CN116286751A - 催化效率提高的双功能纤维素酶突变体及其应用 - Google Patents
催化效率提高的双功能纤维素酶突变体及其应用 Download PDFInfo
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Abstract
本发明公开了一种催化效率提高的双功能纤维素酶突变体及其应用。本发明以GH5‑5家族Bispora sp.MEY‑1来源的具有纤维素酶与甘露聚糖酶活性的双功能纤维素酶BsCel5B作为母本，发现位点Ala261对酶的催化特性有着重要的影响。突变为Ala261Gly后，可获得纤维素酶活性和甘露聚糖酶活性同时提高的突变体。在此改造条件下，纤维素酶突变体对纤维素底物的催化效率比突变前提高了56%，甘露聚糖催化效率提高了21%。突变体更为优越的双功能的特性比野生型有着相对广阔的应用空间，也为其他纤维素酶的改造提供了新的思路。
Description
技术领域
本发明属于基因工程技术领域，具体涉及一种催化效率提高的双功能纤维素酶突变体及其应用。
背景技术
当今世界人口数量增长迅速，石油、煤炭等不可再生资源不仅在储存量上难以满足人们日益增加的需求，也引发气候变暖等环境问题。木质纤维素作为自然界中含量丰富的可再生资源，逐渐成为化石燃料的理想替代品。
糖苷水解酶作为重要的工业用酶，可以有效的将木质纤维素水解成可发酵糖，再转化为生物燃料或商品化学品。尽管随着现代研究的发展，木质纤维素降解能力大大提高，但是酶制剂仍然是纤维素转化过程中最主要的工业成本。植物生物质中，纤维素和半纤维素甘露聚糖含量占70%以上，因此具有同时降解纤维素和甘露聚糖能力的纤维素酶若取代单功能酶，将有效提高糖产量降低成本，对木质纤维素的降解的工业化应用意义重大。迄今为止，研究人员通过各种策略获得多功能酶，除了大量筛选天然酶外，利用蛋白质工程手段对酶分子进行改良也是研究热点。
催化活性是衡量酶工业化应用价值的重要指标，多功能酶催化活性的改造相对于单功能酶存在着更大的挑战。许多单结构域的多功能酶都共享着催化通道内的催化残基与大部分底物结合位点，由于这些位点在功能上存在着平衡与制约，因此如何同时提高多种活性是研究者不断追求的目标。目前，蛋白质工程领域常用的方法主要为理性设计、半理性设计与定向进化。与定向进化和半理性设计相比，理性设计可以更为准确的达到靶向改造多功能酶的目标，成为扩宽多功能酶工业应用潜力的有效改造手段。
酶反应过程中构象变化主要是loop区的变化引起的，loop的运动可以保护酶的疏水核心，还可以使得活性位点靠近底物加速催化。因此，利用理性设计对酶的loop区进行改造，不仅可以探究loop区对酶催化功能的影响机制，还可以指导酶的分子改良，是多功能酶改造的有效途径。
发明内容
本发明的目的在于提供一种催化效率提高的双功能纤维素酶突变体及其应用。
一种催化效率提高的双功能纤维素酶突变体，所述纤维素酶突变体的氨基酸序列如序列表SEQ ID NO：3所示。
所述催化效率提高的双功能纤维素酶突变体基因的核苷酸序列如序列表SEQ IDNO：4所示。
所述双功能纤维素酶突变体基因的载体。
所述双功能纤维素酶突变体基因载体的工程菌。
扩增所述双功能纤维素酶突变体基因的引物。
一种通过优化loop提高双功能纤维素酶催化效率的方法，按照如下步骤进行：
1) 将纤维素酶BsCel5B的野生型序列片段克隆到表达载体pPIC-9r上，重组载体命名pPIC9r-BsCel5B；
2) 以重组载体pPIC9r-BsCel5B为模板，通过携带突变位点的引物对其进行扩增，获得携带突变体序列的重组载体，命名为pPIC9r-BsCel5B-A261G；
3) 将突变体重组载体转化毕赤酵母GS115诱导表达获得突变株GS115/ BsCel5B- A261G。
所述携带突变位点的引物如序列表SEQ ID NO：5、SEQ ID NO：6所示。
本发明的有益效果：本发明通过纤维素酶的BsCel5BA261位点实施定点突变获得A261G突变体，将突变体重组载体转化毕赤酵母GS115，通过对小管水平的发酵液进行酶活测定初步筛选阳性转化子。挑选酶活最高的转化子进行大瓶诱导获得粗酶液，并对粗酶液进行蛋白浓缩及纯化。利用SDS-PAGE电泳检测纯化后突变体和野生型的纯度。以纯化后的蛋白为对象，利用DNS法测定野生型与突变体的基本酶学性质。结果表明，与野生型相比，突变体酶促反应的最适pH值、最适温度并未发生变化；纤维素酶活性以羧甲基纤维素钠为底物，突变体A261G降解羧甲基纤维素钠的催化效率比BsCel5B提高了56%；测定甘露聚糖酶活性时以角豆胶为底物，结果表明，突变体A261G降解角豆胶的催化效率较野生型提高了21%。
附图说明
图1为催化效率提高的双功能纤维素酶与BsCel5B的最适pH。
图2为催化活性提高的纤维素酶突变体与BsCel5B的最适温度。
图3为催化活性提高的纤维素酶突变体与BsCel5B的电泳图。
图4为催化活性提高的纤维素酶突变体与BsCel5B的纤维素酶与甘露聚糖酶比活比较图。
具体实施方式
