CN108913677 A CN 108913677A CN 201810813706 A CN201810813706 A CN 201810813706A CN 108913677 A CN108913677 A CN 108913677A
Authority
CN
China
Prior art keywords
asp
thr
glu
gly
leu
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201810813706.8A
Other languages
English (en)
Other versions
CN108913677B (zh
Inventor
林娟
林云
王国增
许鑫琦
黄晗
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Fuzhou University
Original Assignee
Fuzhou University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fuzhou University filed Critical Fuzhou University
Priority to CN201810813706.8A priority Critical patent/CN108913677B/zh
Publication of CN108913677A publication Critical patent/CN108913677A/zh
Application granted granted Critical
Publication of CN108913677B publication Critical patent/CN108913677B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
    • C12N9/2457—Pullulanase (3.2.1.41)
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38636—Preparations containing enzymes, e.g. protease or amylase containing enzymes other than protease, amylase, lipase, cellulase, oxidase or reductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01041—Pullulanase (3.2.1.41)

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

本发明提供一种定点突变改造的碱性普鲁兰酶及其应用，本发明定点突变改造的碱性普鲁兰酶是在SEQ ID NO.2所示的氨基酸基础上，将第744位苯丙氨酸突变为丙氨酸。对突变体F744A转化大肠杆菌进行异源表达，发现突变体酶活提高了32.18%；热稳定性也得到了提高，T50 30为55.84℃，比原始酶PulSL3△C提高了2.94℃；突变体F744A的pH稳定范围更宽，在pH 5.0~10.0范围内保温1 h仍可保留90%以上的酶活力。作为添加剂应用于洗涤剂行业，可以明显提高洗涤效果。

