CN114854714A - 一种菜豆源环氧化物酶突变体、基因、载体、工程菌及制备方法和应用 - Google Patents
一种菜豆源环氧化物酶突变体、基因、载体、工程菌及制备方法和应用 Download PDFInfo
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- CN114854714A CN114854714A CN202210592666.5A CN202210592666A CN114854714A CN 114854714 A CN114854714 A CN 114854714A CN 202210592666 A CN202210592666 A CN 202210592666A CN 114854714 A CN114854714 A CN 114854714A
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Abstract
本发明涉及分子生物学技术领域,具体涉及一种菜豆源环氧化物酶突变体、基因、载体、工程菌及制备方法和应用,所述环氧化物酶突变体的氨基酸序列如SEQ ID NO.1所示。本发明涉及的突变体是在SEQ ID NO.3所示序列的基础上,将其将第134位上的谷氨酸和第137位的苏氨酸分别突变为赖氨酸与脯氨酸。以工程菌全细胞催化特性作为参考:与表达野生酶的E.coli/pveh3相比,E.coli/pveh3E134K/T137P全细胞的比活力和对映选择性E分别提高了2.7倍和51.1%。
Description
技术领域
本发明涉及分子生物学技术领域,尤其涉及一种菜豆源环氧化物酶突变体、基因、载体、工程菌及制备方法和应用。
背景技术
环氧化物酶(epoxide hydrolase,EC 3.3.2.-)广泛存在于哺乳动物、植物、昆虫和各种微生物细胞内。其中,哺乳动物体内的环氧化物酶不仅具有调节信号通路和代谢途径的作用,还与多种癌症发病机制有关。该酶能够催化外消旋环氧化物的选择性开环,通过加成一个水分子生成相应的手性邻位二醇或选择性保留手性环氧化物。随着绿色化学概念的推广,环氧水解酶作为一种高效且对环境低负担的催化剂也越来越在精细化工领域中受到重视。
手性缩水甘油醚类化合物在医药行业有着巨大的应用前景。其中,芳基缩水甘油醚可被用于合成治疗焦虑症、肥胖症和青光眼等疾病的药物,并且还是生产多种β-阻断剂的中间体。特别是(R)-邻甲苯缩水甘油醚(o-tolyl glycidyl ether,oTGE)是化合物benzazepinone及其衍生物的手性合成砌块,该化合物已作为一种β-肾上腺素受体拮抗剂用于临床上治疗高血压与心绞痛等病症(Bioorg Med Chem,2003 11:1353-1361)。
从菜豆(Phaseolus vulgaris)中鉴定出一种环氧化物酶(命名为PvEH3),但该酶对于芳基缩水甘油醚类的对映选择性及催化活性均不能满足手性拆分反应的需求,其缺陷则主要表现在:一是较低的对映选择性所导致的目标环氧化物对映体较高的消耗比;二是较低的催化活性所造成的反应体系较低的底物起始浓度。以上两个问题均严重的影响拆分反应终产物的光学纯度及产率。
发明内容
有鉴于此,本发明的目的在于提出一种菜豆源环氧化物酶突变体、基因、载体、工程菌及制备方法和应用,以解决较低的催化活性所造成的反应体系较低的底物起始浓度与较低的对映选择性的问题。
基于上述目的,本发明提供了一种菜豆源环氧化物酶突变体,所述环氧化物酶突变体的氨基酸序列如SEQ ID NO.1所示。
本发明中对于突变体的命名方式是:采用“原始氨基酸单字母缩写+位置+替换氨基酸单字母缩写”的准则来表示全文涉及到的所有突变体。
将如SEQ ID NO.3所示氨基酸序列的第134位谷氨酸突变为赖氨酸,将第137位的苏氨酸突变为脯氨酸,因此命名为E134K/T137P。
本发明还提供一种编码所述菜豆源环氧化物酶突变体的基因。
所述基因的核苷酸序列如SEQ ID NO.2所示。
本发明还提供一种含所述菜豆源环氧化物酶突变体基因的表达载体和工程菌。
所述工程菌是以真菌或细菌为宿主构建表达得到的。所述细菌包括大肠杆菌和芽孢杆菌。所述真菌包括酵母菌。
本发明还提供表达所述菜豆源环氧化物酶突变体的工程菌的制备方法,包括如下步骤:
基于环氧化物酶野生酶PvEH3的基因pveh3(如SEQ ID NO.9所示),设计并合成特异性引物,以携带pveh3的重组质粒为模板,利用两步全质粒PCR向pveh3的特定位点引入突变,获得携带编码突变体的基因的重组质粒;转化表达宿主的感受态细胞,培养宿主/工程菌菌株,诱导其表达环氧化物酶突变体E134K/T137P。
