CN118956818A - 一种耐受有机溶剂的脂肪酶突变体及其编码基因与应用 - Google Patents
一种耐受有机溶剂的脂肪酶突变体及其编码基因与应用 Download PDFInfo
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Abstract
本发明公开了一种耐受有机溶剂的脂肪酶突变体及其编码基因与应用。本发明通过对野生型米根霉脂肪酶进行定点突变，筛选获得了有机溶剂耐受性显著提高且比活力更高的脂肪酶突变体G266C，实验表明，该脂肪酶突变体G266C分别在终浓度为50%（v/v）的甲醇、乙醇、异丙醇和丙酮中孵育30 min后，残留酶活分别为野生型米根霉脂肪酶的3.1、2.9、2.1和2.6倍，有机溶剂耐受性显著提高，且相对于野生型其酶活性更好，可应用于脂肪酸甲酯等生物柴油的合成，更适用于工业领域上的应用。
Description
技术领域
本发明属于酶工程领域，具体涉及一种耐受有机溶剂的脂肪酶突变体及其编码基因与应用。
背景技术
脂肪酶（E.C. 3.1.1.3）是一类重要的生物催化剂，广泛应用于油脂加工、食品、医药、饲料添加剂和环境治理等领域。脂肪酶在水环境中能够水解反应，而处于有机溶剂环境中能催化酯化、酸解、醇解和酯交换等反应。酶在有机溶剂介质中的生物转化一直是合成化学领域的热点，因为其比传统水介质中的生物转化具有多种优势，包括增加疏水底物的溶解度、降低微生物污染以及抑制依赖水分解代谢副反应。然而在有机溶剂的作用下，酶容易失活，不利于反应的进行。在实际生物催化过程中，如何在有机介质中保持脂肪酶的高活性，并同时利用其催化底物的广泛多样性，是当前面临的一大技术难题。
米根霉脂肪酶（ROL）能用于富集鱼油的多不饱和脂肪酸和制备生物柴油等方面，这些反应体系均存在有机溶剂。在使用中发现，由于该酶的有机溶剂耐受性较差，在应用过程中容易失活，造成生产成本的增加，不利于产业化应用。因此，提高米根霉脂肪酶的有机溶剂耐受性可以为其产业化应用奠定基础。而在寻找有机溶剂耐受性较好的脂肪酶的时候，由于可突变的位点多且蛋白结构的复杂性，需要既兼顾有机溶剂耐受性又要具有较好的酶活性，能够适应工业用的酶并不容易。
发明内容
本发明针对米根霉脂肪酶有机溶剂耐受性较差的不足，提供了一种有机溶剂耐受性提高的脂肪酶突变体及其编码基因和应用。
本发明的第一个目的是提供一种脂肪酶突变体，所述的脂肪酶突变体为脂肪酶突变体G266C，氨基酸序列如SEQ ID NO.3所示。
本发明的第二个目的是提供所述的脂肪酶突变体的编码基因，其特征在于，所述的编码基因的核酸序列如SEQ ID NO.4所示。
本发明的第三个目的是提供含有所述的脂肪酶突变体的编码基因的重组表达载体。
本发明的第四个目的是提供含有所述的重组表达载体的工程菌。
本发明的第五个目的是提供所述的脂肪酶突变体或所述的脂肪酶突变体的编码基因在脂肪酸甲酯合成中的应用。
本发明具有以下有益效果：
本发明通过对野生型米根霉脂肪酶进行定点突变，筛选获得了有机溶剂耐受性显著提高且比活力更高的脂肪酶突变体G266C。与野生型对比，同一条件下，本发明获得的脂肪酶突变体G266C在终浓度为50%（v/v）的甲醇、乙醇、异丙醇和丙酮中处理30 min后，其残留酶活分别为野生型米根霉脂肪酶的3.1、2.9、2.1和2.6倍，具有显著的提高；并且，该脂肪酶突变体G266C的比活力相对野生型的提高了36%。因此，本发明获得的脂肪酶突变体G266C可应用于生物柴油合成，更适合于工业化应用。
附图说明
图1为有机溶剂耐受突变体的筛选残留酶活结果图。
图2为野生型ROL和脂肪酶突变体G266C在不同有机溶剂中的耐受能力。
图3为野生型ROL和脂肪酶突变体G266C在合成生物柴油（脂肪酸甲酯）中产物的得率。
具体实施方式
以下实施例是对本发明的进一步说明，而不是对本发明的限制。