Description

一种定点突变改造的碱性普鲁兰酶及其应用
技术领域
本发明涉及一种定点突变改造的碱性普鲁兰酶及其应用，属于基因工程与酶工程领域。
背景技术
普鲁兰酶可特异性切开淀粉的α-1, 6糖苷键，从而得到直链淀粉。将其应用于淀粉加工行业，可显著提高淀粉的利用率和生产效率。在饲料行业，目前主要以玉米---豆粕型日粮为原料生产饲料，但其中玉米支链淀粉易形成难以被动物肠道消化的“抗营养因子”，使年幼的动物不能很好的吸收利用饲料，还会形成肠道疾病，因此饲料中添加普鲁兰酶，可促进动物对饲料中营养物质的消化吸收利用，从而提高经济效益。普鲁兰酶作为一类用途广泛的酶制剂，不仅可应用于淀粉深加工和饲料工业，碱性普鲁兰酶还可作为一种有效添加剂应用于洗涤剂行业。但从自然界分离得到的天然菌株，普鲁兰酶分泌能力较低且难以满足工业生产的条件，限制了其在工业上的应用。定点突变作为分子改造的一种有效方式，被广泛应用于提高工业酶制剂的催化效率、改善酶性质等方面。
本发明利用基于结构分析和序列分析相结合的半理性设计，通过定点突变的方法对碱性普鲁兰酶PulSL3△C特定位点的氨基酸进行突变，得到酶活力和稳定性明显提高的突变体，对于提高普鲁兰酶的工业应用具有重要意义。
发明内容
本发明的目的是提供一种定点突变改造的碱性普鲁兰酶及其应用。
为实现上述目的，采用以下技术方案：
本发明采用PCR方法从盐碱湖来源Alkalibacterium sp. SL3菌株中克隆得到碱性普鲁兰酶基因pulSL3△C，核苷酸序列如SEQ ID NO.1所示，全长3555bp，编码1184个氨基酸组成的多肽，序列如SEQ ID NO.2所示，无信号肽序列。
利用在线软件SWISS MODEL 对碱性普鲁兰酶PulSL3△C 3D结构进行同源建模，采用Chiron服务器对模型结构进行在线优化，采用PROCHECK、ERRAT及Verify-3D等方法对经过在线优化后的最终结构进行检测和评价。利用基于结构分析和序列分析相结合的半理性设计，在SEQ ID NO.2所示的氨基酸基础上，将第744位苯丙氨酸突变为丙氨酸，得到突变体F744A；构建得到带有突变基因的重组质粒pET-22b(+)-F744A，转化宿主细胞E. coli BL21(DE3)，获得基因工程菌。
本发明的优点在于：
本发明通过定点突变方法改造碱性普鲁兰酶PulSL3△C的分子结构，得到一株酶活和稳定性明显提高的基因工程菌。突变体F744A酶活提高了32.18%，热稳定性也得到了提高，T50 30为55.84℃，比原始酶PulSL3△C提高了2.94℃；pH稳定范围拓宽为 5.0~10.0，保温1 h仍可保留90%以上的酶活力（原始酶PulSL3△C在pH 7.0~9.0范围内保温1 h可保留80%以上的酶活力）。
将碱性普鲁兰酶作为添加剂添加到洗涤剂中，可以明显提高洗涤效果，复合污垢的去污率提高了14.21%。
附图说明
图1为碱性普鲁兰酶PulSL3△C部分3D结构。
图2为重组质粒pET-22b(+)-pulSL3△C的构建。
图3为突变体F744A与原始酶PulSL3△C的最适反应pH。
图4为突变体F744A与原始酶PulSL3△C的pH稳定性。
图5为突变体F744A与原始酶PulSL3△C的的最适反应温度。
图6为突变体F744A与原始酶PulSL3△C的热稳定性。
具体实施方式
实施例1 普鲁兰酶突变体F744A的构建
根据碱性普鲁兰酶PulSL3△C的3D结构分析（图1），第744位苯丙氨酸导致一个较宽的裂缝和尖锐的凸起，该结构可能会抑制该酶对普鲁兰糖第二个α-1, 6糖苷键的水解，且对支链淀粉、糖原等密集分支型糖的水解抑制作用更严重。苯丙氨酸侧链为位阻较大的苯环，导致该裂缝和凸起的产生，若将Phe744突变成侧链为甲基的丙氨酸，即Ala744，可极大地减小苯环产生的空间位阻。将该突变体命名为F744A。
设计含有突变位点的引物，以重组质粒pET-22b(+)-pulSL3△C（图2）为模板，采用基于改良后寡核苷酸引物所介导的定点突变方法构建得到带有突变基因的表达质粒pET-22b(+)-F744A，转化E. coli BL21(DE3) 感受态细胞，获得突变体重组工程菌。
正向引物：5’-AATCCGGAGCCGGCAGTGAGGGAGAACC-3’；
反向引物：5’- TCACTGCCGGCTCCGGATTTCAGTTCATTTCTG-3’；
带有下划线的碱基为定点突变的位点。
PCR扩增体系：pET-22b(+)-pulSL3△C 1 μL，F744A-F (20 µmol/L) 1 μL，F744A-R (20 µmol/L) 1 μL，PCR Stimulant (5×) 5 μL，High Pure dNTPs(2.5 mmol/L) 4 μL，TransStart FastPfu Fly DNA Polymerase 1 μL，5×TransStart FastPfu Fly Buffer10 μL，MgSO4 1 μL，ddH2O补至50 μL。
PCR扩增条件：98℃预变性5 min；95℃变性30 s，60℃退火30 s，72℃延伸5 min，30个循环；72℃保温5 min；10℃保存。PCR扩增产物用1%琼脂糖凝胶电泳检测，并检测到目的条带正确。
PCR结束后，每50 μL PCR扩增产物加1 μL DMT，混匀，于37℃消化1 h。取1 μL消化产物转化E.coli Top10，涂布到含100 μg/mL Amp的LB固体平板上，过夜培养。挑取单菌落接种至含Amp的LB液体培养基中，吸取部分菌液送测，利用质粒提取试剂盒抽提测序正确的突变质粒，转化E.coli BL21(DE3) 感受态细胞，获得突变体F744A。
突变体核苷酸序列测序结果见序列表中SEQ ID NO.3所示，相应编码蛋白质氨基酸序列见序列表中SEQ ID NO.4所示。
实施例2 普鲁兰酶突变体F744A的诱导表达及纯化
从转化板上挑取单菌落接种到含100 µg/mL Amp的LB液体培养基中进行过夜培养。按1%接种量接种过夜培养液于50 mL含100 µg/mL Amp的 LB培养基中，37℃ 180 rpm培养至OD600达到0.5，加入终浓度为0.1 mmol/L的IPTG和0.5% NaCl，25℃ 180 rpm培养32 h。将发酵液于4℃、13000 rpm离心10 min，取上清液作为粗酶液。
粗酶液首先采用10 kDa的中空纤维柱浓缩，而后进行(NH4)2SO4分级沉淀，透析后的酶液经0.22 µm膜过滤后制成上样样品。首先使用预装柱DEAE FF(Hi TrapTM，5 mL)进行纯化，平衡缓冲液为pH 8.0的标准McIlvaine缓冲液，洗脱缓冲液为溶有1 mol/L NaCl的标准McIlvaine缓冲液(pH 8.0)，进行线性梯度洗脱，280 nm紫外在线监测，分部收集蛋白洗脱液。混合含有目标蛋白且杂质较少的洗脱液，用pH 8.0的标准McIlvaine缓冲液透析过夜，得到用于进一步纯化的样品。往该样品中加入(NH4)2SO4使其终浓度为1 mol/L，并用0.22 µm膜过滤，滤液采用预装柱Phenyl FF(HS)(Hi TrapTM，5 mL)进行纯化，平衡缓冲液为溶有1 mol/L (NH4)2SO4的标准McIlvaine缓冲液(pH 8.0)，洗脱液为pH 8.0的标准McIlvaine缓冲液，对蛋白洗脱液进行分部收集。将酶液进行SDS-PAGE检测分析，确认单一条带，获得纯酶。
实施例3 普鲁兰酶突变体F744A的酶活和性质测定
1. 普鲁兰酶突变体F744A的酶活测定
采用DNS法测定普鲁兰酶活力。将含0.9 mL 0.5%普鲁兰糖（TCI公司）和0.1 mL酶液的混合物，于适当条件下保温10 min，加入1.5 mL DNS终止反应，煮沸5 min显色后，冷却并用蒸馏水定容至10 mL，测定OD540。酶活力单位定义：在一定反应条件下，每min水解普鲁兰糖产生1 μmol还原糖所需的酶量定义为一个酶活力单位U。
普鲁兰酶突变体F744A与原始酶PulSL3△C相比，酶活提高了32.18%。
2. 普鲁兰酶突变体F744A的最适反应pH和pH稳定性
最适反应pH的测定：将普鲁兰酶置于45℃、不同pH(4.0~12.0)的普鲁兰糖溶液中进行酶促反应，将测定的最高酶活力设为100%，计算不同pH下的相对酶活力，测定其最适反应pH。其中缓冲液为：pH 4.0~8.0的标准McIlvaine缓冲液，pH 8.0~10.0的Tris-HCl缓冲液，以及pH 10.0~12.0的Gly-NaOH缓冲液。
pH稳定性的测定：将普鲁兰酶置于不同pH(4.0~12.0)的缓冲溶液中，于37℃保温1h，测定其在45℃、最适反应pH 8.0条件下的酶活力，将测定的最高酶活力设为100%，计算不同pH下的残余酶活力。
突变体F744A的最适反应pH为8.0，与原始酶PulSL3△C相同，但F744A在酸性条件下具有更宽的pH作用范围（图3）；pH稳定性实验表明，F744A在pH 5.0~10.0范围内保温1 h仍可保留90%以上的酶活力，与原始PulSL3△C（在pH 7.0~9.0范围内保温1 h可保留80%以上的酶活力）相比，F744A的pH稳定范围更宽（图4）。
3. 普鲁兰酶突变体F744A的最适反应温度和温度稳定性
最适反应温度的测定：将普鲁兰酶置于最适反应pH 8.0、不同温度的普鲁兰糖溶液中进行酶促反应，将测定的最高酶活力设为100%，计算不同温度下的相对酶活力。
热稳定性及T50 30的测定：将普鲁兰酶置于不同温度的水浴中保温30 min，测定其在最适反应条件（pH 8.0，50℃）下的酶活力，将未处理普鲁兰酶的活力设为100%，计算不同温度下的残余酶活力。酶在一定温度下保温30 min的残余酶活力为50%，即把该温度定义为T50 30；将普鲁兰酶热稳定性的数据利用Origin 8.0软件的Sigm oidal Boltzmann fit进行拟合，即可得普鲁兰酶的T50 30。
突变体F744A的最适反应温度为50℃，与原始酶PulSL3△C相同，其在30℃~40℃范围内相对酶活均较高（图5）。热稳定性实验结果表明，突变体F744A和原始酶PulSL3△C均在47℃以下较稳定，与原始酶PulSL3△C相比，突变体F744A在50℃、52℃和55℃的残余酶活分别提高了6.36%、15.13%、41.16%（图6）。通过计算可得突变体F744A的T50 30为55.84℃，与原始酶PulSL3△C相比，提高了2.94℃，表明突变体F744A的热稳定性更好。
实施例4 普鲁兰酶在洗涤剂中的应用
1. 人工污布的制作
（1）2%马铃薯淀粉溶液：配制2%的马铃薯淀粉水溶液，煮沸10 min。
（2）蛋白污液：用少量水溶解1.2 g阿拉伯树胶粉后，加入1 mL碳素墨水，研磨2min，转移此污液至60 mL含6.9 g全脂奶粉水溶液的玻璃杯中，另加入60 mL蒸馏水，用乳化机均质15 min，然后加入已准备好的含12.5 g鸡蛋液（蛋清∶蛋黄=3:2）的水溶液60 mL，继续均质15 min。
（3）油脂污液：将大豆油与苏丹黑按照质量比1000:0.5配成油污液，搅拌均匀。
（4）人工污布：将上述制备好的2%马铃薯淀粉溶液、蛋白污液、油脂污液按体积比为4:4:1混合，均质15 min，采用刮涂式的方法将混合污液均匀涂布于棉白布上，于60℃烘箱中老化2 h备用。
2. 去污实验
在恒温水浴摇床上进行，一般洗涤条件为：洗涤温度40℃，水质pH为初始pH，摇床转速120 rpm，洗涤时间30 min，水的硬度250 mg/kg。
将制备好的污布裁成5 cm×5 cm的方块片，加入到由250 mg/kg 硬水配制的300mL、2 g/L质量浓度洗衣液的去污溶液中，将其在40℃恒温水浴摇床上预热30 min，每组实验即每个烧杯含4块布片作为平行。比较污布洗涤前后的白度差值，计算洗涤剂的去污率。