优选的,所使用的表达宿主为E.coli BL21(DE3),所述携带编码环氧化物酶野生酶基因pveh3的重组质粒为pET-28a(+)-pveh3,对应的表达宿主为E.coli/pveh3;所述携带编码单位点突变酶基因pveh3E134K的重组质粒为pET-28a(+)-pveh3E134K,对应的表达宿主为E.coli/pveh3E134K。
本发明还提供所述突变体、编码所述菜豆源环氧化物酶突变体的基因、含所述基因的表达载体、含所述基因的工程菌在手性生物催化中的应用。
所述应用是所述环氧化物酶突变体和/或表达环氧化物酶突变体的工程菌为催化剂,在磷酸盐缓冲液中催化外消旋邻甲苯缩水甘油醚。实现环氧化物酶突变体E134K/T137P拆分rac-oTGE。
本发明的有益效果:
(a)以rac-oTGE为底物,纯酶/蛋白质方面,E134K/T137P的比活力为14.5U/mg,是原酶PvEH3(8.41U/mg)的1.7倍。
(b)以rac-oTGE为底物,基因工程菌全细胞方面:E.coli/pveh3E134K/T137P的全细胞的环氧化物酶活性为22.4U/g湿细胞重量(wet cell weight,wcw)时,是E.coli/pveh3全细胞活力(6.03U/g wcw)的3.7倍;在20℃下,E.coli/pveh3E134K/T137P的对映选择率(enantioselective ratio,E)达20.1,而E.coli/pveh3的为13.3;利用E.coli/pveh3E134K /T137P动力学拆分rac-oTGE的最大底物浓度为800mM,(R)-oTGE的产率和时空产率分别为38.5%和7.22g/L/h。
附图说明
为了更清楚地说明本发明或现有技术中的技术方案,下面将对实施例或现有技术描述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,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为本发明单取代缩水甘油醚类底物及产物的缩写与结构式;
图2为本发明E.coli突变转化子菌液PCR验证结果示意图;其中,泳道M:DNAmarker(250bp);泳道1~2:E.coli/pveh3E134K;泳道3~5:E.coli/pveh3E134K/T137P;
图3为本发明表达重组工程菌的SDS-PAGE分析结果示意图;其中,泳道M:蛋白低分子量Marker;泳道1:E.coli/pveh3E134K/T137P全细胞裂解液;泳道2:E.coli/pveh3E134K/T137P破碎上清;泳道3:纯化后的E134K/T137P。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白,以下结合具体实施例,对本发明进一步详细说明。
需要说明的是,除非另外定义,本发明使用的技术术语或者科学术语应当为本发明所属领域内具有一般技能的人士所理解的通常意义。包括”或者“包含”等类似的词语意指出现该词前面的元件或者物件涵盖出现在该词后面列举的元件或者物件及其等同,而不排除其他元件或者物件。
(一)培养基
LB液体培养基(g/L):蛋白胨10,酵母提取物5,NaCl 10。
LB固体培养基(g/L):蛋白胨10,酵母提取物5,NaCl 10,琼脂粉20。
(二)环氧化物酶比活力的测定
取200μL全细胞菌悬液或纯化后的酶液与275μL磷酸钾缓冲液于20℃条件下预热5min,分别加入25μL底物(终浓度为10mM)开始反应,精确反应10min后加入1mL甲醇终止反应,混匀离心后取上清液过0.22μm有机滤膜,样品采用高效液相色谱仪、紫外检测器和色谱柱C18对样品进行分析,测定全细胞催化芳基缩水甘油醚类底物(图1)水解的比活力。分析条件:柱温为35℃,流动相为甲醇/水(70/30,v/v),流速为0.8mL/min,检测波长为220nm。
在此反应条件下,以每分钟消耗1μmol底物所需的湿细胞重量定义为一个酶活力单位U。
全细胞比活力(U/g wcw)=C×v×c/(t×m1),其中C为初始底物浓度,v为反应体积,c为转化率,t为反应时间,m1即为反应体系中湿菌体的质量。
纯化的酶的比活力(U/mg)=C×v×c/(t×m2),式中m2为反应体系中酶的质量。
(三)全细胞对映选择性的测定