以下实施例中所用的材料与试剂：质粒提取试剂盒购自Omega贸易有限公司；Primer STAR试剂盒购自TAKARA公司；Sypro Orange dye购自Sigama公司；TOP10大肠杆菌感受态细胞购自天根生物公司；突变引物由上海生工生物工程公司合成；Pme Ⅰ 限制性内切酶购自New England Biolabs公司；PCR产物纯化回收试剂盒购自大连宝生物公司；电转仪购自Bio-Rad公司；LLB、LLB+Zeocin、YPD、BMGY、BMMY培养基均按照Invitrogen毕赤酵母表达试剂盒操作手册配制，其余试剂均为国内外购买的分析纯级别。
以下实施例中的脂肪酶突变体指的是在野生型ROL中进行定点突变，采用“原始氨基酸-位置-替换的氨基酸”来表示脂肪酶突变体中突变的氨基酸，如脂肪酶突变体G266C，即所述的脂肪酶突变体G266C的突变位点为Gly266Cys。
实施例1：突变脂肪酶表达载体的构建
ROL的表达载体pPICZαA-ROL由本实验前期所构建，即将ROL的基因（其核酸序列如SEQ ID NO.2所示，编码的氨基酸序列如SEQ ID NO.1所示）插入至载体pPICZαA中，限制性酶切位点为EcoR Ⅰ与Sal Ⅰ。分析ROL的晶体结构（PDB ID: 1LGY），根据积累的经验，使用PyMOL软件观察该酶的晶体结构，经过反复分析，最后设计了5个突变体，即脂肪酶突变体I90N、脂肪酶突变体I90T、脂肪酶突变体I254T、脂肪酶突变体L258T或脂肪酶突变体G266C（其氨基酸序列如SEQ ID NO.3所示，是ROL的第266位由甘氨酸Gly突变为半胱氨酸Cys；其编码基因的核酸序列如SEQ ID NO.4所示）。
在QuikChange引物设计网站（http：//www.genomics.agilent.com/primerDesignProgram.jsp）设计脂肪酶突变体的引物。然后以pPICZαA-ROL为模板，进行PCR反应，构建脂肪酶突变体I90N、脂肪酶突变体I90T、脂肪酶突变体I254T、脂肪酶突变体L258T或脂肪酶突变体G266C的表达载体。
PCR扩增条件为：98℃ 3 min；98℃ 10 s、55℃ 30 s、72℃ 5 min，30个循环；72℃延伸5 min。反应体系：引物各为1 μL，Primer Star（2X）为12.5 μL，模板为1 μL，ddH2O为9.5 μL。扩增产物以Dpn Ⅰ 酶消化模板，在琼脂糖凝胶电泳检测突变条带大小后，获得突变质粒，随后使用热激法将突变质粒转入E.coliTOP10感受态细胞，并涂布于LLB（Zeocin浓度为25 μg/mL）固体平板37 ℃过夜培养，挑选阳性转化子进行质粒的测序。
实施例2：突变脂肪酶表达菌株的构建、表达及纯化
将测序正确的阳性转化子于LLB液体培养基（Zeocin浓度为25 μg/mL）过夜扩增培养后，提取质粒，以Pme Ⅰ内切酶线性化处理并纯化回收，以总量为5 μg的质粒线性化产物与X-33毕赤酵母感受态混合电击转化。毕赤酵母感受态制备参照Invitrogen公司操作手册，程序按照Bio-Rad公司推荐参数设置。
电转完毕立刻加入1 mL 1 mol/L山梨醇溶液，将菌液在30℃孵育复苏1 h后，均匀涂布于YPDS+Zeocin（Zeocin浓度为100 μg/mL）抗性平板上筛选，培养72 h后，挑选阳性转化子。
把阳性转化子单菌落接种至50 mL BMGY培养基，30℃ 250 rpm培养至OD 0.8-1.0，按10%接种量接种至400 mL BMMY培养基30℃ 250 rpm发酵96 h，每24 h以终浓度为1%甲醇进行诱导。
将发酵液于4℃ 7000 r/min离心40 min后，收集上清液。上清液用0.45 μm的滤膜抽滤后，以镍柱亲和层析柱进行纯化。层析柱先用含20 mM咪唑的磷酸缓冲液（pH 7.4）进行平衡，流速4 mL/min，平衡结束后再将上清液与层析柱结合，接着用含20 mM咪唑的磷酸缓冲液（pH 7.4）进行洗脱杂蛋白，然后用含300 mM咪唑的磷酸缓冲液（pH 7.4）洗脱目的蛋白，将洗脱下来的目的蛋白进一步以G-25脱盐柱脱除咪唑，获得纯化的野生型ROL、脂肪酶突变体的酶液。