将不同种类的酶制剂添加至含2 g/L洗衣液的洗涤剂中，α-淀粉酶、碱性普鲁兰酶、碱性蛋白酶、脂肪酶的添加量分别为0.17 U/mL、0.74 U/mL、33.33 U/mL、72 U/mL，研究碱性普鲁兰酶、淀粉酶、碱性蛋白酶与脂肪酶复配对复合污垢的去污效果。所用污布为复合污布，按照蛋白污布白度测定方法测定白度，并计算去污率。实验结果见表1，碱性普鲁兰酶的添加可有效提高复合污垢的去污效果，去污率提高了14.21%。
表1 普鲁兰酶、淀粉酶、脂肪酶、碱性蛋白酶复配去除复合污垢的效果
以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所做的均等变化与修饰，皆应属本发明的涵盖范围。
SEQUENCE LISTING
<110> 福州大学
<120> 一种定点突变改造的碱性普鲁兰酶及其应用
<130> 6
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 3555
<212> DNA
<213> 人工序列
<400> 1
atggcagacg gcggaacagc actcctgaca gtggataacg ggtcactgat tgcggaagaa 60
attgagacac cggatgattc agatgaacag ctagaagaaa ccgaggtgga agaaggattt 120
tttcgcatcc attttgcgtc gcttccgtca gatgatcgcg agactcttgg gctctggatc 180
tggaacgatg taaaagagcc ttctgaaaac agaggggcat ggccgaatgg ggcgacctca 240
tttacagagg ctgttcagac cgactacggc tggtatatgg atattgaatt ggaagagaat 300
ccacagtcga ttggcttcct tatcaattcc gtctcgggga acaatctgtc aggagatatc 360
gtcctgcggc ttctgacttc tgagatgaat caggtatggc ttgatcatga gtatgccatg 420
acaccttatg agcctttact tgaaaaggac atgatccgga tcaactacaa acgagacaac 480
aatgactacg atgactgggg cctctggacc tgggacgatg tggcagaacc aacagaaaac 540
tggcctgcag gcgctcagga cagcgatggc gtcgggccta acggaaccta ttttaatctt 600
acgctggcag aagattccga tcagatcggt tttctgttcc tgaataaagc tgatggttcc 660
cagacccggg attacacatt ctcgaacttg tccgctcaca gccagctatt tatgcgtgaa 720
ggagacgata ctatttatac caatccgtac tatgtcagtg aagccggcat gatccgagcc 780
gagctgattt ctgaaactga aatcgaagtg tttttccatt cgacagaagg actggaggag 840
gctgatctgc ttgaattaat tcagctgacc gatgcagaag gacgagacgt gctatttgat 900
gcggctgtcg atcatgaccg acgagtcgtt agtctgaccg gtgacttctc agtggagcat 960
gctccttata ccgtgacttt tgacgaaacg gaagcagaag cgcgaatggg ctggcgcctg 1020
aaggatgcgc tctatgccta tgacggagaa cttggtctga cttttaacga agacgggacc 1080
gctgatctga aagtctggtc tccaagtgcc gacgcagtga cggtcgtttt atatgataaa 1140
gatgatcaga ctgtcgttgt cagagatgat atcgaaatga ctgccgagga gtcaggcgtg 1200
tggcgtgttg ttcttgatga agacacgacc ggactggacg atgtgacggg gtacttctac 1260
cattttgcga ttgaaagaaa tggcgaaacc gtcctggcac tggatcctta tgcccgctca 1320
atggccgcat ggaacagcag tgatccggat aattacatcg gcaaagctgc catcgtgaat 1380
ccgagtgaga tcggtcggga actggattat gctcagatcg aaggctatga caagcgcgaa 1440
gatgcgatca tttatgaaat tcatgtcaga gacttcacat ccgatccgtc aattgaagat 1500
gaactgacca gtcagttcgg gacgttcagt gctttcattg aaaagctcga ttatatcgaa 1560
agtctgggtg tgacccatgt tcagctgctt ccggtcatga gctatttctt tgcaaatgaa 1620
ttcgaaaacg ccgaacggat gttggactac ggatcgactc agaccaacta taactggggt 1680
tacgatccgc agagctactt ctctctgacg ggaatgtatt cagaaaatcc aaaagacccg 1740
gcgagacgca ttgaagaatt caaaaatctg atcgatgcga ttcattccca cggcatgggt 1800
gtcattcttg atgtggtcta taatcacacg gcacgggagc acatttttga agatctggaa 1860
ccgaactatt accatttcat ggatgcggac ggcacgtcac gaacaagttt cggaggcggc 1920
cgactaggca ccactcatga gatggcccgc cgtattctgg tcgattcgat cacatactgg 1980
gtggaagaat ataaagttga cggcttccgc ttcgatatga tgggcgatca cgatgccgaa 2040
agcattcaga tggcatttga cgaagcacag aaactgaatc cgaatatcct gatgatcggt 2100
gagggctggc ggacctttgt cggtgacgaa ggctatgaag acgtcatgcc ggctgaccag 2160
gactggatgc agcacacaca agccgttgga tcgttctcgg atgacttcag aaatgaactg 2220
aaatccggat tcggcagtga gggagaaccg cgctttatta ccggaggcac acggtccatc 2280
caacgaattt ttgacaatct gacagccaat ccacataact ttatggcgac agatccgggc 2340
gatgtggtcc cttatatcgc ggcgcacgac aacctgacgc ttcatgatgt catcgctcaa 2400
agcattcaga aagatccgga ataccaccag gaagagatcc atcagcgtat ccgtctgggc 2460
aatctgatgg tgctgacttc tcagggcacg ccattcgtcc atgccggtca ggaatacgga 2520
cggaccaagc aattccgtga tcctgacttt atcgaacctg tagcaaacga tcaggttcct 2580
tacaagtcga cattcatgac agatgaagac gggaatccgt tcctttaccc gtatttcatt 2640
catgactcat acgattcaac ggatgcggtc aaccgttttg aatgggataa agtgacggat 2700
gctgaagctt atccgattaa tacgcagacc cagtcttaca catctggtct gattgcattg 2760
cggcgcagta cggatgcctt cagtaaagga acgatggaag agatcgcgga catggtgtcg 2820
ctagtggatg cgccggaaat cgaggacgaa gacctggtta tcgtctatcg tgcagaagat 2880
tccaatggcg atcgttacta cgtctttgtg aatgcagatg attccgaacg aacgctgaca 2940
cttgattctg atttgactga agggcacgtc ctggtcgata gtcagcaggc cggcacacga 3000
gcgatcgcca gaccagaagg catcacggtc gaacaggctg gtgtcacgct ggctcctttg 3060
acggcatccg tggttttact tacggataga gaaattgagc cggttgaaga gagtgacgaa 3120
gatggcgatg aaggaactga cccaggcaac ggggaacagc ctggtggaga atctggacca 3180
ggaacagatc agggatccga tggcgacgat cctgtctcag gtggcgaaga aacggctgat 3240
ccagaaagag atgccgaggg tgatgattat ccggaagacg acactgattt gtctgaggat 3300
cctggtgctg gtcaggatag tggagattcg atagctgatg gcgaccaagg tcattcagat 3360
ggtccactag atgggccgga tggtgatgaa acaggcaaag cggaaggcga ttcttctgaa 3420
tcccagactg gtgagcagaa cggagaaagg ctcccctcta cagcaaccct actctggact 3480
gtaggagcag tcggactgat gagcctcttg acaggggtag tggtcagaca gatcaaaaag 3540
aaaaataaga cataa 3555
<210> 2
<211> 1184
<212> PRT
<213> 人工序列
<400> 2
Met Ala Asp Gly Gly Thr Ala Leu Leu Thr Val Asp Asn Gly Ser Leu
1 5 10 15
Ile Ala Glu Glu Ile Glu Thr Pro Asp Asp Ser Asp Glu Gln Leu Glu
20 25 30
Glu Thr Glu Val Glu Glu Gly Phe Phe Arg Ile His Phe Ala Ser Leu
35 40 45
Pro Ser Asp Asp Arg Glu Thr Leu Gly Leu Trp Ile Trp Asn Asp Val
50 55 60
Lys Glu Pro Ser Glu Asn Arg Gly Ala Trp Pro Asn Gly Ala Thr Ser
65 70 75 80
Phe Thr Glu Ala Val Gln Thr Asp Thr Gly Trp Thr Met Asp Ile Glu
85 90 95
Leu Glu Glu Asn Pro Gln Ser Ile Gly Phe Leu Ile Asn Ser Val Ser
