CN103789285B - 一种热稳定性提高的淀粉酶突变体及其构建方法和应用 - Google Patents
一种热稳定性提高的淀粉酶突变体及其构建方法和应用 Download PDFInfo
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Abstract
本发明公开了一种热稳定性提高的淀粉酶突变体及其构建方法和应用，属于遗传工程领域。本发明以SEQ？ID？NO.1所示序列为出发序列，将第193位天冬酰胺替换成苯丙氨酸或第206位蛋氨酸替换为苯丙氨酸或第240位赖氨酸替换成精氨酸或第242位丝氨酸替换成谷氨酰胺或丙氨酸或缺失第180位甘氨酸和181异亮氨酸或其任意组合得到的突变体。在此改造条件下，嗜热脂肪芽孢杆菌淀粉酶在95℃的半衰期由对照(突变前)例的10min提高到240min。利用此策略可以显著提高淀粉酶的热稳定性，为其工业化生产提供了基础。
Description
技术领域
本发明涉及一种淀粉酶突变体及其方法，具体涉及一种热稳定性提高的酸性淀粉酶突变体及其制备方法。
背景技术
淀粉酶是最早用于工业生产的酶制剂，是迄今用于最广，产量最大的酶制剂产品之一。淀粉酶具有相当大的工业应用价值，广泛用于酒精、有机酸、氨基酸及纺织等行业。但是来源于野生菌株未经改造的淀粉酶在热稳定性等方面具有一定局限性，限制了其应用范围。因此基于已得到的酸性淀粉酶在大肠杆菌中的表达系统，利用定点突变的手段，对酸性淀粉酶进行分子改造，以得到酶学性质更适合工业应用的酸性淀粉酶。
发明内容
本发明要解决的技术问题是提供一种热稳定性提高的淀粉酶突变体，对淀粉酶结构域A、B、C内的一个或多个氨基酸进行替换，提高了淀粉酶的热稳定性。
所述淀粉酶突变体以SEQIDNO.1为出发序列，将第193位天冬酰胺替换成苯丙氨酸或第206位蛋氨酸替换为苯丙氨酸或第240位赖氨酸替换成精氨酸或第242位丝氨酸替换成谷氨酰胺或丙氨酸或缺失第180位甘氨酸和181异亮氨酸或其任意组合得到的突变体。
所述氨基酸突变体为第193位天冬酰胺替换成苯丙氨酸的突变体。
所述氨基酸突变体为第242位丝氨酸替换成谷氨酰胺或丙氨酸的突变体。
所述氨基酸突变体为缺失第180位甘氨酸和181异亮氨酸的突变体。
所述氨基酸突变体为第193位天冬酰胺替换成苯丙氨酸、第242位丝氨酸替
换成谷氨酰胺或丙氨酸、缺失第180位甘氨酸和181异亮氨酸的突变体。
能表达上述淀粉酶突变体的载体也属于本专利要求保护的范围。
能表达上述淀粉酶突变体的基因工程菌或转基因细胞系也属于本专利要求保护的范围。
本发明还提供一种制备所述淀粉酶突变体的方法，是将淀粉酶催化部位一个或多个氨基酸通过PCR或化学合成的方法进行替换。
具体而言，是以SEQIDNO.1为出发序列，将第193位天冬酰胺替换成苯丙氨酸、第206位蛋氨酸替换为苯丙氨酸、第240位赖氨酸替换成精氨酸、第242位丝氨酸替换成谷氨酰胺或丙氨酸、缺失第180位甘氨酸和181异亮氨酸或其任意组合。
所述制备所述淀粉酶突变体的方法，具体步骤如下：
1)根据嗜热脂肪芽孢杆菌淀粉酶序列SEQIDNO.1所示，采用化学全合成的方法全合成后克隆到质粒pET-20b(+)中，构建重组质粒pET-20b(+)-Amy(图1)；
2)利用Swiss-model软件对源自嗜热脂肪芽孢杆菌淀粉酶进行模拟，获得淀粉酶空间结构；
3)通过对淀粉酶的序列以及空间结构进行分析，确定要突变的氨基酸为位点，分别为第193位、第206位、第240位、第242位、第180位及第181位；
4)设计突变引物，对淀粉酶基因序列进行定点突变，将所述位点的氨基酸进行替换或缺失，获得含有突变淀粉酶序列的重组载体；
5)将突变后重组载体转化大肠杆菌BL21(DE3)，诱导表达，获得淀粉酶突变体。
表1淀粉酶突变引物序列
本发明提供的酸性淀粉酶突变体热稳定性显著提高，在95℃的半衰期提高到240min，提高了24倍。相对于采用筛菌或诱变等手段，缩短了酶学性质改造时间。将该酸性淀粉酶突变体应用于洗涤、织物退浆、淀粉液化、医药、食品等领域，可以在高温酸性环境下高效降解淀粉，具有广阔的应用前景。
附图说明
图1：pET-20b(+)-Amy质粒图谱
具体实施方式
实施例1：淀粉酶热稳定性突变位点分析与方法
通过对淀粉酶序列（SEQIDNO.1）以及3D空间结构进行分析，确定活性中心与酶的热稳定性提高有关的几个氨基酸残基(Asn193、Met206、Lys240、Ser242、Gly180、Ile181)。
根据嗜热脂肪芽孢杆菌淀粉酶序列，采用化学全合成的方法全合成后，克隆到质粒pET-20b(+)中，构建重组质粒pET-20b(+)-Amy。