用400μL全细胞菌悬液与550μL磷酸钾缓冲液于20℃条件下预热5min,分别加入50μL 1a~5a(终浓度为10mM)反应,定时取样50μL于1mL乙酸乙酯中进行萃取,震荡混匀后离心取上层有机相经无水硫酸镁干燥后过0.22μm有机滤膜。采用高效液相色谱仪、紫外检测器和手性色谱柱OD-H对样品进行分析。分析条件:柱温为30℃,流动相为正己烷/异丙醇,流速为0.8mL/min,检测波长为220nm,底物和产物的出峰时间如表1所示。
表1环氧化物(1a~5a)及其邻位二醇(1b~5b)的检测条件
依据底物和产物的检测峰面积计算反应进程中环氧化物底物的ee和E值等参数。对映体过剩率:ee=(R-S)/(R+S)×100%;对映体选择率:E=ln(1-c)×(1-ee)/ln(1-c)×(1+ee)]。其中,R和S分别表示底物(R)-和(S)-对映体的浓度。底物转化率c可由公式c=Ap×εepoxide/diol/(As+Ap×εepoxide/diol)计算,其中,Ap为产物邻位二醇的峰面积之和,As为底物峰面积之和,εepoxide/diol为摩尔消光系数,以反应进程中两个构型产物峰面积和为横坐标,两个构型底物峰面积和为纵坐标进行线性拟合,斜率即底物在此特定检测波长下的摩尔消光系数εepoxide/diol,本发明所涉及的与单取代芳基缩水甘油醚类环氧化物相关的摩尔消光系数如表1所示。
实施例1
携带野生酶基因pveh3的表达质粒与工程菌的构建
(1)菜豆总RNA的提取
选取饱满的菜豆种子置于培养皿中,加入少许去离子水覆盖培养皿底层,于30℃培养箱中孵育20h,按照Trizol法提取菜豆总RNA。取适量菜豆胚芽于匀浆器中,加入预冷的Trizol溶液迅速研磨20s。静置15min后加入200μL氯仿,剧烈震荡15s后静置,在12000r/min,4℃条件下离心10min。吸取上层水相溶液与等体积的异丙醇混匀,静置20min后同样条件下离心10min,弃上清液。加入1mL 75%乙醇洗涤沉淀,离心3min弃上清,室温干燥5min。加入50μL RNase-Free双蒸水充分溶解即可得到菜豆总RNA。
(2)cDNA的获得
以菜豆总RNA为模板,在逆转录酶AMV Reverse Transcription(Takara公司)的作用下采用逆转录PCR技术逆转录成cDNA。PCR体系及条件如表2所示。
表2逆转录PCR反应体系及条件
(3)野生酶基因pveh3的扩增
以逆转录得到的cDNA为模板进行巢式PCR扩增,PCR体系及条件如表3所示。第一轮PCR条件为:预变性94℃3min;94℃30s,50℃30s,72℃70s,变性-退火-延伸共30个循环;72℃充分延伸10min。第二轮PCR扩增条件除将退火温度调整为55℃外其余均不变。PCR产物经琼脂糖凝胶鉴定后对第二轮产物进行割胶回收。
表3巢式PCR反应体系及条件
表中涉及到的特异性引物Pv3-F(SEQ ID NO.10)和Pv3-R(SEQ ID NO.11):
Pv3-F:5′-CATATGATGGAGGGAATACAGCACAAAG-3′
Pv3-R:5′-CTCGAGTCAAAACTTGTTGATAAAATCATAAATGT-3′
(4)重组质粒pUCm-T-pveh3的构建
根据表4中的体系将割胶回收产物与pUCm-T(上海生工)于17℃条件下连接6h。在E.coli JM109感受态中加入5μL连接液冰浴30min,42℃条件下热激90s后继续冰浴10min,加入400μL无抗LB液体培养基并在37℃摇床中培养1h,4000r/min离心5min弃掉400μL上清后将剩余菌液均匀涂布于含100μg/mL Amp的LB固体培养基上进行蓝白斑筛选(提前涂布20μL 100mM的IPTG和100μL 200mg/mL的X-gal),37℃培养箱中倒置培养过夜。通过蓝白斑常规筛选及序列测序验证获得重组质粒pUCm-T-pveh3及相应的E.coli宿主菌。
表4连接体系
(5)重组质粒pET-28a-pveh3的构建
按照表5中所示体系用Nde I和Xho I双酶切质粒pET-28a(+)和pUCm-T-pveh3,琼脂糖凝胶鉴定后割胶回收目的片段并按照表中连接体系进行连接。连接液转化E.coliBL21(DE3)感受态细胞,将剩余菌液涂布于含100μg/mL硫酸卡那霉素的LB固体培养基上。挑取单菌落进行菌液PCR验证与基因测序,测序正确的工程菌命名为E.coli/pveh3。
表5双酶切体系
实施例2
