CN110468117B - 一种有机溶剂耐受的脂肪酶突变体及其应用 - Google Patents
一种有机溶剂耐受的脂肪酶突变体及其应用 Download PDFInfo
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- CN110468117B CN110468117B CN201910844700.1A CN201910844700A CN110468117B CN 110468117 B CN110468117 B CN 110468117B CN 201910844700 A CN201910844700 A CN 201910844700A CN 110468117 B CN110468117 B CN 110468117B
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Abstract
本发明公开了一种有机溶剂耐受的脂肪酶突变体及其应用，属于基因工程及酶工程技术领域，本发明基于酶蛋白结构分析，设计及构建酶突变库，筛选获得对有机溶剂耐受性增强的脂肪酶突变体，并构建得到高效表达该突变体的毕赤酵母工程菌，该有机溶剂耐受性增强的脂肪酶突变体，相对于野生型在对耐受有机溶剂的能力显著增强，在50％的乙醇处理2小时，其残余活力仍保留是75％，约为是野生型的3倍；该脂肪酶突变体可在生物柴油及风味酯制备加工过程中应用，具有良好的稳定性能，可在应用过程中寿命延长，节约生产成本和提高生产效率。
Description
技术领域
本发明涉及基因工程及酶工程领域，特别是涉及一种有机溶剂耐受的脂肪酶突变体及其应用。
背景技术
脂肪酶，即三酰基甘油酰基水解酶，它催化天然底物油脂水解，生成脂肪酸、甘油和甘油单酯或二酯。脂肪酶基本组成单位仅为氨基酸，通常只有一条多肽链。它的催化活性仅仅决定于它的蛋白质结构(Schmid等,1998)。脂肪酶具有广泛的应用价值，已成为市场上的第三大工业用酶。脂肪酶可以催化解脂、酯交换、酯合成等反应，广泛应用于饲料添加剂、油脂加工、食品、医药、日化等工业。
脂肪酶在水环境中能够催化酯键的水解，当处于有机溶剂环境中能催化酯交换，酯化，醇解，酸解等反应。而脂肪酶作用的底物往往是水不溶性的，在有机相中才有较好的溶解度，但是在有机溶剂中，大多数酶类活力受到抑制甚至失活，所以其在生物催化中的应用受到很大限制。在有机介质中维持较高的脂肪酶活性及脂肪酶催化底物的广泛性是实际生物催化过程中的难题。因此开发耐有机溶剂脂肪酶在理论和工业应用上具有重要的意义。
发明内容
本发明的目的是提供一种有机溶剂耐受的脂肪酶突变体及其应用，以解决上述现有技术存在的问题，该脂肪酶突变体可应用于风味酯的合成及生物柴油的生产，其具有良好的稳定性能，可在应用过程中寿命延长，节约生产成本和提高生产效率。
为实现上述目的，本发明提供了如下方案：
本发明提供一种有机溶剂耐受的脂肪酶突变体，所述有机溶剂耐受的脂肪酶突变体是氨基酸序列为SEQ ID NO.1的脂肪酶的第191位氨基酸由Arg变为Ala。
上述有机溶剂耐受的脂肪酶突变体的氨基酸序列为SEQ ID NO.2。
上述有机溶剂耐受的脂肪酶突变体的编码基因的核酸序列为SEQ ID NO.3。
本发明还提供一种在宿主细胞内表达氨基酸序列为SEQ ID NO.2的有机溶剂耐受的脂肪酶突变体的重组质粒。
进一步地，所述重组质粒含有序列为SEQ ID NO.3的核酸。
本发明还提供一种工程菌株，所述工程菌含有在宿主细胞内表达氨基酸序列为SEQID NO.2的有机溶剂耐受的脂肪酶突变体的重组质粒或含有核酸序列为SEQ ID NO.3的重组质粒。
进一步地，所述工程菌株为巴斯德毕赤酵母Pichia pastoris。
本发明还提供上述的有机溶剂耐受的脂肪酶突变体、上述的重组质粒、上述的工程菌株在风味酯物质及生物柴油合成中的应用。
本发明公开了以下技术效果：