实施例3：脂肪酶突变体有机溶剂耐受性测定
首先使用无水甲醇对实施例2获得的纯化的野生型ROL、脂肪酶突变体的酶液分别进行处理和测定，即将纯化的酶液以20 mM pH 7.0磷酸盐缓冲液稀释到0.2 mg/mL，然后添加至甲醇中，使甲醇的终浓度为50%（v/v），于30 ℃中孵育30 min，得到用甲醇处理过的酶溶液；并以pNP比色法测定残留酶活。pNP比色法测定残留酶活：吸取24.23 µL的4-硝基苯基辛酸酯（p-Nitrophenylcaprylate，C8）溶于10 mL无水乙醇中，涡旋振荡充分溶解后作为底物。反应体系为10 µL 10 mM 底物、10 µL用甲醇处理过的酶溶液、80 µL 20 mM pH 7.0磷酸盐缓冲液，于室温孵育5 min后添加100 µL异丙醇终止反应的进行，并且在405 nm波长测定其吸光值。如图1所示，其中脂肪酶突变体G266C耐受甲醇能力较好，其残留酶活为71%，为野生型ROL（23%）的3.1倍。
然后再以上述相同方法替换掉其中的甲醇使用乙醇、异丙醇或丙酮处理野生型ROL或脂肪酶突变体G266C，测定其残留酶活。如图2所示，脂肪酶突变体G266C对其他有机溶剂也表现出更优的耐受性能。脂肪酶突变体G266C在乙醇、异丙醇和丙酮中残留酶活分别为野生型ROL的2.9、2.1和2.6倍。
实施例4：脂肪酶的比活力测定
以富含橄榄油的乳化液为底物测定野生型ROL和脂肪酶突变体G266C的酶活性：称取质量分数4% 聚乙烯醇（PVA）水溶液150 g，橄榄油50 g，用高速匀浆机处理6 min制成乳化液。于100 mL锥形瓶中，加入4 g乳化液和5 mL 20 mM磷酸缓冲液（pH为7.5）于30℃孵育5min。将实施例2获得的纯化的野生型ROL、脂肪酶突变体G266C纯化的酶液稀释，向锥形瓶中加入1 mL稀释的酶溶液反应10 min，以15 mL体积分数95%乙醇水溶液终止反应。加入酚酞，用0.05 M氢氧化钠溶液进行滴定，以测定反应过程中释放的游离脂肪酸的量。对照组：将上述加入的稀释的酶溶液用相同体积的20 mM磷酸缓冲液（pH为7.5）代替即可。酶比活力U定义为每分钟产生1 μmol脂肪酸所消耗酶量。比活力测定结果如表1所示，与野生型ROL对比，脂肪酶突变体G266C的比活力提高了36%。
表1 比活测定结果
| 酶 | 比活力(U/mg) |
| 野生型ROL | 1180.2 |
| 脂肪酶突变体G266C | 1605.1 |
实施例5 ：脂肪酸甲酯合成中的应用
使用以大豆油与甲醇溶液为底物的反应体系来考察脂肪酶突变体G266C合成生物柴油（脂肪酸甲酯）的效果。具体反应体系如下：废弃大豆油（3 g）、甲醇（甲醇：大豆油的物质的量比的比例为4.5：1）和1 mL 0.5 mg/mL脂肪酶（野生型ROL或脂肪酶突变体G266C）溶液，35℃反应24小时。检测产物及反应物的含量变化，并计算转化率。如图3所示，脂肪酶突变体G266C的脂肪酸甲酯的得率达到92%左右，而野生型ROL的约为61%。
Claims (5)
1. 一种脂肪酶突变体，其特征在于，所述的脂肪酶突变体为脂肪酶突变体G266C，氨基酸序列如SEQ ID NO.3所示。
2. 一种权利要求1所述的脂肪酶突变体的编码基因，其特征在于，所述的编码基因的核酸序列如SEQ ID NO.4所示。
3.一种含有权利要求2所述的脂肪酶突变体的编码基因的重组表达载体。
4.一种含有权利要求3所述的重组表达载体的工程菌。
5.权利要求1所述的脂肪酶突变体或权利要求2所述的脂肪酶突变体的编码基因在脂肪酸甲酯合成中的应用。
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