为了便于理解本发明，下面将对本发明进行更全面的描述。但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
下述实施例采用的试验材料和试剂：
1、菌株及载体：表达宿主Pichia pastoris GS115，表达质粒载体pPIC-9r为本实验室保存。
2、生化试剂：限制性内切酶购自NEB公司，连接酶购自Promaga公司，点突变试剂盒购自全式金公司，羧甲基纤维素钠购自Sigma公司。其它都为国产分析纯试剂（均可从普通生化试剂公司购买得到）。
3、培养基：LB培养基：0.5% 酵母提取物，1% 蛋白胨，1% NaCl，pH 7.0 。
YPD培养基：1% 酵母提取物，2% 蛋白胨，2% 葡萄糖 。
MD固体培养基：2% 葡萄糖，1.5% 琼脂糖，1.34% YNB，0.00004% Biotin。
BMGY培养基：1% 酵母提取物，2% 蛋白胨，1% 甘油(V/V)，1.34% YNB，0.00004%Biotin。
BMMY培养基：1% 酵母提取物，2% 蛋白胨，1.34%YNB，0.00004% Biotin，0.5%甲醇(V/V)。
4、本实施例中未做详细具体说明的分子生物学实验方法，均参照《分子克隆实验指南》（第三版）J.萨姆布鲁克一书中所列的具体方法进行，或者按照试剂盒和产品说明书进行。
实施例1 催化活性提高的纤维素酶突变体重组载体pPIC9r-BsCel5B-A261G的制备
将纤维素酶野生型序列片段（蛋白和基因序列分别如序列表SEQ ID NO：1、SEQ IDNO：2所示）克隆到表达载体pPIC-9r上，重组载体命名pPIC9r-BsCel5B-A261G；以重组载体pPIC9r-BsCel5B为模板，通过携带突变位点的引物对其进行扩增，获得携带突变体序列的重组载体，命名为pPIC9r-BsCel5B-A261G。
表1催化活性提高的纤维素酶突变体特异性引物
实施例2 催化活性提高的纤维素酶突变体的制备
（1）催化活性提高的纤维素酶突变体BsCel5B-A261G在毕赤酵母中摇瓶水平的大量表达。
将获得的含有催化活性提高的突变体基因BsCel5B-A261G的重组质粒pPIC9r- BsCel5B-A261G转化毕赤酵母GS115，获得重组酵母菌株GS115/BsCel5B-A261G。 取含有重组质粒的GS115菌株，接种于300 mL BMGY培养基的1 L三角瓶中，置于30℃，220 rpm摇床培养48 h；培养液4000 g离心5 min，弃上清，沉淀用200 mL含有0.5%甲醇的BMMY培养基重悬，并再次置于30℃，220 rpm条件下诱导培养。每隔12 h补加1 mL甲醇，同时取上清用于酶活性检测。
（2）重组蛋白酶的纯化
收集摇瓶表达的重组纤维素酶上清液，通过10 kDa膜包进行浓缩，同时用低盐缓冲液置换其中的培养基，最后剩余约20 ml蛋白浓缩液。浓缩的重组纤维素酶BsCel5B-A261G利用离子交换层析法进行纯化。具体地，取纤维素酶BsCel5B及突变体BsCel5B-A261G浓缩液10.0 mL经预先用10 mmol/L Tris-HCl (pH 8.0)平衡过的HiTrap Q HP阴离子柱，然后用含有1 mol/L NaCl的10 mmol/L Tris-HCl (pH 8.0)进行线性梯度洗脱，利用DNS法对梯度洗脱的蛋白液进行酶活性检测，同时利用SDS-PAGE凝胶电泳对梯度洗脱的蛋白液进行纯度的检测。
实施例3 重组催化活性提高的纤维素酶突变体和野生型的活性分析
采用二硝基水杨酸 (DNS) 法测定重组内切纤维素酶及甘露聚糖酶的基本酶学性质。具体方法如下：在pH 4.0，80℃条件下，1 mL的反应体系包括100 µL适当的稀释酶液，900 µL底物，反应10 min，加入1.5 mL DNS终止反应；沸水浴煮5 min后冷却至室温，在540nm波长下测定OD值。内切纤维素酶活性单位定义：在一定条件下，每分钟分解底物生成1 μmoL 葡萄糖所需要的酶量为1个活性单位(U)。甘露聚糖酶活性单位定义：在一定条件下，每分钟分解底物生成1 μmoL 甘露糖糖所需要的酶量为1个活性单位 (U)。酶学性质研究所用酶液均需达到电泳纯。
（1）最适pH分析比较
经纯化的（实施例2）表达的纤维素酶突变体BsCel5B及突变体BsCel5B-A261G在不同的pH下进行酶促反应以测定其最适pH。所用缓冲液为pH2.0-8.0的柠檬酸一磷酸氢二钠系列缓冲体系。纯化的纤维素酶突变体BsCel5B及突变体BsCel5B-A261G在不同pH的缓冲体系、80℃下测定的最适pH结果（图1）表明：BsCel5B和BsCel5B-A261G的最适pH均为4.0。
（2）最适温度分析比较
纯化的内切纤维素酶在pH 4.0（以羧甲基纤维素钠为底物）条件下，测定不同温度（30-90℃）下的酶活性，以确定重组酶最适温度。实验结果表明，该酶的最适反应温度为80℃（图2）。
（3）催化效率分析比较
纯化后（实施例2）的纤维素酶突变体BsCel5B，与突变体BsCel5B-A261G，80 ℃件下进行酶促反应以测定其酶活性及动力学参数。