本发明基于酶蛋白结构分析，设计及构建酶突变库，筛选获得对有机溶剂耐受性增强的脂肪酶突变体，并构建得到高效表达该突变体的毕赤酵母工程菌，该有机溶剂耐受性增强的脂肪酶突变体，相对于野生型在对耐受有机溶剂的能力显著增强，在50％的乙醇处理2小时，其残余活力仍保留75％，约为是野生型的3倍；该脂肪酶突变体可在生物柴油及风味酯制备加工过程中应用，具有良好的稳定性能，可在应用过程中寿命延长，节约生产成本和提高生产效率。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为实施例3有机溶剂耐受脂肪酶突变体的筛选酶活对比图；
图2为实施例3中R191A突变体在不同溶剂中耐受能力对比图；
图3为实施例5的MAS1及R191A突变体合成己酸乙酯的合成效率对比图。
具体实施方式
现详细说明本发明的多种示例性实施方式，该详细说明不应认为是对本发明的限制，而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式，并非用于限制本发明。另外，对于本发明中的数值范围，应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明，否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料，但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入，用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时，以本说明书的内容为准。
在不背离本发明的范围或精神的情况下，可对本发明说明书的具体实施方式做多种改进和变化，这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本申请说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等，均为开放性的用语，即意指包含但不限于。
实施例1突变体表达载体的构建
根据Stratagene公司的QuikChange方案，在QuikChange引物设计网站(http：//www.genomics.agilent.com/primerDesignProgram.jsp)设计了突变体R191A、Q201A、V203A、V199F、H139R、V48D及P252D引物。采取的定点突变(QuikChange Site-directedMutagenesis)中对脂肪酶MAS1基因进行定点突变。具体如表1所示：
表1脂肪酶基因定点突变的反应体系
PCR反应条件为：98℃预变性3min；94℃变性10s，68℃延伸4min，20个循环；72℃7min。PCR产物采用1％的琼脂糖凝胶电泳检验。PCR产物经检测确认后，加入DpnI处理去除带甲基化的原始模板链。酶切体系如下：酶切反应条件：37℃，1h。
将酶切产物转化到大肠杆菌DH5α感受态中，涂布在含Amp的LB平板，在37℃恒温培养箱中过夜培养。挑取单克隆接种于5mL的LB培养基中，在37℃下过夜培养，提取质粒进行基因序列测定。
实施例2突变体酶蛋白制备与纯化
将含有MAS1脂肪酶(氨基酸序列为SEQ ID NO：1)及其突变体基因的pET22b表达载体转化到BL21(DE3)菌种中，并进行诱导表达：
(1)接种200μL阳性克隆子甘油菌到50mL低盐LB(含100μg/mL氨苄青霉素)液体培养基中，37℃、200rpm过夜培养15～18h。
(2)按2％接种量将种子液接种到500mL LB(含100μg/mL氨苄青霉素)液体培养基中，37℃、200rpm培养OD600至0.6～0.8，然后加入1M IPTG溶液至IPTG终浓度为0.4mM，15℃、200rpm诱导培养18h。
诱导结束后进行重组蛋白的纯化：
(1)收菌：将菌液置于冰上预冷约15min，然后于4℃、10000rpm条件下离心10min，弃上清。
(2)破菌收集上清：用预冷的PBS溶液重悬细胞，超声破碎15min，然后在4℃，10000rpm条件下离心10min收集细胞裂解液。所得细胞裂解液通过0.45μm滤膜抽滤以进一步除去菌体和悬浮杂质。
