CN111826367B - 酶活性提升的纤维素酶 - Google Patents
酶活性提升的纤维素酶 Download PDFInfo
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- CN111826367B CN111826367B CN201910303853.5A CN201910303853A CN111826367B CN 111826367 B CN111826367 B CN 111826367B CN 201910303853 A CN201910303853 A CN 201910303853A CN 111826367 B CN111826367 B CN 111826367B
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- cellulase
- thr
- ser
- gly
- ala
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Abstract
本案系关于一种具提升酶活性的纤维素酶，其氨基酸序列系为将序列编号2第120个位置或与序列编号2具有80％以上序列相似度的序列中对应位置的甲硫氨酸突变成天冬酰胺的序列。
Description
【技术领域】
本案系关于一种纤维素酶，尤指一种具提升酶活性的纤维素酶。
【现有技术】
纤维素是植物细胞壁的主要成分，其占植物干质量的35-50％，故为地球上最丰沛的可再生生物质能(biomass)。纤维素是以葡萄糖为单位，以1,4-β-醣苷键(1,4-β-glycosidic bonds)所键连而成的长链多醣。纤维素微纤维的高度紧密结构有助于生物质能抗降解屏障(biomass recalcitrance)及抵抗微生物攻击。纤维素可以降解成葡萄糖，并作为细菌、酵母菌及真菌等微生物的能量来源。纤维素的完全降解需要几种纤维素酶，例如内切葡聚醣酶(endoglucanase,EC 3.2.1.4)、外切葡聚醣酶(cellobiohydrolase,EC3.2.1.91)、以及葡萄醣苷酶(β-glucosidase,EC 3.2.1.21)。在这些纤维素酶中，内切葡聚醣酶为关键的纤维素分解酶，能够随机水解β-醣苷键而将长链纤维素切成许多小片段的寡糖。内切葡聚糖酶存在于各种微生物中，包括真菌和细菌。基于它们的氨基酸序列相似性，纤维素酶被分类为不同的糖苷水解酶(glycoside hydrolase,GH)家族，包括GH 5、6、10、12、18、45、及74。
近年来，纤维素酵素水解作用的利用被大量研究，且内切葡聚糖酶被广泛应用在各种工业，例如动物饲料、食品制造、纺织工业、及生物燃料生产。根据不同的工业需求，纤维素酶也必须适用于不同的工作条件，因此许多科学家也试图透过在自然界中寻找新基因或修饰现有的酶来找出更好的工业用纤维素酶。在许多修饰酶的策略中，基于结构分析的合理设计的蛋白质工程是改善工业酶的主要策略之一。在此策略中，增加酶活性是改善工业酶的关键点，因为较高的酶活性即代表了工业过程的成本降低，更进而提升了商业利润。
因此，本案即欲藉由分析纤维素酶的酶结构来找出对酶活性具关键性的氨基酸，并进一步修饰酶以提升纤维素酶的酶活性，进而增加纤维素酶的工业应用价值。
【发明内容】
本案之目的在于利用结构分析及点突变技术来改造现有纤维素酶，以有效提升纤维素酶的酶活性，进而增加纤维素酶的工业应用价值。
为达上述目的，本案之一较广义实施方案为提供一种纤维素酶，其氨基酸序列系为将序列编号2第120位置或与序列编号2具有80％以上序列相似度的序列中对应位置的甲硫氨酸突变成天冬酰胺的序列。
在一实施例中，编码该序列编号2的基因系从棘孢曲霉F-50中所分离出来的FI-羧甲基纤维素酶基因。
在一实施例中，该纤维素酶系为一内切葡聚糖酶。
在一实施例中，该纤维素酶的氨基酸序列如序列编号5所示。
本案之另一较广义实施方案为提供一种编码前述纤维素酶之核酸分子。
本案之又一较广义实施方案为提供一种重组质粒，其系包含前述核酸分子。
【附图说明】
图1显示野生型FI-羧甲基纤维素酶的核苷酸序列以及氨基酸序列。
图2显示野生型FI-羧甲基纤维素酶与纤维四糖复合的蛋白质结构。
图3显示点突变技术所采用的引物序列。
图4显示M120N突变型FI-羧甲基纤维素酶的核苷酸序列以及氨基酸序列。
图5显示野生型及M120N突变型FI-羧甲基纤维素酶的纤维素酶活性分析。
【实施方式】
体现本案特征与优点的一些典型实施例将在后段的说明中详细叙述。应理解的是本案能够在不同的态样上具有各种的变化，其皆不脱离本案的范围，且其中的说明及图式在本质上系当作说明之用，而非用以限制本案。