比活测定结果如表2所示，BsCel5B以羧甲基纤维素钠为底物时的比活为941 ±18 U/mg，突变体BsCel5B-A261G的比活为1350 ± 27相较于野生型，突变体BsCel5B-A261G的比活力较野生型提高了43%（表1）。BsCel5B的Km值为6.41±0.4 mg/ml，突变体BsCel5B-A261G的Km值为5.42 ± 0.4 mg/ml。BsCel5B的Vmax为1445 ± 126 μmol/min/mg，突变体BsCel5B-A261G提高至1783 ± 123 μmol/min/mg。野生型的转换数为842 ± 73 S-1，BsCel5B-A261G提高至1040 ± 89 S-1，kcat/Km由野生型的122 ± 10提高至BsCel5B-A261G的191 ± 14 ml/s/mg。与野生型相比，突变体BsCel5B-A261G对纤维素酶底物CMC-Na的比活力和催化效率分别是野生型的1.43倍和1.56倍。动力学参数值中，Km值的降低反映了突变体增强了对纤维素底物的亲和力，Vmax与转换数的提高表明突变体加快了产物释放的速度，最终导致突变体的催化效率优于野生型。
表2
发明人测定了以角豆胶为底物的甘露聚糖比活力，野生型为1718 ± 24 U/mg，突变体突变体BsCel5B-A261G比活力为2035 ± 33 U/mg，相较于野生型提高了18%。BsCel5B的Km值为2.32 ± 0.1 mg/ml，Vmax为2445 ± 131 μmol/min/mg，突变体BsCel5B-A261G的Km值为2.24 ± 0.1 mg/ml，Vmax为2849 ± 143 μmol/min/mg，因此野生型的转换数由1426 ± 76 S-1提高至1661 ± 73 S-1，最终催化效率kcat/Km由614 ± 53 ml/s/mg提高为741 ± 41 ml/s/mg。与野生型相比，突变体的比活力和催化效率分别提高了18%和21%。综合以上酶学数据，双功能纤维素酶BsCel5B突变体比野生型有着更好的应用前景。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
Claims (7)
1.一种催化效率提高的双功能纤维素酶突变体，其特征在于，所述纤维素酶突变体的氨基酸序列如序列表SEQ ID NO：3所示。
2.一种催化效率提高的双功能纤维素酶突变体的基因，其特征在于，所述纤维素酶突变体基因的核苷酸序列如序列表SEQ ID NO：4所示。
3.包含权利要求2所述双功能纤维素酶突变体基因的载体。
4.包含权利要求3所述双功能纤维素酶突变体基因载体的工程菌。
5.扩增权利要求2所述双功能纤维素酶突变体基因的引物。
6.一种通过优化loop提高双功能纤维素酶催化效率的方法，其特征在于，按照如下步骤进行：
1) 将纤维素酶BsCel5B的野生型序列片段克隆到表达载体pPIC-9r上，重组载体命名pPIC9r-BsCel5B；
2) 以重组载体pPIC9r-BsCel5B为模板，通过携带突变位点的引物对其进行扩增，获得携带突变体序列的重组载体，命名为pPIC9r-BsCel5B-A261G；
3) 将突变体重组载体转化毕赤酵母GS115诱导表达获得突变株GS115/BsCel5B- A261G。
7.根据权利要求6所述通过优化loop提高双功能纤维素酶催化效率的方法，其特征在于，所述携带突变位点的引物如序列表SEQ ID NO：5、SEQ ID NO：6所示。
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Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103361331A (zh) * | 2013-07-04 | 2013-10-23 | 杭州佳惠农业开发有限公司 | 一种纤维素酶、编码基因、载体、工程菌及其应用 |
| US20140329283A1 (en) * | 2011-09-14 | 2014-11-06 | Dupont Nutrition Biosciences Aps | Enzymes |
| CN104630185A (zh) * | 2015-02-03 | 2015-05-20 | 中国农业科学院饲料研究所 | 对纤维素底物特异性提高的突变双功能木聚糖酶/纤维素酶及其编码基因和应用 |
| US20180371442A1 (en) * | 2015-11-12 | 2018-12-27 | Council Of Scientific & Industrial Research | Cellulase derived from metagenomics |
| CN110656099A (zh) * | 2019-10-14 | 2020-01-07 | 江苏科技大学 | 一种40℃下高比活木聚糖酶突变体及其构建方法与应用 |