针对不同位点的定点突变，设计相应的定点突变引物(表1)。利用定点突变引物及重组质粒pET-20b(+)-Amy，对淀粉酶进行定点突变。采用PCR酶，利用突变引物对重组质粒pET-20b(+)-Amy进行扩增。将扩增后的PCR产物用DpnⅠ处理，消化模板。经DpnⅠ处理后的PCR产物直接转化大肠杆菌宿主BL21(DE3)，获得重组大肠杆菌，诱导表达，获得单点突变后的重组淀粉酶。以单点突变后获得的重组质粒为模板进行下一轮的突变，获得组合突变后的重组淀粉酶。
SEQIDNO.1：
1AlaAlaProPheAsnGlyThrMetMetGlnTyrPheGluTrpTyrLeuProAspAsp
20GlyThrLeuTrpThrLysValAlaAsnGluAlaAsnAsnLeuSerSerLeuGlyIle
39ThrAlaLeuTrpLeuProProAlaTyrLysGlyThrSerArgSerAspValGlyTyr
58GlyValTyrAspLeuTyrAspLeuGlyGluPheAsnGlnLysGlyThrValArgThr
77LysTyrGlyThrLysAlaGlnTyrLeuGlnAlaIleGlnAlaAlaHisAlaAlaGly
96MetGlnValTyrAlaAspValValPheAspHisLysGlyGlyAlaAspGlyThrGlu
115TrpValAspAlaValGluValAsnProSerAspArgAsnGlnGluIleSerGlyThr
134TyrGlnIleGlnAlaTrpThrLysPheAspPheProGlyArgGlyAsnThrTyrSer
153SerPheLysTrpArgTrpTyrHisPheAspGlyValAspTrpAspGluSerArgLys
172LeuSerArgIleTyrLysPheArgGlyLysAlaTrpAspTrpGluValAspThrGlu
191PheGlyAsnTyrAspTyrLeuMetTyrAlaAspLeuAspMetAspHisProGluVal
210ValThrGluLeuLysAsnTrpGlyLysTrpTyrValAsnThrThrAsnIleAspGly
229PheArgLeuAspAlaValLysHisIleLysPheGlnPhePheProAspTrpLeuSer
248TyrValArgSerGlnThrGlyLysProLeuPheThrValGlyGluTyrTrpSerTyr
267AspIleAsnLysLeuHisAsnTyrIleThrLysThrAspGlyThrMetSerLeuPhe
286AspAlaProLeuHisAsnLysPheTyrThrAlaSerLysSerGlyGlyAlaPheAsp
305MetArgThrLeuMetThrAsnThrLeuMetLysAspGlnProThrLeuAlaValThr
324PheValAspAsnHisAspThrGluProValGlnAlaLeuGlnSerTrpValAspPro
343TrpPheLysProLeuAlaTyrAlaPheIleLeuThrArgGlnGluGlyTyrProCys
362ValPheTyrGlyAspTyrTyrGlyIleProGlnTyrAsnIleProSerLeuLysSer
381LysIleAspProLeuLeuIleAlaArgArgAspTyrAlaTyrGlyThrGlnHisAsp
400TyrLeuAspHisSerAspIleIleGlyTrpThrArgGluGlyGlyThrGluLysPro
419GlySerGlyLeuAlaAlaLeuIleThrAspGlyProGlyGlySerLysTrpMetTyr
438ValGlyLysGlnHisAlaGlyLysValPheTyrAspLeuThrGlyAsnArgSerAsp
457ThrValThrIleAsnSerAspGlyTrpGlyGluPheLysValAsnGlyGlySerVal
476SerValTrpValProArgLysThrThrValSerThrIleAlaArgProIleThrThr
495ArgProTrpThrGlyGluPheValArgTrpHisGluProArgLeuValAlaTrpPro
实施例2：淀粉酶活力测定方法
DNS法测定淀粉酶酶活：
1)DNS试剂的配制：称取6.5g3,5-二硝基水杨酸溶于少量水中，移入1L容量瓶中，加入2mol/L氢氧化钠溶液262mL，再加入185g酒石酸钾钠及5g苯酚和5g无水亚硫酸钠，定容至1L，储存至棕色瓶中，放置于4℃冰箱待用。
2)葡萄糖标准曲线的制作：配制0.1g/L-1.0g/L不同浓度的葡萄糖溶液。取2mL不同浓度的葡萄糖与3mL的DNS溶液混合，放入沸水浴中，水浴7min。用冷水冷却，定容至15mL，A540测定吸光值。以葡萄糖浓度为横坐标，以吸光值为纵坐标，制作标准曲线。