本案所采用的纤维素酶基因是从可产生许多纤维素分解酶的棘孢曲霉(Aspergillus aculeatus)F-50中所分离出来的FI-羧甲基纤维素酶(FI-carboxymethylcellulase,简称FI-CMCase)基因。FI-羧甲基纤维素酶基因被表达在大肠杆菌(Escherichia coli)及啤酒酵母菌(Saccharomyces cerevisiae)中，并被进一步定性。所表达的FI-羧甲基纤维素酶蛋白为内切葡聚糖酶，且其最适pH及温度分别为5.0及50℃。在本案中发现，FI-羧甲基纤维素酶可在工业上常用的毕赤酵母(Pichia pastoris)中有效表达及生产。此外，在毕赤酵母中表达的FI-羧甲基纤维素酶重组蛋白对羧甲基纤维素(carboxymethyl cellulose,CMC)和滤纸显示出相当高的酶活性。这些结果证实FI-羧甲基纤维素酶是商业利用上具有发展潜力的候选酶。为了提高此纤维素酶的工业应用价值，本案利用X射线晶体学解出了FI-羧甲基纤维素酶与其底物复合的蛋白质结构。
如前所述，FI-羧甲基纤维素酶基因(基因库登录号为X52525.1)是从棘孢曲霉(A.aculeatus)F-50中所分离出来。1图1即显示野生型FI-羧甲基纤维素酶的核苷酸序列以及氨基酸序列，其中，野生型FI-羧甲基纤维素酶基因包含666个碱基(含终止密码子，核苷酸序列以序列编号1标示)，且编码221个氨基酸(氨基酸序列以序列编号2标示)。首先，将FI-羧甲基纤维素酶基因利用EcoRI以及NotI限制酶切引入到pPICZαA载体中，再以PmeI限制酶将该质粒DNA线性化后，以电转染方式转形入毕赤酵母X33菌株中。接着将转形株菌液涂于含有100μg/ml Zeocin抗生素的YPD培养基(含1％酵母提取物、2％蛋白胨、2％葡萄糖、2％琼脂)，并于30℃下进行培养两天。接着利用以下小规模表达来测试转染株的蛋白质表达。将所挑选的菌落接种到5ml YPD进行培养，并放大接种到50ml BMGY中于30℃下培养24小时。接着利用离心收成细胞，并重新浮悬于20ml BMMY中以诱导蛋白质表达，之后选择具有较高表达水平的转染株进行放大表达。将所选转染株细胞接种到5ml YPD进行培养，并放大接种到500ml BMGY中于30℃下培养24小时。接着收成细胞并重新浮悬于500ml BMMY中，然后连续4天每24小时补充总浓度为0.5％的甲醇来诱导蛋白质表达。至于蛋白质纯化，则是利用离心收集上清液，然后对5L含有25mM Tris，pH 7.5的缓冲液进行两次透析，再利用FPLC系统以DEAE管柱纯化蛋白质。最后将纯化的蛋白质在含25mM Tris-HCl，pH 7.5及150mM氯化钠的溶液中浓缩至10mg/ml，再利用SDS-PAGE分析确认蛋白纯度。
为了利用X射线晶体学解析FI-羧甲基纤维素酶的蛋白质结构，本案以坐滴蒸气扩散法(sitting-drop vapor diffusion)取得蛋白质结晶。先以市售的结晶筛选套组(Crystal Screen Kit)在10天内获得FI-羧甲基纤维素酶的初始晶体，再从含有0.3M乙酸锌二水合物、0.1M二甲胂酸钠pH 6.5、及19％(w/v)聚乙二醇8000的室温储存溶液中在10天内获得更好的晶体。之后以分子置换方法解决相位问题，并将晶体浸泡在10mM纤维四糖中，以获得FI-羧甲基纤维素酶的复合晶体。
图2显示野生型FI-羧甲基纤维素酶与纤维四糖复合的蛋白质结构。FI-羧甲基纤维素酶的蛋白质结构显示出β-果冻卷(jelly roll)蛋白质折叠，这是GH 12家族酶的典型特征。根据酶-底物复合结构，可观察到在第120位置的甲硫氨酸(methionine)残基(Met120)系位于活性区并与纤维四糖有相互作用，故此残基被认为可能对FI-羧甲基纤维素酶的催化反应有重要性。因此，本案选择Met120为突变位点，并利用点突变技术将其突变为天冬酰胺(asparagine)，以期提升其酶活性。
以下将详述本案之纤维素酶改造方法及其所得到之改良纤维素酶。突变体系以市售的点突变套组来制备，并以野生型FI-羧甲基纤维素酶基因为模板。图3显示点突变技术所采用的引物序列，以将FI-羧甲基纤维素酶氨基酸序列第120位置的甲硫氨酸突变成天冬酰胺，其中，M120N即意指第120位置的氨基酸由甲硫氨酸(methionine,M)突变成天冬酰胺(asparagine,N)，且该突变引物之序列以序列编号3标示。原始的模板则利用DpnI限制酶于37℃进行消化而去除。接着把突变质粒送入大肠杆菌，并藉由DNA测序确认突变的基因序列。
图4显示M120N突变型FI-羧甲基纤维素酶的核苷酸序列以及氨基酸序列，其中M120N突变基因包含666个碱基(含终止密码子，核苷酸序列以序列编号4标示)，且编码221个氨基酸(氨基酸序列以序列编号5标示)，且第120位置的氨基酸由甲硫氨酸突变成天冬酰胺。
最后如前述方法所述，将突变基因转染入毕赤酵母进行蛋白质表达，且野生型及突变型FI-羧甲基纤维素酶的上清液接着在相同蛋白浓度下进行纤维素酶活性检测。纤维素酶活性测试方法系采用二硝基水杨酸(dinitrosalicylic acid,DNS)方法。在此实施例中，将等量的酶溶液(50mM柠檬酸钠缓冲液，pH 4.8)和1％(w/v)羧甲基纤维素共同置于50℃水浴中10分钟，再将反应物与1％DNS混合，然后置于100℃沸水中10分钟以除去残留的酶活性。在冷水浴中冷却5分钟后，测量反应溶液的540nm吸光度以计算酶活性。一个活性单位定义为每分钟释放1μM产物的酶量。