100 105 110
Gly Asn Asn Leu Ser Gly Asp Ile Val Leu Arg Leu Leu Thr Ser Glu
115 120 125
Met Asn Gln Val Trp Leu Asp His Glu Thr Ala Met Thr Pro Thr Glu
130 135 140
Pro Leu Leu Glu Lys Asp Met Ile Arg Ile Asn Thr Lys Arg Asp Asn
145 150 155 160
Asn Asp Thr Asp Asp Trp Gly Leu Trp Thr Trp Asp Asp Val Ala Glu
165 170 175
Pro Thr Glu Asn Trp Pro Ala Gly Ala Gln Asp Ser Asp Gly Val Gly
180 185 190
Pro Asn Gly Thr Thr Phe Asn Leu Thr Leu Ala Glu Asp Ser Asp Gln
195 200 205
Ile Gly Phe Leu Phe Leu Asn Lys Ala Asp Gly Ser Gln Thr Arg Asp
210 215 220
Thr Thr Phe Ser Asn Leu Ser Ala His Ser Gln Leu Phe Met Arg Glu
225 230 235 240
Gly Asp Asp Thr Ile Thr Thr Asn Pro Thr Thr Val Ser Glu Ala Gly
245 250 255
Met Ile Arg Ala Glu Leu Ile Ser Glu Thr Glu Ile Glu Val Phe Phe
260 265 270
His Ser Thr Glu Gly Leu Glu Glu Ala Asp Leu Leu Glu Leu Ile Gln
275 280 285
Leu Thr Asp Ala Glu Gly Arg Asp Val Leu Phe Asp Ala Ala Val Asp
290 295 300
His Asp Arg Arg Val Val Ser Leu Thr Gly Asp Phe Ser Val Glu His
305 310 315 320
Ala Pro Thr Thr Val Thr Phe Asp Glu Thr Glu Ala Glu Ala Arg Met
325 330 335
Gly Trp Arg Leu Lys Asp Ala Leu Thr Ala Thr Asp Gly Glu Leu Gly
340 345 350
Leu Thr Phe Asn Glu Asp Gly Thr Ala Asp Leu Lys Val Trp Ser Pro
355 360 365
Ser Ala Asp Ala Val Thr Val Val Leu Thr Asp Lys Asp Asp Gln Thr
370 375 380
Val Val Val Arg Asp Asp Ile Glu Met Thr Ala Glu Glu Ser Gly Val
385 390 395 400
Trp Arg Val Val Leu Asp Glu Asp Thr Thr Gly Leu Asp Asp Val Thr
405 410 415
Gly Thr Phe Thr His Phe Ala Ile Glu Arg Asn Gly Glu Thr Val Leu
420 425 430
Ala Leu Asp Pro Thr Ala Arg Ser Met Ala Ala Trp Asn Ser Ser Asp
435 440 445
Pro Asp Asn Thr Ile Gly Lys Ala Ala Ile Val Asn Pro Ser Glu Ile
450 455 460
Gly Arg Glu Leu Asp Thr Ala Gln Ile Glu Gly Thr Asp Lys Arg Glu
465 470 475 480
Asp Ala Ile Ile Thr Glu Ile His Val Arg Asp Phe Thr Ser Asp Pro
485 490 495
Ser Ile Glu Asp Glu Leu Thr Ser Gln Phe Gly Thr Phe Ser Ala Phe
500 505 510
Ile Glu Lys Leu Asp Thr Ile Glu Ser Leu Gly Val Thr His Val Gln
515 520 525
Leu Leu Pro Val Met Ser Thr Phe Phe Ala Asn Glu Phe Glu Asn Ala
530 535 540
Glu Arg Met Leu Asp Thr Gly Ser Thr Gln Thr Asn Thr Asn Trp Gly
545 550 555 560
Thr Asp Pro Gln Ser Thr Phe Ser Leu Thr Gly Met Thr Ser Glu Asn
565 570 575
Pro Lys Asp Pro Ala Arg Arg Ile Glu Glu Phe Lys Asn Leu Ile Asp
580 585 590
Ala Ile His Ser His Gly Met Gly Val Ile Leu Asp Val Val Thr Asn
595 600 605
His Thr Ala Arg Glu His Ile Phe Glu Asp Leu Glu Pro Asn Thr Thr
610 615 620
His Phe Met Asp Ala Asp Gly Thr Ser Arg Thr Ser Phe Gly Gly Gly
625 630 635 640
Arg Leu Gly Thr Thr His Glu Met Ala Arg Arg Ile Leu Val Asp Ser
645 650 655
Ile Thr Thr Trp Val Glu Glu Thr Lys Val Asp Gly Phe Arg Phe Asp
660 665 670
Met Met Gly Asp His Asp Ala Glu Ser Ile Gln Met Ala Phe Asp Glu
675 680 685
Ala Gln Lys Leu Asn Pro Asn Ile Leu Met Ile Gly Glu Gly Trp Arg
690 695 700
Thr Phe Val Gly Asp Glu Gly Thr Glu Asp Val Met Pro Ala Asp Gln
705 710 715 720
Asp Trp Met Gln His Thr Gln Ala Val Gly Ser Phe Ser Asp Asp Phe
725 730 735
Arg Asn Glu Leu Lys Ser Gly Phe Gly Ser Glu Gly Glu Pro Arg Phe
740 745 750
Ile Thr Gly Gly Thr Arg Ser Ile Gln Arg Ile Phe Asp Asn Leu Thr
755 760 765
Ala Asn Pro His Asn Phe Met Ala Thr Asp Pro Gly Asp Val Val Pro
770 775 780
Thr Ile Ala Ala His Asp Asn Leu Thr Leu His Asp Val Ile Ala Gln
785 790 795 800
Ser Ile Gln Lys Asp Pro Glu Thr His Gln Glu Glu Ile His Gln Arg
805 810 815
Ile Arg Leu Gly Asn Leu Met Val Leu Thr Ser Gln Gly Thr Pro Phe
820 825 830
Val His Ala Gly Gln Glu Thr Gly Arg Thr Lys Gln Phe Arg Asp Pro
835 840 845
Asp Phe Ile Glu Pro Val Ala Asn Asp Gln Val Pro Thr Lys Ser Thr
850 855 860
Phe Met Thr Asp Glu Asp Gly Asn Pro Phe Leu Thr Pro Thr Phe Ile
865 870 875 880
His Asp Ser Thr Asp Ser Thr Asp Ala Val Asn Arg Phe Glu Trp Asp
885 890 895
Lys Val Thr Asp Ala Glu Ala Thr Pro Ile Asn Thr Gln Thr Gln Ser
900 905 910
Thr Thr Ser Gly Leu Ile Ala Leu Arg Arg Ser Thr Asp Ala Phe Ser
915 920 925
Lys Gly Thr Met Glu Glu Ile Ala Asp Met Val Ser Leu Val Asp Ala
930 935 940
Pro Glu Ile Glu Asp Glu Asp Leu Val Ile Val Thr Arg Ala Glu Asp
945 950 955 960
Ser Asn Gly Asp Arg Thr Thr Val Phe Val Asn Ala Asp Asp Ser Glu
965 970 975
Arg Thr Leu Thr Leu Asp Ser Asp Leu Thr Glu Gly His Val Leu Val
980 985 990
Asp Ser Gln Gln Ala Gly Thr Arg Ala Ile Ala Arg Pro Glu Gly Ile
995 1000 1005
Thr Val Glu Gln Ala Gly Val Thr Leu Ala Pro Leu Thr Ala Ser
1010 1015 1020
Val Val Leu Leu Thr Asp Arg Glu Ile Glu Pro Val Glu Glu Ser
1025 1030 1035
Asp Glu Asp Gly Asp Glu Gly Thr Asp Pro Gly Asn Gly Glu Gln
1040 1045 1050
Pro Gly Gly Glu Ser Gly Pro Gly Thr Asp Gln Gly Ser Asp Gly
1055 1060 1065
Asp Asp Pro Val Ser Gly Gly Glu Glu Thr Ala Asp Pro Glu Arg
1070 1075 1080
Asp Ala Glu Gly Asp Asp Thr Pro Glu Asp Asp Thr Asp Leu Ser
1085 1090 1095
Glu Asp Pro Gly Ala Gly Gln Asp Ser Gly Asp Ser Ile Ala Asp
1100 1105 1110
Gly Asp Gln Gly His Ser Asp Gly Pro Leu Asp Gly Pro Asp Gly
1115 1120 1125
Asp Glu Thr Gly Lys Ala Glu Gly Asp Ser Ser Glu Ser Gln Thr
1130 1135 1140
Gly Glu Gln Asn Gly Glu Arg Leu Pro Ser Thr Ala Thr Leu Leu
1145 1150 1155
Trp Thr Val Gly Ala Val Gly Leu Met Ser Leu Leu Thr Gly Val