携带突变酶基因pveh3E134K/T137P的表达质粒与工程菌的构建
(1)单位点突变质粒及工程菌的构建
取过夜培养的E.coli/pveh3菌液(由实施例1制备得到)置于1.5mL的离心管中,8000×g离心2min收集菌体沉淀,并用质粒提取试剂盒Pure Plasmid Mini Kit(康为世纪生物科技有限公司)提取pET-28a(+)-pveh3质粒,-20℃保存。
采用全质粒两步PCR技术扩增突变体基因,即以pET-28a(+)-pveh3重组质粒为模板,以E134K-F(见SEQ ID NO.4)和pET-28a(+)-R(见SEQ ID NO.5)为上、下游引物,在DNA聚合酶PrimeSTAR HS DNA polymerase的作用下进行第一轮PCR扩增,反应体系及条件如表6所示。
表6第一轮PCR扩增的反应体系及条件
第二轮PCR扩增与第一轮一样均是以pET-28a-pveh3重组质粒为模板,但第二轮PCR扩增引物为第一轮PCR产物,反应体系及PCR条件如下表7所示。
表7第二轮PCR扩增的反应体系及条件
使用限制性内切酶Dpn I识别并切割二轮PCR反应体系内的从E.coli/pveh3中提取的模板质粒pET-28a(+)-pveh3,消化体系及条件如表8所示。
表8消化体系及条件
吸取5μL消化液于已在4℃环境下保存30min以上的E.coli BL21(DE3)感受态细胞中轻轻混匀,进行细胞转化操作,并在含硫酸卡那霉素(Kan)抗性的LB固体培养基上筛选阳性克隆。挑取突变体单菌落接种于2mL LB抗性液体培养基中,37℃培养4h,以引物T7-F(SEQID NO.6)和T7-R(SEQ ID NO.7)为上下游引物按照表9中的反应体系及条件进行菌液PCR扩增,扩增产物用1%琼脂糖凝胶进行鉴定(图2,泳道1~2)。将鉴定结果与测序结果均正确的重组菌株命名为E.coli/pveh3E134K。
表9菌液PCR扩增体系及条件
(2)双位点突变质粒及工程菌的构建
以突变体PvEH3E134K的重组质粒pET-28a(+)-pveh3E134K为模板,以T137P-F(SEQ IDNO.8)和pET-28a(+)-R为引物参照实施例2(1)所述方法采用全质粒两步PCR技术构建双位点突变体基因pveh3E134K/T137P、pET-28a(+)-pveh3E134K/T137P以及重组工程菌E.coli/pveh3E134K/T137P。其菌液PCR鉴定结果如附图2泳道3~5。
E134K/T137P重组蛋白在E.coli中的诱导表达及纯化
将甘油管中的E.coli/pveh3E134K/T137P在LB固体平板(含100μg/mL的Kan)上划线并在37℃培养箱中倒置培养12~14h。挑取单菌落于2mL LB液体抗性培养基中,37℃,220r/min培养过夜。按2%的接种量(2mL)接种于100mL含相同Kan浓度的液体培养基中,37℃,220r/min培养至OD600达到0.6~0.8(约2.5h),添加IPTG至终浓度为0.05mM,20℃条件下诱导8h。8000r/min离心5min收集菌体,用pH 7.0,100mM的磷酸钾缓冲液将其重悬制备成50mg/mL湿细胞的菌悬液进行超声破碎。0℃条件下11000r/min离心15min,将破碎上清即粗酶液上样至经结合液预处理的镍NTA亲核层析柱(Ni-NTA)分离纯化。将含有目的蛋白的洗脱液上样至Sephadex G-25凝胶层析柱除盐,从而获得E134K/T137P的纯酶液,其SDS-PAGE结果如附图3所示。以rac-oTGE为底物,测得E134K/T137P的比活力为14.5U/mg,是原酶PvEH3(8.41U/mg)的1.7倍。
E134K/T137P对于单取代芳基缩水甘油醚类催化特性分析
按照上文所述方法测定E.coli/pveh3E134K/T137P对于五种单取代芳基环氧化物的全细胞环氧化物酶比活性与对映选择性。与表达野生酶的E.coli/pveh3相比,除5a之外,E.coli/pveh3E134K/T137P全细胞的比活力均有不同程度的提高,其提高倍数为0.2~2.7倍;E.coli/pveh3E134K/T137P全细胞催化3a(即rac-oTGE)的对映选择性E较E.coli/pveh3提高了51.1%。
表10 E.coli/pveh3E134K/T137P的催化性质分析
表10的括号中为E.coli/pveh3的数据。
底物rac-oTGE的初始浓度对于拆分反应的影响