图5显示野生型及M120N突变型FI-羧甲基纤维素酶的纤维素酶活性分析，其中，这些试样的蛋白浓度有经过标准化，且野生型酶的纤维素酶活性设定为100％。根据图5的分析可知，M120N突变型酶的比活性(unit/mg)高于野生型酶。特别是，与野生型酶相比，M120N突变型酶的比活性显著增加了26％。此外，M120N突变型酶的表达水平与野生型酶相似。这些结果表明，当Met120突变为天冬酰胺时，可提升酶的总活性。这意味着与野生型酶相比，M120N突变型酶具有更高的工业应用价值。
此外，酶在不同物种间通常存在一些变异，但仍具有相同功能，且彼此间大多具有80％以上的氨基酸序列相似度，显见要保有酶功能，亦可容许有部分氨基酸序列的变异。换言之，本案改造之纤维素酶序列当不仅限于将序列编号2第120位置的氨基酸由甲硫氨酸突变成天冬酰胺的序列，亦可包含将与序列编号2具有80％以上序列相似度的序列中对应位置的甲硫氨酸突变成天冬酰胺的序列。
由于工业过程通常涉及各种苛刻条件，因此工业酶须进一步改进以满足工业要求，例如优异的热稳定性，更宽范围的pH适应性，以及更高的酶活性。为了达到这些目标，需要酶的结构信息以更理解催化机制并设计随后的蛋白质工程，从而提高酶的性质和效率。因此，为了提高FI-羧甲基纤维素酶的工业价值，本案解析其酶-底物复合结构，并选择位于FI-羧甲基纤维素酶活性区的残基Met120进行进一步修饰。结果显示M120N突变型酶的纤维素酶活性显著高于野生型酶的纤维素酶活性，也就是说，本案成功地提升了纤维素酶的酶活性，并进一步增加了纤维素酶的工业应用价值。
纵使本发明已由上述实施例详细叙述而可由熟悉本技艺人士任施匠思而为诸般修饰，然皆不脱如所附权利要求书所欲保护者。
序列表
<110> 湖北大学
<120> 酶活性提升的纤维素酶
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Claims (3)
1.一种纤维素酶，其氨基酸序列系为将序列编号2第120个位置的甲硫氨酸突变成天冬酰胺的序列。
2.一种编码如权利要求1所述之纤维素酶之核酸分子。
3.一种重组质粒，其系包含如权利要求2所述之核酸分子。
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| US16/406,255 US10479985B1 (en) | 2019-04-16 | 2019-05-08 | Cellulase having improved enzymatic activity |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106906198A (zh) * | 2015-12-23 | 2017-06-30 | 东莞泛亚太生物科技有限公司 | 提升耐温性的纤维素酶 |
| CN107663520A (zh) * | 2016-07-29 | 2018-02-06 | 东莞泛亚太生物科技有限公司 | 提升活性的纤维素酶 |
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2019
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106906198A (zh) * | 2015-12-23 | 2017-06-30 | 东莞泛亚太生物科技有限公司 | 提升耐温性的纤维素酶 |
| CN107663520A (zh) * | 2016-07-29 | 2018-02-06 | 东莞泛亚太生物科技有限公司 | 提升活性的纤维素酶 |
Non-Patent Citations (3)
| Title |
|---|
| Crystal structure and genetic modifications of FI-CMCase from Aspergillus aculeatus F-50;Jian-Wen Huang et al.;《Biochemical and Biophysical Research Communications》;20161231;全文 * |
| Enhanced activity of Thermotoga maritima cellulase 12A by mutating a unique surface loop;Ya-Shan Cheng et al.;《Appl Microbiol Biotechnol》;20121231;全文 * |
| 基于C-端氨基酸突变改造提高内切型纤维素酶活性和稳定性的研究;戚耀之;《中国优秀博硕士学位论文全文数据库(硕士) 基础科学辑》;20140215;全文 * |
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