| WO2020160126A1 (en) * | 2019-01-31 | 2020-08-06 | Novozymes A/S | Polypeptides having xylanase activity and use thereof for improving the nutritional quality of animal feed |
| CN111676209A (zh) * | 2020-08-05 | 2020-09-18 | 中国农业科学院北京畜牧兽医研究所 | 一种提高双功能纤维素酶的甘露聚糖酶活的方法及纤维素酶突变体rmx-m和应用 |
| US20210189297A1 (en) * | 2018-06-29 | 2021-06-24 | Novozymes A/S | Subtilase variants and compositions comprising same |
| CN113684198A (zh) * | 2021-10-27 | 2021-11-23 | 中国农业科学院北京畜牧兽医研究所 | 一种提高纤维素酶催化效率的方法及突变体5i77-m2 |
| CN113980940A (zh) * | 2021-12-23 | 2022-01-28 | 中国农业科学院北京畜牧兽医研究所 | 一种提高双功能纤维素酶的催化效率的方法及突变体、基因和应用 |
-
2023
- 2023-05-15 CN CN202310541291.4A patent/CN116286751B/zh active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140329283A1 (en) * | 2011-09-14 | 2014-11-06 | Dupont Nutrition Biosciences Aps | Enzymes |
| CN103361331A (zh) * | 2013-07-04 | 2013-10-23 | 杭州佳惠农业开发有限公司 | 一种纤维素酶、编码基因、载体、工程菌及其应用 |
| CN104630185A (zh) * | 2015-02-03 | 2015-05-20 | 中国农业科学院饲料研究所 | 对纤维素底物特异性提高的突变双功能木聚糖酶/纤维素酶及其编码基因和应用 |
| US20180371442A1 (en) * | 2015-11-12 | 2018-12-27 | Council Of Scientific & Industrial Research | Cellulase derived from metagenomics |
| US20210189297A1 (en) * | 2018-06-29 | 2021-06-24 | Novozymes A/S | Subtilase variants and compositions comprising same |
| WO2020160126A1 (en) * | 2019-01-31 | 2020-08-06 | Novozymes A/S | Polypeptides having xylanase activity and use thereof for improving the nutritional quality of animal feed |
| CN110656099A (zh) * | 2019-10-14 | 2020-01-07 | 江苏科技大学 | 一种40℃下高比活木聚糖酶突变体及其构建方法与应用 |
| CN111676209A (zh) * | 2020-08-05 | 2020-09-18 | 中国农业科学院北京畜牧兽医研究所 | 一种提高双功能纤维素酶的甘露聚糖酶活的方法及纤维素酶突变体rmx-m和应用 |
| CN113684198A (zh) * | 2021-10-27 | 2021-11-23 | 中国农业科学院北京畜牧兽医研究所 | 一种提高纤维素酶催化效率的方法及突变体5i77-m2 |
| CN113980940A (zh) * | 2021-12-23 | 2022-01-28 | 中国农业科学院北京畜牧兽医研究所 | 一种提高双功能纤维素酶的催化效率的方法及突变体、基因和应用 |
Non-Patent Citations (2)
| Title |
|---|
| FEI ZHENG 等: "Improvement of the catalytic performance of a Bispora antennata cellulase by replacing the N-terminal semi-barrel structure", 《BIORESOUR TECHNOL》, vol. 218, pages 279 - 285 * |
| 郑菲: "真菌第五家族纤维素酶的基因挖掘与分子改良研究", 《中国博士学位论文全文数据库 基础科学辑》, no. 04, pages 006 - 52 * |
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