按照表11中反应体系测定E.coli/pveh3E134K/T137P全细胞拆分rac-oTGE的最大底物浓度反应限。在20℃反应条件下,取一定量的全细胞菌悬液与磷酸钾缓冲液混匀后预热5min,加入终浓度分别为50~1000mM的rac-oTGE开始反应,定时取样按照本发明前文所述方法对样品进行处理分析,HPLC监测反应进程直至(S)-oTGE完全被水解,计算c和ee等参数。时空产率(space-time-yield,STY)这里指在一定催化反应条件下,单位时间单位体积所获得的目的产物的总量。STY(g/h/L)=Mw×n×Y/(t×v),其中Mw为底物rac-oTGE的相对分子质量,n为底物的物质的量,Y为目的产物(R)-oTGE的得率,t为反应时间,v为反应体系总体积。
表11底物rac-oTGE的初始浓度对于E.coli/pveh3E134K/T137P拆分反应的影响
在最适水解温度(20℃)和pH(7.0)条件下测定E.coli/pveh3E134K/T137P水解动力学拆分rac-oTGE的最大底物浓度。如表12所示,当底物浓度为50~800mM时,E.coli/pveh3E134K/T137P能在2~7h内催化(S)-oTGE水解完全使(R)-oTGE的ee值均大于99%,此时(R)-oTGE的得率为38.5~41.3%。但当底物浓度提高至1000mM时,反应进行14h后(R)-oTGE的ee值仅为74.8%,说明在此反应体系下E.coli/pveh3E134K/T137P不能将(S)-oTGE的完全水解。
表12 E.coli/pveh3E134K/T137P水解动力学拆分不同浓度底物的相关参数
利用E.coli/pveh3E134K/T137P制备(R)-oTGE
以E.coli/pveh3E134K/T137P湿菌体为全细胞催化剂水解动力学拆分800mM rac-oTGE制备(R)-oTGE,反应进行至8h时ee为99.5%,利用乙酸乙酯反复萃取后旋转蒸发得到(R)-oTGE与对应邻位二醇3b的混合物(浅黄色油状物质),将混合物与硅胶混匀吹干后干法上样硅胶柱,利用石油醚/乙酸乙酯为5:1的展开剂进行薄层层析检测流分,合并带有(R)-oTGE流分并再次旋蒸,最后得到0.629g(R)-oTGE(ee>99.5%),其终产率为24.0%。
所属领域的普通技术人员应当理解:以上任何实施例的讨论仅为示例性的,并非旨在暗示本发明的范围(包括权利要求)被限于这些例子;在本发明的思路下,以上实施例或者不同实施例中的技术特征之间也可以进行组合,步骤可以以任意顺序实现,并存在如上所述的本发明的不同方面的许多其它变化,为了简明它们没有在细节中提供。
本发明旨在涵盖落入所附权利要求的宽泛范围之内的所有这样的替换、修改和变型。因此,凡在本发明的精神和原则之内,所做的任何省略、修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
<110> 安徽工程大学
<120> 一种菜豆源环氧化物酶突变体、基因、载体、工程菌及制备方法和应用
<141> 2022-05-27
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 318
<212> PRT
<213> 菜豆(Phaseolus vulgaris)
<400> 1
Met Glu Gly Ile Gln His Lys Glu Val Glu Val Asn Gly Ile Lys Met
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His Val Ala Glu Lys Gly Glu Gly Pro Val Val Leu Phe Leu His Gly
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Thr Glu Ala Pro Ala Ser Met Ser Ser Tyr Ser Cys Phe Asp Ile Val
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Gly Asp Leu Val Ala Leu Ile Asp Leu Leu Gly Val Asp Gln Val Phe
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Leu Val Ala His Asp Trp Gly Ala Ile Ile Gly Trp Tyr Leu Cys Met
