CN110540980B - 一种海洋链霉菌脂肪酶突变体及其应用 - Google Patents
一种海洋链霉菌脂肪酶突变体及其应用 Download PDFInfo
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- CN110540980B CN110540980B CN201910844698.8A CN201910844698A CN110540980B CN 110540980 B CN110540980 B CN 110540980B CN 201910844698 A CN201910844698 A CN 201910844698A CN 110540980 B CN110540980 B CN 110540980B
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- lipase
- mutant
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Abstract
本发明公开了一种海洋链霉菌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,筛选获得偏好偏甘油酯底物的脂肪酶突变体,并构建得到高效表达该突变体的毕赤酵母工程菌,从而为该突变体的广泛应用奠定基础。本发明提供的海洋链霉菌脂肪酶突变体,与野生型的脂肪酶对比,其更高效催化合成富含n‑3脂肪酸的偏甘油酯,在最优的反应条件下,催化产物中偏甘油酯的含量为86.48%(其中49.01%PUFA‑DAG,37.47%PUFA‑MAG),相对于野生型(PUFA‑DAG 3.8%)提高了约23倍。
Description
技术领域
本发明涉及基因工程及酶工程技术领域,特别是涉及一种海洋链霉菌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。它的催化活性仅仅取决于它的蛋白质结构(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。
酶学特性和酶的稳定性是影响其应用的关键因素之一;同时,很多酶学性质较好的蛋白在实际应用条件下,实际使用效果也不好。
寻找和克隆合适脂肪酶的途径有2个:一是筛选产新型酶的微生物;二是对酶进行适当的蛋白质工程改造。微生物菌种选育是工业生产和学术研究最常用和最简单的手段之一;但是,其缺点是工作量大,随机性强;因此往往很难筛选到理想菌株。而蛋白质工程是这些年新兴的、借用分子生物学和生物信息学的方法对蛋白进行定向突变和理性改造,而获得理想蛋白或酶的方法,其优点是工作量相对较小和概率大;缺点是多数突变体蛋白酶学性质没有改变或者变得更差。因此,获得更好效果的脂肪酶突变体一直是本领域的研究热点。
发明内容
本发明的目的是提供一种海洋链霉菌脂肪酶突变体及其应用,以解决上述现有技术存在的问题,本发明基于酶蛋白结构分析,设计及构建酶突变库,筛选获得偏好偏甘油酯底物的脂肪酶突变体,并构建得到高效表达该突变体的毕赤酵母工程菌,从而为该突变体的广泛应用奠定基础。
为实现上述目的,本发明提供了如下方案:
本发明提供一种海洋链霉菌脂肪酶突变体,所述海洋链霉菌脂肪酶突变体是氨基酸序列为SEQ ID NO.1的脂肪酶的第40位氨基酸由Gly变为Phe。
上述海洋链霉菌脂肪酶突变体的氨基酸序列为SEQ ID NO.2。
上述海洋链霉菌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的海洋链霉菌脂肪酶突变体的重组质粒。
进一步地,所述重组质粒含有核酸序列为SEQ ID NO.3的核酸。
本发明还提供一种工程菌株,所述工程菌株含有在宿主细胞内表达氨基酸序列为SEQ ID NO.2的海洋链霉菌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海洋链霉菌突变体、上述的重组质粒、上述的工程菌株在制备富含n-3PUFA甘油二酯中的应用。
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,筛选获得偏好偏甘油酯底物的脂肪酶突变体,并构建得到高效表达该突变体的毕赤酵母工程菌,从而为该突变体的广泛应用奠定基础。
本发明提供的海洋链霉菌脂肪酶突变体,与野生型的脂肪酶对比,其更高效催化合成富含n-3脂肪酸的偏甘油酯,在最优的反应条件下,催化产物中偏甘油酯的含量为86.48%(其中49.01%PUFA-DAG,37.47%PUFA-MAG),相对于野生型(PUFA-DAG 3.8%)提高了约23倍。
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2中MAS1及其突变体对不同种类三油酸甘油酯的水解特性比较。
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。
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海洋链霉菌脂肪酶突变体的构建及表达
对MAS1脂肪酶(氨基酸序列为SEQ ID NO:1)结构分析发现,除催化三联体(S109-H232-D200)外,其催化口袋由T38,F39,G49,H108,T141,V202,V203,T237等氨基酸组成。为了增强口袋对TAG底物的位阻效应,改变脂肪酶底物选择性特性,设计了单突变体T38R/F,G40E/F,H108W,T141F/R,V202F,V203L/F,V233F/R,T237Y及双突变H108W-T38R/F,H108W-G40F,H108W-T141F/R,H108W-V202L/F,H108WV233R/F,H108W-T237Y等系列的酶突变体。以上突变体的基因克隆于pET22b载体中,并转化于BL21(DE3)中表达。