1160 1165 1170
Val Val Arg Gln Ile Lys Lys Lys Asn Lys Thr
1175 1180
<210> 3
<211> 3555
<212> DNA
<213> 人工序列
<400> 3
atggcagacg gcggaacagc actcctgaca gtggataacg ggtcactgat tgcggaagaa 60
attgagacac cggatgattc agatgaacag ctagaagaaa ccgaggtgga agaaggattt 120
tttcgcatcc attttgcgtc gcttccgtca gatgatcgcg agactcttgg gctctggatc 180
tggaacgatg taaaagagcc ttctgaaaac agaggggcat ggccgaatgg ggcgacctca 240
tttacagagg ctgttcagac cgactacggc tggtatatgg atattgaatt ggaagagaat 300
ccacagtcga ttggcttcct tatcaattcc gtctcgggga acaatctgtc aggagatatc 360
gtcctgcggc ttctgacttc tgagatgaat caggtatggc ttgatcatga gtatgccatg 420
acaccttatg agcctttact tgaaaaggac atgatccgga tcaactacaa acgagacaac 480
aatgactacg atgactgggg cctctggacc tgggacgatg tggcagaacc aacagaaaac 540
tggcctgcag gcgctcagga cagcgatggc gtcgggccta acggaaccta ttttaatctt 600
acgctggcag aagattccga tcagatcggt tttctgttcc tgaataaagc tgatggttcc 660
cagacccggg attacacatt ctcgaacttg tccgctcaca gccagctatt tatgcgtgaa 720
ggagacgata ctatttatac caatccgtac tatgtcagtg aagccggcat gatccgagcc 780
gagctgattt ctgaaactga aatcgaagtg tttttccatt cgacagaagg actggaggag 840
gctgatctgc ttgaattaat tcagctgacc gatgcagaag gacgagacgt gctatttgat 900
gcggctgtcg atcatgaccg acgagtcgtt agtctgaccg gtgacttctc agtggagcat 960
gctccttata ccgtgacttt tgacgaaacg gaagcagaag cgcgaatggg ctggcgcctg 1020
aaggatgcgc tctatgccta tgacggagaa cttggtctga cttttaacga agacgggacc 1080
gctgatctga aagtctggtc tccaagtgcc gacgcagtga cggtcgtttt atatgataaa 1140
gatgatcaga ctgtcgttgt cagagatgat atcgaaatga ctgccgagga gtcaggcgtg 1200
tggcgtgttg ttcttgatga agacacgacc ggactggacg atgtgacggg gtacttctac 1260
cattttgcga ttgaaagaaa tggcgaaacc gtcctggcac tggatcctta tgcccgctca 1320
atggccgcat ggaacagcag tgatccggat aattacatcg gcaaagctgc catcgtgaat 1380
ccgagtgaga tcggtcggga actggattat gctcagatcg aaggctatga caagcgcgaa 1440
gatgcgatca tttatgaaat tcatgtcaga gacttcacat ccgatccgtc aattgaagat 1500
gaactgacca gtcagttcgg gacgttcagt gctttcattg aaaagctcga ttatatcgaa 1560
agtctgggtg tgacccatgt tcagctgctt ccggtcatga gctatttctt tgcaaatgaa 1620
ttcgaaaacg ccgaacggat gttggactac ggatcgactc agaccaacta taactggggt 1680
tacgatccgc agagctactt ctctctgacg ggaatgtatt cagaaaatcc aaaagacccg 1740
gcgagacgca ttgaagaatt caaaaatctg atcgatgcga ttcattccca cggcatgggt 1800
gtcattcttg atgtggtcta taatcacacg gcacgggagc acatttttga agatctggaa 1860
ccgaactatt accatttcat ggatgcggac ggcacgtcac gaacaagttt cggaggcggc 1920
cgactaggca ccactcatga gatggcccgc cgtattctgg tcgattcgat cacatactgg 1980
gtggaagaat ataaagttga cggcttccgc ttcgatatga tgggcgatca cgatgccgaa 2040
agcattcaga tggcatttga cgaagcacag aaactgaatc cgaatatcct gatgatcggt 2100
gagggctggc ggacctttgt cggtgacgaa ggctatgaag acgtcatgcc ggctgaccag 2160
gactggatgc agcacacaca agccgttgga tcgttctcgg atgacttcag aaatgaactg 2220
aaatccggag gcggcagtga gggagaaccg cgctttatta ccggaggcac acggtccatc 2280
caacgaattt ttgacaatct gacagccaat ccacataact ttatggcgac agatccgggc 2340
gatgtggtcc cttatatcgc ggcgcacgac aacctgacgc ttcatgatgt catcgctcaa 2400
agcattcaga aagatccgga ataccaccag gaagagatcc atcagcgtat ccgtctgggc 2460
aatctgatgg tgctgacttc tcagggcacg ccattcgtcc atgccggtca ggaatacgga 2520
cggaccaagc aattccgtga tcctgacttt atcgaacctg tagcaaacga tcaggttcct 2580
tacaagtcga cattcatgac agatgaagac gggaatccgt tcctttaccc gtatttcatt 2640
catgactcat acgattcaac ggatgcggtc aaccgttttg aatgggataa agtgacggat 2700
gctgaagctt atccgattaa tacgcagacc cagtcttaca catctggtct gattgcattg 2760
cggcgcagta cggatgcctt cagtaaagga acgatggaag agatcgcgga catggtgtcg 2820
ctagtggatg cgccggaaat cgaggacgaa gacctggtta tcgtctatcg tgcagaagat 2880
tccaatggcg atcgttacta cgtctttgtg aatgcagatg attccgaacg aacgctgaca 2940
cttgattctg atttgactga agggcacgtc ctggtcgata gtcagcaggc cggcacacga 3000
gcgatcgcca gaccagaagg catcacggtc gaacaggctg gtgtcacgct ggctcctttg 3060
acggcatccg tggttttact tacggataga gaaattgagc cggttgaaga gagtgacgaa 3120
gatggcgatg aaggaactga cccaggcaac ggggaacagc ctggtggaga atctggacca 3180
ggaacagatc agggatccga tggcgacgat cctgtctcag gtggcgaaga aacggctgat 3240
ccagaaagag atgccgaggg tgatgattat ccggaagacg acactgattt gtctgaggat 3300
cctggtgctg gtcaggatag tggagattcg atagctgatg gcgaccaagg tcattcagat 3360
ggtccactag atgggccgga tggtgatgaa acaggcaaag cggaaggcga ttcttctgaa 3420
tcccagactg gtgagcagaa cggagaaagg ctcccctcta cagcaaccct actctggact 3480
gtaggagcag tcggactgat gagcctcttg acaggggtag tggtcagaca gatcaaaaag 3540
aaaaataaga cataa 3555
<210> 4
<211> 1184
<212> PRT
<213> 人工序列
<400> 4
Met Ala Asp Gly Gly Thr Ala Leu Leu Thr Val Asp Asn Gly Ser Leu
1 5 10 15
Ile Ala Glu Glu Ile Glu Thr Pro Asp Asp Ser Asp Glu Gln Leu Glu
20 25 30
Glu Thr Glu Val Glu Glu Gly Phe Phe Arg Ile His Phe Ala Ser Leu
35 40 45
Pro Ser Asp Asp Arg Glu Thr Leu Gly Leu Trp Ile Trp Asn Asp Val
50 55 60
Lys Glu Pro Ser Glu Asn Arg Gly Ala Trp Pro Asn Gly Ala Thr Ser
65 70 75 80
Phe Thr Glu Ala Val Gln Thr Asp Thr Gly Trp Thr Met Asp Ile Glu
85 90 95
Leu Glu Glu Asn Pro Gln Ser Ile Gly Phe Leu Ile Asn Ser Val Ser
100 105 110
Gly Asn Asn Leu Ser Gly Asp Ile Val Leu Arg Leu Leu Thr Ser Glu
115 120 125
Met Asn Gln Val Trp Leu Asp His Glu Thr Ala Met Thr Pro Thr Glu
130 135 140
Pro Leu Leu Glu Lys Asp Met Ile Arg Ile Asn Thr Lys Arg Asp Asn