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Phe Arg Pro Asp Arg Val Lys Ala Tyr Val Cys Leu Ser Val Pro Leu
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Leu His Arg Asn Pro Lys Ile Arg Pro Val Asp Ala Met Arg Ala Met
130 135 140
Tyr Gly Asp Asp Tyr Tyr Ile Cys Arg Phe Gln Lys Pro Gly Glu Met
145 150 155 160
Glu Ala Gln Met Ala Glu Val Gly Thr Gly Tyr Val Leu Lys Asn Ile
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Leu Thr Thr Arg Lys Pro Gly Pro Pro Ile Phe Pro Lys Gly Glu Tyr
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Gly Thr Gly Phe Asn Pro Asp Met Thr Asn Ser Leu Pro Ser Trp Leu
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Ser Gln His Asp Leu Ala Tyr Tyr Val Ser Lys Phe Gln Lys Thr Gly
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Phe Thr Gly Pro Leu Asn Tyr Tyr Arg Asn Met Asn Pro Asn Trp Glu
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Leu Thr Ala Pro Trp Ser Gly Ala Lys Ile Lys Val Pro Val Lys Phe
245 250 255
Ile Thr Gly Asp Leu Asp Met Val Tyr Thr Ser Leu Asn Met Lys Glu
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Tyr Ile His Gly Gly Gly Phe Lys Glu Asp Val Pro Asn Leu Glu Glu
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Val Ile Val Gln Lys Gly Val Ala His Phe Asn Asn Gln Glu Ala Ala
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Glu Glu Ile Asn Thr His Ile Tyr Asp Phe Ile Asn Lys Phe
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<210> 2
<211> 957
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<213> 菜豆(Phaseolus vulgaris)
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atggagggaa tacagcacaa agaagtggaa gtaaatggca tcaaaatgca tgttgcagag 60
aaaggagagg gtcctgtggt cttgttcctc catggcttcc ctgaactgtg gtattcctgg 120
cgccaccaga ttctggctct cagttcccga ggatatcgcg ctgttgcacc agatctacgt 180
ggctacggtg acacagaggc accagcttca atgagcagct acagctgctt tgacatagtg 240
ggtgatctgg ttgcgcttat agaccttctg ggtgttgatc aagtcttcct tgtggctcat 300