实施例2海洋链霉菌脂肪酶突变体的筛选
利用大肠杆菌表达系统制备了MAS1脂肪酶突变体,并以乳化橄榄油(TAG:98%、DAG:2%)和花王甘油二酯(TAG:65%、DAG:32%、MAG:2%)为底物,测定裂解液酶活力的差异。结果如图1所示,从图1可以看出,位点T38和T141突变后导致MAS1脂肪酶活力丧失,而V202和V203位的突变也使酶水解活力大幅下降。只有单突变G40E/F,H108W,V233F,T237Y和双突变H108W-G40E/F,H108W-V233F,H108W-T237Y对酶的活力没有明显的影响。其中G40F突变体对DAG底物的水解活性高于甘油三酯底物,提示G40F位点的突变可能导致了MAS1脂肪酶的底物选择性发生改变。
G40F单点突变体的氨基酸序列为SEQ ID NO:2,参照该序列合成一个编码核苷酸序列SEQ ID NO:3。
结构分析发现,野生型MAS1脂肪酶拥有一个长凹槽型的催化口袋,而将Gly40位点突变成苯丙氨酸(Phe)后,底物结合位点则由于引入大侧链的苯丙氨酸而被截断,使其催化口袋变短。这将导致TAG中的一条脂肪酸链无法稳定地与蛋白结合,从而使得突变体G40F对TAG的结合能力下降,水解活性下降,但突变DAG的结合影响较小,因此不影响酶对DAG水解能力。
实施例3海洋链霉菌脂肪酶突变体的表达制备
(1)毕赤酵母表达工程菌的构建:将MAS1-G40F基因克隆到毕赤酵母表达载体pPICZαA载体中,筛选并测序验证基因正确。pPICZαA-MAS1-G40F载体进行线性化,并电转化到毕赤酵母X-33菌种中,利用博来霉素进行筛选,获得阳性重组表达菌种。
(2)一级种子液的制备:将上述得到的单菌落接种到已灭菌的含有50mL YPD液体培养基的锥形瓶中,再将锥形瓶置于温度为30℃和转速为200rpm的摇床中振荡培养18-24h,即得一级种子液;
(4)二级种子液的制备:将5mL一级种子液接入到已灭菌的100mL新鲜YPD液体培养基中,于转速为200rpm的30℃摇床中振荡培养24h;
(5)发酵罐发酵:将二级种子液按照10%接种量接到发酵罐培养基中,酵母生长阶段,即未开始诱导阶段,其发酵条件为30℃,pH 5.0,600rpm;当菌体湿重达到180g/L左右时,葡萄糖消耗完,开始补加甲醇进行诱导表达,在温度为24℃和pH值为6.0的条件下,诱导培养6天,在整个发酵过程中的溶氧量均控制在20%和60%之间;
(6)脂肪酶MAS1发酵液制备:诱导结束后,将发酵液置于温度为4℃和转速为12000rpm的离心机中离心15min,用0.45mm的滤膜过滤所得的上清液,再用膜包浓缩,浓缩完后,分别利用橄榄油乳化法和考马斯亮蓝(Bradford)法蛋白浓度试剂盒测定发酵液的水解酶活和蛋白浓度,并将其保存于4℃冰箱中备用。
实施例4海洋链霉菌脂肪酶突变体固定化酶的制备
浓缩后的MAS1-G40F脂肪酶发酵粗酶液与二乙烯基苯/丙烯酸酯大孔吸附树脂ECR1030按酶与载体比例为60mg/g在锥形瓶中混合后,加入与酶液等体积的100mM pH 8.0的Tris-HCl缓冲液。随后将锥形瓶置于恒温(30℃)气浴摇床,在转速200rpm的条件下震荡吸附8h。固定化酶通过布氏漏斗过滤回收,随后将回收得到的固定化酶置于30℃真空恒温干燥箱中干燥8h。按此方法多次制备得到固定化MAS1-G40F,其酯化活力为2856U/g。
实施例5含n-3脂肪酸的偏甘油酯制备
将固定化MAS1-G40F应用于催化甘油与富含n-3PUFA的脂肪酸(n-3PUFA含量为90.37%)反应制备PUFA-DAG。分别研究了底物摩尔比(底物总质量20g,甘油与富含n-3PUFA的脂肪酸的摩尔比分别为4:1、2:1、1:1、1:2、1:3、1:4)、固定化MAS1-G40F脂肪酶添加量(100、200、300、400、500U/g底物)、反应温度(30、35、40、45、50℃)和反应真空度(200Pa、2000Pa、常压)对富含n-3PUFA的脂肪酸的转化率及PUFA-DAG生成量的影响。在优化的反应条件下(甘油与富含n-3PUFA的脂肪酸的摩尔比为1:2,反应温度为40℃,加酶量为400U/g底物,真空度200Pa,反应时间48h),富含n-3PUFA的脂肪酸的转化率达到90.22%。此时,产物中含有49.01%PUFA-DAG,37.47%PUFA-MAG,3.74%PUFA-TAG。
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
<110> 华南理工大学
<120> 一种海洋链霉菌脂肪酶突变体及其应用
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tga 783
Claims (6)
1.一种海洋链霉菌(Streptomyces marinensis)脂肪酶突变体,其特征在于,所述海洋链霉菌脂肪酶突变体是氨基酸序列为SEQ ID NO.1的脂肪酶的第40位氨基酸由Gly变为Phe。
2.权利要求1所述的海洋链霉菌脂肪酶突变体的编码基因,其特征在于,该编码基因的核酸序列为SEQ ID NO.3。
3.一种重组质粒,其特征在于,所述重组质粒含有权利要求2所述的海洋链霉菌脂肪酶突变体的编码基因。
4.一种工程菌株,其特征在于,所述工程菌株含有权利要求3所述的重组质粒。
5.根据权利要求4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)。
6.权利要求1所述的海洋链霉菌突变体、权利要求2所述的海洋链霉菌脂肪酶突变体的编码基因、权利要求3所述的重组质粒、权利要求4或5所述的工程菌株在制备含n-3PUFA甘油二酯中的应用。
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