145 150 155 160
Asn Asp Thr Asp Asp Trp Gly Leu Trp Thr Trp Asp Asp Val Ala Glu
165 170 175
Pro Thr Glu Asn Trp Pro Ala Gly Ala Gln Asp Ser Asp Gly Val Gly
180 185 190
Pro Asn Gly Thr Thr Phe Asn Leu Thr Leu Ala Glu Asp Ser Asp Gln
195 200 205
Ile Gly Phe Leu Phe Leu Asn Lys Ala Asp Gly Ser Gln Thr Arg Asp
210 215 220
Thr Thr Phe Ser Asn Leu Ser Ala His Ser Gln Leu Phe Met Arg Glu
225 230 235 240
Gly Asp Asp Thr Ile Thr Thr Asn Pro Thr Thr Val Ser Glu Ala Gly
245 250 255
Met Ile Arg Ala Glu Leu Ile Ser Glu Thr Glu Ile Glu Val Phe Phe
260 265 270
His Ser Thr Glu Gly Leu Glu Glu Ala Asp Leu Leu Glu Leu Ile Gln
275 280 285
Leu Thr Asp Ala Glu Gly Arg Asp Val Leu Phe Asp Ala Ala Val Asp
290 295 300
His Asp Arg Arg Val Val Ser Leu Thr Gly Asp Phe Ser Val Glu His
305 310 315 320
Ala Pro Thr Thr Val Thr Phe Asp Glu Thr Glu Ala Glu Ala Arg Met
325 330 335
Gly Trp Arg Leu Lys Asp Ala Leu Thr Ala Thr Asp Gly Glu Leu Gly
340 345 350
Leu Thr Phe Asn Glu Asp Gly Thr Ala Asp Leu Lys Val Trp Ser Pro
355 360 365
Ser Ala Asp Ala Val Thr Val Val Leu Thr Asp Lys Asp Asp Gln Thr
370 375 380
Val Val Val Arg Asp Asp Ile Glu Met Thr Ala Glu Glu Ser Gly Val
385 390 395 400
Trp Arg Val Val Leu Asp Glu Asp Thr Thr Gly Leu Asp Asp Val Thr
405 410 415
Gly Thr Phe Thr His Phe Ala Ile Glu Arg Asn Gly Glu Thr Val Leu
420 425 430
Ala Leu Asp Pro Thr Ala Arg Ser Met Ala Ala Trp Asn Ser Ser Asp
435 440 445
Pro Asp Asn Thr Ile Gly Lys Ala Ala Ile Val Asn Pro Ser Glu Ile
450 455 460
Gly Arg Glu Leu Asp Thr Ala Gln Ile Glu Gly Thr Asp Lys Arg Glu
465 470 475 480
Asp Ala Ile Ile Thr Glu Ile His Val Arg Asp Phe Thr Ser Asp Pro
485 490 495
Ser Ile Glu Asp Glu Leu Thr Ser Gln Phe Gly Thr Phe Ser Ala Phe
500 505 510
Ile Glu Lys Leu Asp Thr Ile Glu Ser Leu Gly Val Thr His Val Gln
515 520 525
Leu Leu Pro Val Met Ser Thr Phe Phe Ala Asn Glu Phe Glu Asn Ala
530 535 540
Glu Arg Met Leu Asp Thr Gly Ser Thr Gln Thr Asn Thr Asn Trp Gly
545 550 555 560
Thr Asp Pro Gln Ser Thr Phe Ser Leu Thr Gly Met Thr Ser Glu Asn
565 570 575
Pro Lys Asp Pro Ala Arg Arg Ile Glu Glu Phe Lys Asn Leu Ile Asp
580 585 590
Ala Ile His Ser His Gly Met Gly Val Ile Leu Asp Val Val Thr Asn
595 600 605
His Thr Ala Arg Glu His Ile Phe Glu Asp Leu Glu Pro Asn Thr Thr
610 615 620
His Phe Met Asp Ala Asp Gly Thr Ser Arg Thr Ser Phe Gly Gly Gly
625 630 635 640
Arg Leu Gly Thr Thr His Glu Met Ala Arg Arg Ile Leu Val Asp Ser
645 650 655
Ile Thr Thr Trp Val Glu Glu Thr Lys Val Asp Gly Phe Arg Phe Asp
660 665 670
Met Met Gly Asp His Asp Ala Glu Ser Ile Gln Met Ala Phe Asp Glu
675 680 685
Ala Gln Lys Leu Asn Pro Asn Ile Leu Met Ile Gly Glu Gly Trp Arg
690 695 700
Thr Phe Val Gly Asp Glu Gly Thr Glu Asp Val Met Pro Ala Asp Gln
705 710 715 720
Asp Trp Met Gln His Thr Gln Ala Val Gly Ser Phe Ser Asp Asp Phe
725 730 735
Arg Asn Glu Leu Lys Ser Gly Phe Gly Ser Glu Gly Glu Pro Arg Phe
740 745 750
Ile Thr Gly Gly Thr Arg Ser Ile Gln Arg Ile Phe Asp Asn Leu Thr
755 760 765
Ala Asn Pro His Asn Ala Met Ala Thr Asp Pro Gly Asp Val Val Pro
770 775 780
Thr Ile Ala Ala His Asp Asn Leu Thr Leu His Asp Val Ile Ala Gln
785 790 795 800
Ser Ile Gln Lys Asp Pro Glu Thr His Gln Glu Glu Ile His Gln Arg
805 810 815
Ile Arg Leu Gly Asn Leu Met Val Leu Thr Ser Gln Gly Thr Pro Phe
820 825 830
Val His Ala Gly Gln Glu Thr Gly Arg Thr Lys Gln Phe Arg Asp Pro
835 840 845
Asp Phe Ile Glu Pro Val Ala Asn Asp Gln Val Pro Thr Lys Ser Thr
850 855 860
Phe Met Thr Asp Glu Asp Gly Asn Pro Phe Leu Thr Pro Thr Phe Ile
865 870 875 880
His Asp Ser Thr Asp Ser Thr Asp Ala Val Asn Arg Phe Glu Trp Asp
885 890 895
Lys Val Thr Asp Ala Glu Ala Thr Pro Ile Asn Thr Gln Thr Gln Ser
900 905 910
Thr Thr Ser Gly Leu Ile Ala Leu Arg Arg Ser Thr Asp Ala Phe Ser
915 920 925
Lys Gly Thr Met Glu Glu Ile Ala Asp Met Val Ser Leu Val Asp Ala
930 935 940
Pro Glu Ile Glu Asp Glu Asp Leu Val Ile Val Thr Arg Ala Glu Asp
945 950 955 960
Ser Asn Gly Asp Arg Thr Thr Val Phe Val Asn Ala Asp Asp Ser Glu
965 970 975
Arg Thr Leu Thr Leu Asp Ser Asp Leu Thr Glu Gly His Val Leu Val
980 985 990
Asp Ser Gln Gln Ala Gly Thr Arg Ala Ile Ala Arg Pro Glu Gly Ile
995 1000 1005
Thr Val Glu Gln Ala Gly Val Thr Leu Ala Pro Leu Thr Ala Ser
1010 1015 1020
Val Val Leu Leu Thr Asp Arg Glu Ile Glu Pro Val Glu Glu Ser
1025 1030 1035
Asp Glu Asp Gly Asp Glu Gly Thr Asp Pro Gly Asn Gly Glu Gln
1040 1045 1050
Pro Gly Gly Glu Ser Gly Pro Gly Thr Asp Gln Gly Ser Asp Gly
1055 1060 1065
Asp Asp Pro Val Ser Gly Gly Glu Glu Thr Ala Asp Pro Glu Arg
1070 1075 1080
Asp Ala Glu Gly Asp Asp Thr Pro Glu Asp Asp Thr Asp Leu Ser
1085 1090 1095
Glu Asp Pro Gly Ala Gly Gln Asp Ser Gly Asp Ser Ile Ala Asp
1100 1105 1110
Gly Asp Gln Gly His Ser Asp Gly Pro Leu Asp Gly Pro Asp Gly
1115 1120 1125
Asp Glu Thr Gly Lys Ala Glu Gly Asp Ser Ser Glu Ser Gln Thr
1130 1135 1140
Gly Glu Gln Asn Gly Glu Arg Leu Pro Ser Thr Ala Thr Leu Leu
1145 1150 1155
Trp Thr Val Gly Ala Val Gly Leu Met Ser Leu Leu Thr Gly Val
1160 1165 1170
Val Val Arg Gln Ile Lys Lys Lys Asn Lys Thr
1175 1180
<210> 5
<211> 28
<212> DNA
<213> 人工序列
<400> 5
aatccggagc cggcagtgag ggagaacc 28
<210> 6
<211> 33
<212> DNA
<213> 人工序列
<400> 6
tcactgccgg ctccggattt cagttcattt ctg 33