gactggggtg ccatcatagg ttggtacctc tgcatgtttc gccccgacag agtcaaggcc 360
tatgtctgcc tcagtgtgcc tttactccac cgaaacccca agatcagacc agtcgatgcc 420
atgcgtgcta tgtacggaga cgactactac atctgcagat ttcagaaacc aggagaaatg 480
gaagctcaga tggctgaagt tgggactggg tatgtgctca aaaacatcct cacaactcgc 540
aaacctggtc ctccaatctt tcccaaggga gagtacggaa ctggattcaa cccagatatg 600
actaattcct taccctcttg gctctcacaa catgatcttg cttattatgt ttccaaattt 660
cagaaaacgg gcttcactgg acccttgaac tattacagaa atatgaaccc aaattgggag 720
ctgacagcac cgtggagtgg agcgaaaata aaagtgccgg taaagttcat cacaggtgat 780
ttggacatgg tatacacctc actgaacatg aaggagtaca tccatggtgg aggtttcaaa 840
gaagatgtgc caaatctaga agaagtgatt gtgcagaaag gagtagctca cttcaataat 900
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<210> 3
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<212> PRT
<213> 菜豆(Phaseolus vulgaris)
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Met Glu Gly Ile Gln His Lys Glu Val Glu Val Asn Gly Ile Lys Met
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Claims (9)
1.一种菜豆源环氧化物酶突变体,其特征在于,所述环氧化物酶突变体的氨基酸序列如SEQ ID NO.1所示。
2.一种编码权利要求1所述菜豆源环氧化物酶突变体的基因。
3.根据权利要求2所述基因,其特征在于,所述基因的核苷酸序列如SEQ ID NO.2所示。
4.一种含权利要求2或3基因的表达载体。
5.一种含权利要求2或3所述基因的工程菌。
6.根据权利要求5所述基因的工程菌,其特征在于,所述工程菌是以真菌或细菌为宿主构建表达得到的。
7.表达权利要求1所述菜豆源环氧化物酶突变体的工程菌的制备方法,其特征在于,所述制备方法包括如下步骤:
基于环氧化物酶野生酶的基因pveh3,设计并合成特异性引物,以携带pveh3的重组质粒为模板,利用两步全质粒PCR向pveh3的特定位点引入突变,获得携带编码突变体的基因的重组质粒;转化表达宿主的感受态细胞,培养宿主/工程菌菌株,诱导其表达环氧化物酶突变体E134K/T137P。
8.根据权利要求1所述突变体、编码权利要求1所述菜豆源环氧化物酶突变体的基因、含所述基因的表达载体、含所述基因的工程菌在手性生物催化中的应用。
9.根据权利要求8所述应用,其特征在于,所述应用是以权利要求1所述环氧化物酶突变体和/或表达权利要求1环氧化物酶突变体的工程菌为催化剂,在磷酸盐缓冲液中催化外消旋邻甲苯缩水甘油醚。
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CN114891763A (zh) * | 2022-05-31 | 2022-08-12 | 安徽工程大学 | 一种费氏链霉菌来源的环氧水解酶、基因、载体、工程菌、制备方法及应用 |
CN114891763B (zh) * | 2022-05-31 | 2023-10-20 | 安徽工程大学 | 一种费氏链霉菌来源的环氧水解酶、基因、载体、工程菌、制备方法及应用 |
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