(3)镍柱亲和层析：重组MAS1及其突变体蛋白的纯化采用镍柱亲和层析的方法，柱子选用His trapTm 5mL预装柱。首先用超纯水除去用于保护填料的乙醇，再用平衡缓冲液(pH7.4 20mM磷酸盐缓冲液，含20mM咪唑和0.5M NaCl)平衡镍柱填料，待A280处吸光值、电导率等参数显示平衡即完成平衡过程。接着将抽滤后的细胞裂解液上样到镍柱中，待上样完成后继续用平衡缓冲液洗去未与填料结合的蛋白。待A280处吸光值、电导率等参数显示平衡后，用含有20～500mM咪唑的洗脱液(20mM磷酸盐缓冲液，pH7.4，含1M NaCl)进行分步洗脱，全过程流速保持在5mL/min。分管收集洗脱液样品置于4℃保存并进行SDS-PAGE检测。
实施例3耐受有机溶剂突变体的筛选
酶活力测定采用碱式滴定法，具体方法为：在100mL具塞锥形瓶中加入4mL橄榄油聚乙烯醇乳化液和5mL缓冲液于恒温水浴摇床下预热5min，然后实验组加入1mL适当稀释的酶液，对照组加入1mL蛋白所在的缓冲液，200rpm反应5min后，加入15mL 95％乙醇终止反应。反应结束后加入2滴酚酞溶液，用0.05mol/L NaOH标准溶液滴定。在一定的反应条件下，每分钟催化底物水解生成1μmol脂肪酸所需要的酶量定义为1个酶活力单位，用U表示。
有机溶剂对酶稳定性的影响：加入甲醇、乙醇、异丙醇和丙酮，使有机溶剂终浓度为50％(v/v)，置于37℃孵育2h，在最适反应条件下以橄榄油聚乙烯醇乳化液为底物测定酶活力，所有实验重复三次。有机溶剂对MAS1及其突变体酶活力的影响用相对酶活力表示，以未加入有机溶剂蛋白样品测定的酶活力定为100％。。首先利用乙醇对MAS1及其突变体进行筛选，结果如图1所示，其中R191A(氨基酸序列为SEQ ID NO：2)突变体耐受乙醇能力较好，仍残留75％的活力，野生型的仅为25％的活力。利用不同溶剂对R191A进行酶活力测定，结果如图2所示，R191A突变体对其他有机溶剂也表现出良好的耐受性能。
实施例4酶蛋白在毕赤酵母中的表达制备
将MAS1-R119A基因(核苷酸序列SEQ ID NO：3)克隆到毕赤酵母表达载体pPICZαA载体中，获得pPICZαA-MAS1-R191A表达载体。测序正确后，将载体进行线性化并电转化到毕赤酵母X-33菌种中，利用高浓度Zeocin抗生素进行筛选，获得阳性重组表达菌种，并通过少量验证表达。
在发酵罐中进行培养发酵重组菌，诱导的表达条件设定为：诱导温度24℃和pH值6.0，并诱导培养8天，发酵的酶活单位超过5000U/mL。发酵过程结束后，离心获得发酵液上清，通过30KDa的膜包浓缩，获得酶制剂。
实施例5风味酯合成中的应用
以己酸与乙醇为底物的酯化反应体系来考察MAS1脂肪酶及MAS1-突变体合成风味酯己酸乙酯的能力。具体反应体系如下：己酸：乙醇摩尔比为1:2，加酶量为60U/mL(反应体系体积)，初始加水量为0.5％，45度反应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，有机溶剂耐受性增强有效提升合成风味内酯能力，MAS1-突变体24小时后合成己酸乙酯的转化率为96％，而野生型的为67％。
以上所述的实施例仅是对本发明的优选方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案做出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。
序列表
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<120> 一种有机溶剂耐受的脂肪酶突变体及其应用
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Claims (5)
1.一种有机溶剂耐受的脂肪酶突变体，其特征在于，所述有机溶剂耐受的脂肪酶突变体的氨基酸序列为SEQ ID NO.2。
2.一种重组质粒，其特征在于，其用于在宿主细胞内表达权利要求1所述的有机溶剂耐受的脂肪酶突变体。
3.一种工程菌株，其特征在于，所述工程菌含有权利要求2所述的重组质粒。
4.根据权利要求3所述的工程菌株，其特征在于，所述工程菌株为巴斯德毕赤酵母Pichia pastoris。
5.权利要求1所述的有机溶剂耐受的脂肪酶突变体、权利要求2所述的重组质粒、权利要求3或4所述的工程菌株在风味酯物质及生物柴油合成中的应用。
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