Claims (6)

1.一种定点突变改造的碱性普鲁兰酶，其特征在于：所述碱性普鲁兰酶是在SEQ IDNO.2所示的氨基酸基础上，第744位苯丙氨酸突变为丙氨酸。
2.根据权利要求1所述的一种定点突变改造的碱性普鲁兰酶，其特征在于：其氨基酸序列如SEQ ID NO.4所示。
3.编码权利要求1所述的一种定点突变改造的碱性普鲁兰酶的基因，其序列如SEQ IDNO.3所示。
4.包含权利要求1所述碱性普鲁兰酶的载体。
5.包含权利要求4所述载体的基因工程菌。
6.如权利要求1所述的定点突变改造的碱性普鲁兰酶在制备洗涤剂中的应用。
CN201810813706.8A 2018-07-23 2018-07-23 一种定点突变改造的碱性普鲁兰酶及其应用 Active CN108913677B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810813706.8A CN108913677B (zh) 2018-07-23 2018-07-23 一种定点突变改造的碱性普鲁兰酶及其应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810813706.8A CN108913677B (zh) 2018-07-23 2018-07-23 一种定点突变改造的碱性普鲁兰酶及其应用

Publications (2)

Publication Number Publication Date
CN108913677A true CN108913677A (zh) 2018-11-30
CN108913677B CN108913677B (zh) 2021-06-29