酶活力单位定义：在pH6.0，温度70℃条件下，在1min降解可溶性淀粉产生1μmol还原糖物质(以葡萄糖计)所需要的酶量为1个酶活力单位(U)。
实施例3：淀粉酶在95℃的热稳定性测定与分析
将淀粉酶在含有20%甘油、pH6.0、95℃条件下处理，定期取样，采用实施例2的方法测定其残留酶活。淀粉酶的半衰期T1/2即为残留酶活力为初始酶活力一半时的时间。
表2单点突变重组淀粉酶在95℃的热稳定性
通过测定发现，单突变体N193F、M206F、K240R、S242Q、S242A、ΔIG在95℃的半衰期(表2)都有提高，其中ΔIG突变体提高的效果最显著，半衰期提高至原来的6倍。在此基础上优选N193F、S242Q、ΔIG进行组合突变，获得N193F/S242Q，N193F/ΔIG，S242Q/ΔIG，N193F/S242Q/ΔIG四个突变体，通过测定发现，他们在95℃的半衰期(表3)都有提高，其中N193F/S242Q/ΔIG三突变体效果最显著，半衰期提高至原来的24倍。该淀粉酶在酸性条件下具有较强的热稳定性。
表3优选位点组合突变重组酶在95℃的热稳定性
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (6)
1.一种热稳定提高的淀粉酶突变体，其特征在于以SEQIDNO.1所示序列为出发序列，将第193位天冬酰胺替换成苯丙氨酸。
2.能表达生产权利要求1所述淀粉酶突变体的载体。
3.能表达生产权利要求1所述淀粉酶突变体的基因工程菌或转基因细胞系。
4.权利要求1所述淀粉酶突变体的制备方法，其特征在于，将SEQIDNO.1所示淀粉酶活性位点内的一个氨基酸通过PCR或化学合成的方法进行替换，将第193位天冬酰胺替换成苯丙氨酸得到的突变体。
5.权利要求4所述的方法，其特征在于，具体步骤如下：
1)根据嗜热脂肪芽孢杆菌淀粉酶序列SEQIDNO.1所示，采用化学全合成的方法全合成后克隆到质粒pET-20b(+)中，构建重组质粒；
2)利用Swiss-model软件对源自嗜热脂肪芽孢杆菌淀粉酶进行模拟，获得淀粉酶空间结构；
3)通过对淀粉酶的序列以及空间结构进行分析，确定要突变的氨基酸位点，为第193位；
4)设计突变引物，对淀粉酶基因序列进行定点突变，将所述位点的氨基酸进行替换，获得含有突变淀粉酶序列的重组载体；
5)将突变后重组载体转化大肠杆菌BL21(DE3)，诱导表达，获得淀粉酶突变体。
6.权利要求1所述淀粉酶突变体在洗涤、织物退浆、淀粉液化、医药、食品领域的应用。
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| CN101528923A (zh) * | 2006-08-04 | 2009-09-09 | 天野酶株式会社 | 变异型酶的设计法、制备法以及变异型酶 |
| CN101857858A (zh) * | 2000-08-01 | 2010-10-13 | 诺维信公司 | 具有改变特性的α-淀粉酶突变体 |
| CN102057040A (zh) * | 2008-06-06 | 2011-05-11 | 丹尼斯科美国公司 | 具有改良特性的嗜热脂肪土芽孢杆菌α-淀粉酶(AMYS)变体 |
| CN102378813A (zh) * | 2009-04-01 | 2012-03-14 | 丹尼斯科美国公司 | 包含具有改变的性质的α-淀粉酶变体的组合物和方法 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101857858A (zh) * | 2000-08-01 | 2010-10-13 | 诺维信公司 | 具有改变特性的α-淀粉酶突变体 |
| CN101242853A (zh) * | 2005-06-24 | 2008-08-13 | 诺维信公司 | 用于药物用途的淀粉酶 |
| CN101528923A (zh) * | 2006-08-04 | 2009-09-09 | 天野酶株式会社 | 变异型酶的设计法、制备法以及变异型酶 |
| CN102057040A (zh) * | 2008-06-06 | 2011-05-11 | 丹尼斯科美国公司 | 具有改良特性的嗜热脂肪土芽孢杆菌α-淀粉酶(AMYS)变体 |
| CN102378813A (zh) * | 2009-04-01 | 2012-03-14 | 丹尼斯科美国公司 | 包含具有改变的性质的α-淀粉酶变体的组合物和方法 |
Non-Patent Citations (1)
| Title |
|---|
| PRF: 227885;GenBank;《GenBank》;19960410 * |
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