Family

ID=64415991

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810813706.8A Active CN108913677B (zh) 2018-07-23 2018-07-23 一种定点突变改造的碱性普鲁兰酶及其应用

Country Status (1)

Country Link
CN (1) CN108913677B (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110317263A (zh) * 2019-07-04 2019-10-11 华北电力大学 基于同源建模的高效检测PAEs的牛血清蛋白改造方法
CN115029336A (zh) * 2022-01-10 2022-09-09 昆明理工大学 一种具有洗涤剂抗性的普鲁兰酶PulY103B及其应用

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1183808A (zh) * 1995-05-10 1998-06-03 花王株式会社 具有碱性支链淀粉酶和碱性α－淀粉酶两种活性的酶的基因
JP2002112776A (ja) * 2000-10-03 2002-04-16 Kao Corp アルカリプルラナーゼ
US20090238923A1 (en) * 2007-11-05 2009-09-24 Danisco Us Inc., Genencor Division Variants of bacillus licheniformis alpha-amylase with increased thermostability and/or decreased calcium dependence
CN102796751A (zh) * 2012-08-21 2012-11-28 广西科学院 一种长野芽孢杆菌普鲁兰酶突变体Pul324及其应用
CN103194434A (zh) * 2013-04-15 2013-07-10 山东天力药业有限公司 新型麦芽寡糖基海藻糖水解酶、酶基因、含该基因的重组表达载体及重组工程菌及酶的制备
CN104357427A (zh) * 2014-11-11 2015-02-18 福州大学 一种高温碱性耐盐木聚糖酶XynSL4及其基因和应用
WO2017139697A1 (en) * 2016-02-10 2017-08-17 Synlogic, Inc. Bacteria engineered to treat diseases associated with hyperammonemia
CN107164346A (zh) * 2017-07-18 2017-09-15 福州大学 一种碱性耐盐普鲁兰酶PulA及其基因和应用
CN107267483A (zh) * 2017-07-18 2017-10-20 福州大学 一种木聚糖酶XynMF10及其基因和应用
CN107922933A (zh) * 2015-07-21 2018-04-17 诺维信公司 适合用于液化中的具有普鲁兰酶活性的多肽

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1183808A (zh) * 1995-05-10 1998-06-03 花王株式会社 具有碱性支链淀粉酶和碱性α－淀粉酶两种活性的酶的基因
JP2002112776A (ja) * 2000-10-03 2002-04-16 Kao Corp アルカリプルラナーゼ
US20090238923A1 (en) * 2007-11-05 2009-09-24 Danisco Us Inc., Genencor Division Variants of bacillus licheniformis alpha-amylase with increased thermostability and/or decreased calcium dependence
CN102796751A (zh) * 2012-08-21 2012-11-28 广西科学院 一种长野芽孢杆菌普鲁兰酶突变体Pul324及其应用
CN103194434A (zh) * 2013-04-15 2013-07-10 山东天力药业有限公司 新型麦芽寡糖基海藻糖水解酶、酶基因、含该基因的重组表达载体及重组工程菌及酶的制备
CN104357427A (zh) * 2014-11-11 2015-02-18 福州大学 一种高温碱性耐盐木聚糖酶XynSL4及其基因和应用
CN107922933A (zh) * 2015-07-21 2018-04-17 诺维信公司 适合用于液化中的具有普鲁兰酶活性的多肽
WO2017139697A1 (en) * 2016-02-10 2017-08-17 Synlogic, Inc. Bacteria engineered to treat diseases associated with hyperammonemia
CN107164346A (zh) * 2017-07-18 2017-09-15 福州大学 一种碱性耐盐普鲁兰酶PulA及其基因和应用
CN107267483A (zh) * 2017-07-18 2017-10-20 福州大学 一种木聚糖酶XynMF10及其基因和应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
QIAO Y.等: "Gene cloning and enzymatic characterization of alkali-tolerant type I pullulanase from Exiguobacterium acetylicum", 《LETTERS IN APPLIED MICROBIOLOGY》 *
严芬等: "一株碱性普鲁兰酶菌株的筛选、鉴定及酶学性质研究", 《福州大学学报(自然科学版)》 *
陈逍遥等: "嗜热地芽胞杆菌(Geobacillus kaustophilus)DY115普鲁兰酶基因的克隆、表达和酶学性质", 《生物资源》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110317263A (zh) * 2019-07-04 2019-10-11 华北电力大学 基于同源建模的高效检测PAEs的牛血清蛋白改造方法
CN110317263B (zh) * 2019-07-04 2021-03-23 华北电力大学 基于同源建模的高效检测PAEs的牛血清蛋白改造方法
CN115029336A (zh) * 2022-01-10 2022-09-09 昆明理工大学 一种具有洗涤剂抗性的普鲁兰酶PulY103B及其应用

Also Published As

Publication number Publication date
CN108913677B (zh) 2021-06-29

Similar Documents

Publication Publication Date Title
CN110777136B (zh) 一种洗涤用的碱性蛋白酶突变体及其在液体洗涤剂中的应用
CN108559739B (zh) 耐热性提高的甘露聚糖酶PMan5A突变体及其基因和应用
CN109355275B (zh) 高热稳定性β-葡萄糖苷酶突变体及其应用
CN113862233B (zh) 提高葡萄糖氧化酶的酸稳定性的方法及突变体q241e/r499e、基因和应用
CN112813054A (zh) 低温耐盐性改变的菊粉酶突变体MutS117N及其应用
CN112708607A (zh) 热适应性改变的菊粉酶突变体MutS120R及其应用
CN112646793A (zh) 低温适应性和盐适应性改良的菊粉酶突变体MutS120D及其应用
CN112725306A (zh) 热盐性改变的菊粉酶突变体MutY119T及其应用
CN112725305A (zh) 热盐性敏感的菊粉酶突变体MutY119D及其制备方法
CN112813053A (zh) 菊粉酶突变体MutY119H及其制备方法
CN108660145A (zh) 耐热型普鲁兰酶的编码基因及其重组表达和应用
CN112813051A (zh) 热适应性改良的低温外切菊粉酶突变体MutP124G及应用
CN112980813A (zh) 低温改良的外切菊粉酶突变体MutS117G
CN104130988A (zh) 一种1,3-1,4-β-葡聚糖酶突变体
CN112391365A (zh) 一种催化活力提高的淀粉分支酶突变体及其应用
CN112980814A (zh) 低温适应性提高的外切菊粉酶突变体MutV268Δ13
CN108841809A (zh) 具有高比活及热稳定性的淀粉酶突变体及其基因和应用
CN108588061A (zh) 一种比酶活及热稳定性提高的低温碱性果胶酶突变体
CN111676210B (zh) 一种提高纤维素酶活性的方法及纤维素酶突变体5i77-m和应用
CN110423737B (zh) 来源于嗜热脂肪土芽孢杆菌的耐热型α-淀粉酶及其应用
CN111004794B (zh) 一种热稳定性提高的枯草蛋白酶e突变体及其应用
CN109679937B (zh) 一种具有高比酶活力的生淀粉水解酶、其编码基因及其应用
CN110373403B (zh) 耐高温中性普鲁兰酶及其应用
CN108913677A (zh) 一种定点突变改造的碱性普鲁兰酶及其应用
CN115838704A (zh) 脂肪酶突变体及其应用

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant