CN112908417A - 功能序列和结构模拟相结合的基因挖掘方法、nadh偏好型草铵膦脱氢酶突变体及应用 - Google Patents
功能序列和结构模拟相结合的基因挖掘方法、nadh偏好型草铵膦脱氢酶突变体及应用 Download PDFInfo
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Abstract
本发明公开了一种功能序列和结构模拟相结合的基因挖掘方法、NADH偏好型草铵膦脱氢酶突变体及应用。所述基因挖掘方法包括:(1)分析NADH型谷氨酸脱氢酶应具备的特性序列;(2)根据特征序列搜索基因库;(3)对搜索获得的基因进行聚类分析和蛋白结构模拟;(4)选择基因聚合度高且蛋白结构与已知草铵膦脱氢酶结构类似的基因作为候选基因。经基因挖掘得到来源于堆肥赖氨酸杆菌(Lysinibacillus composti)氨基酸序列如SEQ ID No.2所示的野生型草铵膦脱氢酶,并经过突变筛选到NADH偏好型草铵膦脱氢酶突变体,突变位点选自以下一种:(1)A144G‑V375F‑M91A;(2)A144G‑V345A‑M91A;(3)A144G,该突变体酶能够利用廉价的辅酶NAD进行催化反应。
Description
技术领域
本发明涉及生物技术领域,特别是涉及一种功能序列和结构模拟相结合的基因挖掘方法、NADH偏好型草铵膦脱氢酶突变体及应用。
背景技术
草铵膦(phosphinothricin,也叫glufosinate,简称PPT)的化学名称为2-氨基-4-[羟基(甲基)膦酰基]-丁酸,是世界第二大转基因作物耐受除草剂,由赫斯特公司(几经合并后现归属拜耳公司)开发生产,又称草铵膦铵盐、Basta、Buster等,属膦酸类除草剂,非选择性(灭生性)触杀型除草剂是谷氨酰胺合成酶抑制剂。
草铵膦有两种光学异构体,为L-草铵膦和D-草铵膦。但只有L-型具有生理活性,且在土壤中易分解,对人类和动物的毒性较小,除草谱广,对环境的破坏力小。
目前,市场上销售的草铵膦一般都是外消旋混合物。若草铵膦产品能以L-构型的纯光学异构体形式使用,可显著降低草铵膦的使用量,这对于提高原子经济性、降低使用成本、减轻环境压力具有重要意义。
手性纯L-草铵膦的制备方法主要有三种:手性拆分法,化学合成法和生物催化法。生物催化法生产草铵膦则具有立体选择性严格、反应条件温和、收率高等优点,是生产L-草铵膦的优势方法。主要包括以下三类:
1)以L-草铵膦的衍生物为底物,通过酶法直接水解获得,主要的优点转化率高,产物e.e.值较高,但需要昂贵且不易获得的手性原料作为前体,成本加高,不利于工业化生产。例如生物法制备L-草铵膦最简单的方法就是利用蛋白酶直接水解双丙氨膦。双丙氨膦是一种天然的三肽化合物,在蛋白酶的催化下,双丙氨膦脱去2分子L-丙氨酸,生成L-草铵膦。
2)以外消旋草铵膦的前体为底物,通过酶的选择性拆分获得。主要优点为原料相对易得,催化剂活力高,但其理论收率只能达到50%,会造成原料的浪费。例如Cao等人(CaoC-H,Cheng F,Xue Y-P,Zheng Y-G(2020)Efficient synthesis of L-phosphinothricinusing a novel aminoacylase mined from Stenotrophomonas maltophilia.Enzyme andMicrobial Technology 135doi:10.1016/j.enzmictec.2019.109493)采用一种来源于Stenotrophomonas maltophilia的新型的氨基酰化酶手性拆分N-乙酰-PPT,得到L-草铵膦。用全细胞进行催化,在4小时内转化率>49%,获得了光学纯的L-PPT(>99.9%e.e.)。
3)以α-酮酸-2-羰基-4-(羟基甲基膦酰基)丁酸(PPO)为底物,通过酶的不对称合成获得,主要涉及的酶包括转氨酶与草铵膦脱氢酶。Bartsch(Bartsch K(2005)Processfor the preparation of 1-phosphinothrcine by enzymatic transamination withaspartate.US Patent no.US6936444B1)等利用PPO为底物,L-天冬氨酸为氨基供体,利用从土壤微生物中筛选分离出来的对PPO和L-天冬氨酸有特异性酶活的转氨酶进行催化,当底物浓度为552mM时,在非常高的温度(80℃)下反应4小时,转化率仍达到52%,时空产率为4.5g L-PPT/g·L-1·d-1。但利用转氨酶制备L-草铵膦有两大缺陷,其一是这是一个可逆反应,原料PPO不能完全转化为L-PPT,转化率无法达到100%;其二是要使可逆反应向生成L-PPT的方向进行,需要加入至少2倍以上的L-天冬氨酸作为氨基供体,过量的天冬氨酸给L-PPT的分离带来了很大的麻烦。
在诸多草铵膦的酶法合成路线中,酮酸中间体的酮羰基是潜手性官能团,能够通过酶法合成途径构建手性中心,酮酸路线也因原料价廉易得,且可以避免使用剧毒氰化物,而成为适宜L-草铵膦工业化开发生产的路线。
氨基酸脱氢酶(EC 1.4.1.X,AADH)是一类能将氨基酸可逆的脱氨生成对应酮酸的氨基酸脱氢酶,其反应需要核苷类辅酶的参与(NAD+),被广泛的应用于天然与非天然α-氨基酸的合成。根据其底物特异性,可分为谷氨酸脱氢酶、亮氨酸脱氢酶、丙氨酸脱氢酶、缬氨酸脱氢酶等。如果其表现出对草铵膦前体具有一定的活力,就可称之为“草铵膦脱氢酶(PPTDH)”。
葡萄糖脱氢酶(EC1.1.1.47,GDH)是生物催化的重要辅助酶,用于氧化还原催化反应中辅酶NADH的再生循环。
尽管目前NADPH偏好型草铵膦脱氢酶的酶活略高于NADH偏好型草铵膦脱氢酶(大于50倍),然而NADPH的市场价格(约每吨2万元)是NADH的5倍,在实际应用中外源添加NADPH会导致产品L-草铵膦的成本显著增加。因此,发明出一种NADH偏好型高活力草铵膦脱氢酶,配合外源添加廉价的NADH或NAD,具有很好的应用前景。
发明内容
本发明目的是针对现有草铵膦脱氢酶对2-羰基-4-(羟基甲基膦酰基)-丁酸不对称胺化还原活性不高的问题,提供了一种NADH偏好型草铵膦脱氢酶突变体及利用该NADH偏好型草铵膦脱氢酶突变体基因重组菌及其粗酶液作为生物催化剂,用于L-草铵膦手性生物合成。
一种NADH偏好型草铵膦脱氢酶突变体,由来源于堆肥赖氨酸杆菌(Lysinibacillus composti)的野生型草铵膦脱氢酶突变所得,野生型草铵膦脱氢酶的氨基酸序列如SEQ ID No.2所示,所述NADH偏好型草铵膦脱氢酶突变体的突变位点选自以下一种:
(1)A144G-V375F-M91A;
(2)A144G-V345A-M91A;
(3)A144G。
本发明又公开了编码所述NADH偏好型草铵膦脱氢酶突变体的基因。
本发明又公开了一种基因工程菌,包括宿主细胞和转入宿主细胞的目的基因,所述目的基因包含所述的基因。
优选的,所述的基因工程,目的基因还包括葡萄糖脱氢酶的编码基因。这样可以进行草铵膦脱氢酶突变体与葡萄糖脱氢酶的共表达。更优选的,葡萄糖脱氢酶的编码基因序列对应的GenBank登录号为KM817194.1。
本发明还公开了所述NADH偏好型草铵膦脱氢酶突变体、所述基因或所述基因工程菌在制备L-草铵膦中的应用。
本发明还公开了一种L-草铵膦的制备方法,以2-羰基-4-(羟基甲基膦酰基)-丁酸为底物,在无机氨基供体、辅酶再生循环系统及对应辅助底物存在的条件下,利用催化剂催化底物反应获得L-草铵膦;
所述催化剂为以下一种:
(1)所述NADH偏好型草铵膦脱氢酶突变体;
(2)能够表达所述NADH偏好型草铵膦脱氢酶突变体的基因工程菌或该基因工程菌经裂解得到的粗酶液。
优选的,所述辅酶再生循环系统使用葡萄糖脱氢酶、甲酸脱氢酶或醇脱氢酶偏好型的辅酶再生循环系统。
本发明还提供了一种功能序列和结构模拟相结合的基因挖掘方法,包括以下步骤:
(1)分析NADH型谷氨酸脱氢酶应具备的特性序列,特征序列包括:
(1.1)蛋白质大小:候选蛋白质的长度为300-500个氨基酸,
(1.2)草铵膦脱氢酶的两段必要特性序列:第一段为GGGKGG;同时第二段为VVTG、FVTG、VLTG、VFTG、FITG、FFTG、VVFG、FVFTG、VLFG、VFFG、FLFG、FFFG其中之一,
(1.3)NADH结合的特性序列:GXRVXXG,其中X位置表示任意一种氨基酸;
(2)根据特征序列搜索基因库;
(3)对步骤(2)搜索获得的基因进行聚类分析和蛋白结构模拟;
(4)选择基因聚合度高且蛋白结构与已知草铵膦脱氢酶结构类似的基因作为候选基因。
步骤(2)搜索基因库时,利用以上特性序列对NCBI微生物基因组资源中然后对NCBI NR序列数据库(含有约1亿个蛋白基因)进行迭代PSI-BLAST搜索并进行聚类分析,获得了15个簇,15个簇的聚合度分别为0.82、0.76、0.71、0.66、0.65、0.58、0.43、0.42、0.40、0.39、0.38、0.34、0.33、0.32、0.30(从高到低排列)。
步骤(3)选取聚合度最高的6个簇中具有代表性的36个蛋白(每簇6个蛋白)进行3维结构模拟(可以采用腾讯tFold蛋白结构服务器),对模拟的结构与已知的草铵膦脱氢酶(PDB数据库编号:1LEH、1BW9、5IJZ)进行结构比对,其中,来源堆肥赖氨酸杆菌(Lysinibacillus composti)的LcGDH和这三个已知的草铵膦脱氢酶结构标准偏差(RMSD)都小于,故选择LcGDH基因作为出发基因(氨基酸序列SEQ ID No.2所示)。
与现有技术相比,本发明具有以下有益效果:
(1)本发明草铵膦脱氢酶突变体具有更好的催化效率,以2-羰基-4-(羟基甲基膦酰基)-丁酸为底物进行催化反应时,转化率远高于野生型酶,PPO产率也大幅提升。
(2)本发明利用草铵膦脱氢酶突变体和辅酶循环系统将2-羰基-4-[羟基(甲基)膦酰基]丁酸催化还原为L-草铵膦,进而实现不对称合成L-草铵膦。
(3)本发明能够直接以2-羰基-4-[羟基(甲基)膦酰基]丁酸为底物进行不对称合成,无需昂贵的化学拆分试剂,也无需合成草铵膦衍生物,利用廉价的辅酶NAD进行催化反应,相比之前利用辅酶NADP的生产过程,成本显著降低,具有较高的工业化应用前景。
附图说明
图1为草铵膦脱氢酶突变体偶联葡萄糖脱氢酶不对称胺化还原中间产物2-羰基-4-(羟基甲基膦酰基)-丁酸制备L-草铵膦的反应示意图。
图2为实施例3中LcGDH和EsGDH双酶偶联SDS-PAGE图。其中泳道1:标准蛋白分子量;泳道2:含有EsGDH的重组大肠杆菌细胞。泳道3:EsGDH未表达的重组大肠杆菌细胞。
图3为草铵膦脱氢酶突变体LcGDH(A144G)偶联葡萄糖脱氢酶不对称胺化还原2-羰基-4-(羟基甲基膦酰基)-丁酸反应进程图,反应体系中外加了1mM NAD+辅酶。
图4为草铵膦脱氢酶突变体LcGDH(A144G-V345A-M91A)偶联葡萄糖脱氢酶不对称胺化还原2-羰基-4-(羟基甲基膦酰基)-丁酸反应进程图,反应体系中外加了1mM NAD+辅酶。
图5为草铵膦脱氢酶突变体LcGDH(A144G-V375F-M91A)偶联葡萄糖脱氢酶不对称胺化还原2-羰基-4-(羟基甲基膦酰基)-丁酸反应进程图,反应体系中外加了1mM NAD+辅酶。
具体实施方式
实施例1
步骤1:分析NADH型谷氨酸脱氢酶应具备的特性序列:①蛋白质大小:候选蛋白质的长度(300-500个氨基酸),②草铵膦脱氢酶的两段必要特性序列:第一段为GGGKGG,其中X位置表示任意一种氨基酸,同时第二段为VVTG、FVTG、VLTG、VFTG、FITG、FFTG、VVFG、FVFTG、VLFG、VFFG、FLFG、FFFG其中之一,③NADH结合的特性序列:GXRVXXG。
步骤2:搜索基因库:利用以上特性序列对NCBI微生物基因组资源中然后对NCBINR序列数据库(含有约1亿个蛋白基因)进行迭代PSI-BLAST搜索并进行聚类分析,获得了15个簇,15个簇的聚合度分别为0.82、0.76、0.71、0.66、0.65、0.58、0.43、0.42、0.40、0.39、0.38、0.34、0.33、0.32、0.30(从高到低排列)。
步骤3:选取聚合度最高的6个簇中具有代表性的36个蛋白(每簇6个蛋白)进行3维结构模拟(采用腾讯tFold蛋白结构服务器),对模拟的结构与已知的草铵膦脱氢酶(1LEH、1BW9、5IJZ)进行结构比对,其中,来源堆肥赖氨酸杆菌(Lysinibacillus composti)的LcGDH和这三个已知的草铵膦脱氢酶结构标准偏差(RMSD)都小于,故选择LcGDH基因作为出发基因(氨基酸序列SEQ ID No.2所示)。
实施例2:草铵膦脱氢酶突变体文库的构建及筛选
将实施例1的LcGDH氨基酸序列进行密码子优化(密码子优化后的核苷酸序列为SEQ ID No.1所示),由杭州擎科生物技术有限公司进行基因合成获得的LcGDH基因,克隆到质粒pETDuet的MCS1(多克隆位点1)的NcoI上,构建重组表达载体pETDuet-LcGDH,保留质粒本身的His-Tag基因,转化至大肠杆菌E.coli BL21(DE3),送杭州擎科生物技术有限公司合成野生型草铵膦脱氢酶工程菌E.coli BL21(DE3)/pETDuet-LcGDH。
再从微小杆菌(Exiguobacterium sibiricum)ZJBML01011中克隆得到葡萄糖脱氢酶基因EsGDH(核苷酸序列为GenBank登录号为KM817194.1所示),通过Vazyme公司的OneStep Cloning Kit构建至重组表达载体pETDuet-LcGDH的MCS2(多克隆位点2)的NdeI上,获得共表达载体pETDuet-LcGDH-EsGDH,转化至大肠杆菌E.coli BL21(DE3),获得野生型草铵膦脱氢酶与葡萄糖脱氢酶出发共表达菌株E.coli BL21(DE3)/pETDuet-LcGDH-EsGDH。图1为该菌株不对称胺化还原中间产物2-羰基-4-(羟基甲基膦酰基)-丁酸制备L-草铵膦的反应示意图。
草铵膦脱氢酶突变体文库的制备通过4轮定点饱和突变来实现,引物设计如表1(其中引物序列中涉及的简并碱基中,N表示A、C、G或T;K表示G或T;M表示A或C),以载体pETDuet-LcGDH-EsGDH为模板,以表1中序列(A144)为引物,经饱和突变PCR,后用DpnI消化,并转化E.coli BL21(DE3),涂布含50μg/mL氨苄霉素的LB平板,挑取菌株使用高通量筛选方法进行优势菌株筛选,再以上述步骤重复进行第二轮、第三轮、第四轮的定点饱和突变,筛选活力较高的优势菌株。
表1草铵膦脱氢酶定点饱和突变引物设计
突变PCR体系(100μL)为:2倍Phanta Max缓冲液25μL,dNTPs 1μL,突变上下引物各1μL,模板1μL,Phanta Super-Fidelity DNA聚合酶0.5μL,补ddH2O至50μL。PCR条件为:95℃预变性5分钟,经30个循环:90℃ 30秒,62℃ 30秒,72℃ 7分钟,最后72℃终延伸5分钟。PCR结果分别进行DNA琼脂糖凝胶电泳阳性验证,将PCR产物进行DpnI酶消化模板,37℃,1小时,220转/分钟,65℃,1分钟灭活,将PCR产物热击转化,并将大肠杆菌E.coli BL21(DE3)活化,置于37℃、220转/分钟,培养1小时,涂布于含50μg/mL氨苄霉素抗性的LB平板上,37℃倒置培养过夜,对获得的突变体按实施例3方法进行优势突变体的筛选,将获得的优势菌株送杭州擎科生物技术有限公司进行测序确认,并保存。共筛选到如下带有突变草铵膦脱氢酶基因的共表达菌株:
(1)E.coli BL21(DE3)/pETDuet-1-LcGDH(A144G)-EsGDH:表达出来的草铵膦脱氢酶(LcGDH)具有突变A144G。
(2)E.coli BL21(DE3)/pETDuet-1-LcGDH(A144G-V345A-M91A)-EsGDH:表达出来的草铵膦脱氢酶(LcGDH)具有三个氨基酸残基的突变,分别为A144G、V345A和M91A。
(3)E.coli BL21(DE3)/pETDuet-1-LcGDH(A144G-V375F-M91A)-EsGDH:表达出来的草铵膦脱氢酶(LcGDH)具有三个氨基酸残基的突变,分别为A144G、V375F和M91A。
实施例3:草铵膦脱氢酶突变体工程菌的诱导表达
将实施例2中的野生型草铵膦脱氢酶与葡萄糖脱氢酶出发共表达菌株E.coliBL21(DE3)/pETDuet-1-LcGDH-EsGDH和草铵膦脱氢酶突变体与葡萄糖脱氢酶共表达菌株:
E.coli BL21(DE3)/pETDuet-1-LcGDH(A144G)-EsGDH、
E.coli BL21(DE3)/pETDuet-1-LcGDH(A144G-V345A-M91A)-EsGDH、
E.coli BL21(DE3)/pETDuet-1-LcGDH(A144G-V375F-M91A)-EsGDH,
分别接种到含终浓度50μg/mL氨苄霉素的LB液体培养基中,37℃培养8小时,以体积浓度2%的接种量接种到新鲜的含终浓度50μg/mL氨苄霉素的LB液体培养基中,37℃、180转/分钟培养2小时,再向培养液中加入终浓度为0.1mM IPTG,18℃培养14小时后,4℃、8000转/分钟离心10分钟,获得相应的湿菌体。
以上获得的细胞产有相应的蛋白,可用于蛋白纯酶液的制备,也可用于粗酶液不对称胺化2-羰基-4-(羟基甲基膦酰基)-丁酸制备L-草铵膦。图2为LcGDH和EsGDH双酶偶联SDS-PAGE图。其中泳道1:标准蛋白分子量;泳道2:含有EsGDH的重组大肠杆菌细胞。泳道3:EsGDH未表达的重组大肠杆菌细胞。
实施例4:突变文库筛选
以实施例3方法制备的野生型草铵膦脱氢酶与葡萄糖脱氢酶出发共表达湿菌体或草铵膦脱氢酶突变体与葡萄糖脱氢酶共表达湿菌体为催化剂,以中间产物2-羰基-4-(羟基甲基膦酰基)-丁酸为底物,以葡萄糖为辅酶再生底物,加入硫酸铵,外源添加微量NADH,以pH 7.4、100mM磷酸盐缓冲液为反应介质构成1mL反应体系,催化剂用量以湿菌体终浓度计20g/L,底物终浓度100mM,葡萄糖终浓度125mM,硫酸铵终浓度150mM,35℃、600转/分钟反应5min,取反应液50μL,加5μL盐酸终止反应,反应液稀释100倍,取200μl稀释后的反应液+400μL衍生化试剂(含15mM邻苯二甲醛、15mM N-乙酰-L-半胱氨酸的pH=9.8的硼酸缓冲液)30℃衍生化5min,再加400μL超纯水补足至1mL,12000转/分钟离心1分钟,取上清,过0.22μM微滤膜,收集滤液作为液相样品,HPLC检测2-羰基-4-(羟基甲基膦酰基)-丁酸、L-草铵膦、D-草铵膦及e.e.值。以产物L-草铵膦浓度和对映体过量e.e.为指标,筛选优势突变体,实验结果示于表2。
2-羰基-4-(羟基甲基膦酰基)-丁酸液相检测条件:色谱柱C18(4.6×250mm,Acchrom,China)柱,流动相乙腈:50mM磷酸二氢铵溶液(pH3.8,含10%四丁基氢氧化铵)体积比为12:88。流速为1mL/min,检测波长为232nm,进样量10μL,柱温30℃,2-羰基-4-(羟基甲基膦酰基)-丁酸保留时间为:9.7分钟。
草铵膦液相检测条件:色谱柱C18(4.6×250mm,Acchrom,China)柱,流动相甲醇:0.05M乙酸铵(pH 5.7)体积比为10∶90,流速1.0mL/min,检测波长Ex=340nm、Em=450nm,进样量10μL,柱温35℃。L-草铵膦、D-草铵膦、保留时间分别为:10.6分钟,12.6分钟。
表2野生型LcGDH及其突变体全细胞的催化性能和立体选择性
注:A144G-V375F-M91A代表的是LcGDH的144位氨基酸残基A突变为G、375位氨基酸残基V突变为F、91位M突变为A。
实施例4:野生型草铵膦脱氢酶及其突变体的纯化
将实施例1构建的草铵膦脱氢酶工程菌及优势突变体按实施例2方法制备相应湿菌体。分别取野生型草铵膦脱氢酶工程菌及草铵膦脱氢酶突变体工程菌的湿菌体各0.2g分别用10ml结合缓冲液(含0.3M NaCl的pH 7.4、100mM磷酸钠缓冲液)悬浮,超声破碎15分钟(冰浴,功率400W,破碎1秒、暂停5秒),4℃、12000转/分钟离心20min,取上清,作为样品。使用Ni亲和柱(1.6×10cm,Bio-Rad公司,美国)纯化蛋白,具体操作如下:①用5倍柱体积的结合缓冲液(含0.3M NaCl的pH 7.4、50mM磷酸钠缓冲液)平衡Ni柱,至基线稳定;②样品上样,流速1mL/min,上样量在25-40mg/mL蛋白,使目标蛋白吸附于Ni柱上;③用6倍柱体积的缓冲液A(含0.3M NaCl、30mM咪唑的pH7.4、50mM磷酸钠缓冲液)冲洗杂蛋白,流速1mL/min,至基线稳定;④用缓冲液B(含0.3M NaCl、500mM咪唑的pH 7.4、50mM磷酸钠缓冲液)洗脱,流速1mL/min,收集目的蛋白。将目的蛋白置于pH 7.4、20mM磷酸盐缓冲液中透析过夜,收集截留液,分别获得野生型草铵膦脱氢酶纯酶10ml和草铵膦脱氢酶突变体纯酶10ml;⑤5倍柱体积的结合缓冲液(含0.3M NaCl的pH 8.0、50mM磷酸钠缓冲液)冲洗Ni柱直至基线稳定,用5倍柱体积含20%乙醇的超纯水保存Ni柱。
实施例5:野生型草铵膦脱氢酶及其突变体比酶活测定
酶活单位(U)定义为:在35℃、pH 7.4条件下,每分钟每生成1μmol的L-草铵膦所需的酶量定义为一个酶活单位,U。比酶活定义为每毫克酶蛋白所具有的活力单位数,U/mg。
酶活检测标准条件:100mM 2-羰基-4-(羟基甲基膦酰基)-丁酸,10mM NADH,0.02μg/μL的酶液(实施例4方法制备),30℃、pH 7.4,600转/分钟条件下反应10分钟,采用实施例3方法进行HPLC检测分析。
蛋白浓度用BCA蛋白测定试剂盒(南京凯基生物科技发展有限公司,南京)测定,如表3所示。
表3野生型草铵膦脱氢酶及其突变体比酶活
a:在标准条件下,每个野生型草铵膦脱氢酶的初始酶活均指定为100%。
实施例6:野生型草铵膦脱氢酶及其突变体动力学参数测定
考察野生型草铵膦脱氢酶及其突变体的动力学参数,以2-羰基-4-(羟基甲基膦酰基)-丁酸作为底物,浓度设置为2-10mM(2、4、6、8、10mM),添加足量辅酶(10mM),加入100μL的纯酶液(实施例4方法收集)。
反应体系选择为500μL,实施例4收集的纯酶液以pH7.4、100mM磷酸盐缓冲液稀释10倍后取100μL,加入底物和外源性辅酶NADPH,pH 7.4、100mM磷酸盐缓冲液为反应介质,35℃、600转/分钟反应10min取样,取反应液HPLC检测L-草铵膦浓度(同实施例3)。
通过双倒数作图可计算得出Kcat、vmax、Km,结果如表4所示,通过比较kcat和Km,可以发现,LcGDH对2-羰基-4-(羟基甲基膦酰基)-丁酸的Km值是8.56mM,其余突变体对2-羰基-4-(羟基甲基膦酰基)-丁酸亲和力有增加趋势。突变体LcGDH-(A144G-V375F-M91A)-EsGDH对2-羰基-4-(羟基甲基膦酰基)-丁酸的催化效率kcat/Km达到169.25mM-1,母本(kcat/Km=1.15·mM-1)提高147.17倍。
表4母本LcGDH及其突变体动力学参数比较
酶 | k<sub>cat</sub>(s<sup>-1</sup>)<sup>a</sup> | K<sub>m</sub>(mM) | k<sub>cat</sub>/K<sub>m</sub>(s<sup>-1</sup>*mM) |
LcGDH | 9.88 | 8.56 | 1.15 |
LcGDH(A144G) | 428.12 | 4.54 | 94.30 |
LcGDH(A144G+V345A+M91A) | 533.25 | 4.22 | 126.36 |
LcGDH(A144G+V375F+M91A) | 736.22 | 4.35 | 169.25 |
实施例7:草铵膦脱氢酶突变体LcGDH-A144G偶联葡萄糖脱氢酶不对称胺化还原2-羰基-4-(羟基甲基膦酰基)-丁酸
将实施例2方法制备的E.coli BL21(DE3)/LcGDH(A144G)-EsGDH湿菌体1g,用40mL、pH 7.4、磷酸缓冲液(100mM)重悬,加入终浓度100mM的2-羰基-4-(羟基甲基膦酰基)丁酸,终浓度125mM的葡萄糖,终浓度125mM硫酸铵构成反应体系50mL,在35℃、磁力搅拌转速为600rpm下进行反应,流加氨水使反应液pH维持在7.4。以实施例3所示液相方法检测反应过程中产物L-草铵膦的生成和e.e.值的变化,反应进程曲线如图3所示,该图显示,产物浓度随时间的推移而逐渐升高,6h内反应完成,底物转化率大于99%,产物e.e.值始终保持在99.5%以上。
实施例8:草铵膦脱氢酶突变体LcGDH(A144G-V345A-M91A)-EsGDH偶联葡萄糖脱氢酶不对称胺化还原2-羰基-4-(羟基甲基膦酰基)-丁酸
将实施例2方法制备的E.coli BL21(DE3)/LcGDH-(A144G-V345A-M91A)-EsGDH湿菌体1g,用40mL、pH 7.4、磷酸缓冲液(100mM)重悬,加入终浓度100mM的2-羰基-4-(羟基甲基膦酰基)丁酸,终浓度125mM的葡萄糖,终浓度125mM硫酸铵构成反应体系50mL,在35℃、磁力搅拌转速为600rpm下进行反应,流加氨水使反应液pH维持在7.4。以实施例3所示液相方法检测反应过程中产物L-草铵膦的生成和e.e.值的变化,反应进程曲线如图4所示,该图显示,产物浓度随时间的推移而逐渐升高,5.5h内反应完成,底物转化率大于99%,产物e.e.值始终保持在99.5%以上。
实施例9:草铵膦脱氢酶突变体LcGDH-(A144G-V375F-M91A)-EsGDH偶联葡萄糖脱氢酶不对称胺化还原2-羰基-4-(羟基甲基膦酰基)-丁酸
将实施例2方法制备的E.coli BL21(DE3)/LcGDH-(A144G-V375F-M91A)-EsGDH湿菌体1g,用40mL、pH 7.4、磷酸缓冲液(100mM)重悬,加入终浓度100mM的2-羰基-4-(羟基甲基膦酰基)丁酸,终浓度125mM的葡萄糖,终浓度125mM硫酸铵构成反应体系50mL,在35℃、磁力搅拌转速为600rpm下进行反应,流加氨水使反应液pH维持在7.4。以实施例3所示液相方法检测反应过程中产物L-草铵膦的生成和e.e.值的变化,反应进程曲线如图5所示,该图显示,产物浓度随时间的推移而逐渐升高,5h内反应完成,底物转化率大于99%,产物e.e.值始终保持在99.5%以上。
序列表
<110> 浙江工业大学
<120> 功能序列和结构模拟相结合的基因挖掘方法、NADH偏好型草铵膦脱氢酶突变体及应用
<160> 24
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1242
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atggcagaaa acctgaactt atttacgagc acccaggagg ttgtgaaaga agcgctgaac 60
aaactgggtt atgatgaggc aatgtacgaa ctgctgaaag aaccgctgcg cctgctgaaa 120
gtgcgtattc ctgtgaagat ggacgatggc accacacagg tgtttacggg ttatcgcgca 180
caacattccg atgcagtagg tcccaccaaa ggtggcgtgc gttttcatcc tatggtttct 240
gaagacgaag ttaaagcact gagcatgtgg atgaccctga agtgcgggat tgtagatctg 300
ccttatggtg gtggtaaagg tggcattatt tgtgatccgc gtcagatgag catgggggaa 360
ttagaacgtc tgagccgtgg atatgttcgg gcaattagtc agattgttgg gccgaccaaa 420
gatataccgg caccggatgt ttttaccaat gcacaaatta tggcatggat gatggatgag 480
tatagccgta tggatgaatt taatagtccg ggttttataa ccggtaaacc tctggtgctg 540
ggcggtagta aagggcgtga tcgggcgacg gcagaaggtg ttacgattgt tattcaggag 600
gcagcaaaaa agagaaatat cgatatcaaa ggtgcacgcg ttgttattca agggttcggt 660
aatgccggca gttttttagc aaagtttatg agtgatctgg gcgcgaaggt tataggaata 720
agtgatgcat acggggccct gcacgatccg aatggtttag atattgatta tctgctggac 780
agacgtgata gttttggtac cgttaccacg ctgtttgaaa atacaattac gaatcaggag 840
ctgctggaac tggattgtga tattctggtg ccggccgcaa ttgagaatca gattacggca 900
gaaaatgcac ataatattaa ggcaaccata gttgtggaag cagcgaacgg cccaaccacc 960
tctgaagcaa ccaaaattct gaccgaacgt ggtattctgt tagtgccaga cgttttagca 1020
agcgcaggtg gggttacagt tagctacttt gagtgggttc aaaataatat gggctattac 1080
tgggaagaag aagaggttca agaaaaactg tacaaaaaaa tggtggatag ctttgaagca 1140
gtatatacaa ccgcaaccac gcgcaatata gatatgcgtc tggcagcgta tatggtggga 1200
gtgagaagaa cagcagaagc gagccgtttc cggggctggg tg 1242
<210> 2
<211> 414
<212> PRT
<213> 堆肥赖氨酸杆菌(Lysinibacillus composti)
<400> 2
Met Ala Glu Asn Leu Asn Leu Phe Thr Ser Thr Gln Glu Val Val Lys
1 5 10 15
Glu Ala Leu Asn Lys Leu Gly Tyr Asp Glu Ala Met Tyr Glu Leu Leu
20 25 30
Lys Glu Pro Leu Arg Leu Leu Lys Val Arg Ile Pro Val Lys Met Asp
35 40 45
Asp Gly Thr Thr Gln Val Phe Thr Gly Tyr Arg Ala Gln His Ser Asp
50 55 60
Ala Val Gly Pro Thr Lys Gly Gly Val Arg Phe His Pro Met Val Ser
65 70 75 80
Glu Asp Glu Val Lys Ala Leu Ser Met Trp Met Thr Leu Lys Cys Gly
85 90 95
Ile Val Asp Leu Pro Tyr Gly Gly Gly Lys Gly Gly Ile Ile Cys Asp
100 105 110
Pro Arg Gln Met Ser Met Gly Glu Leu Glu Arg Leu Ser Arg Gly Tyr
115 120 125
Val Arg Ala Ile Ser Gln Ile Val Gly Pro Thr Lys Asp Ile Pro Ala
130 135 140
Pro Asp Val Phe Thr Asn Ala Gln Ile Met Ala Trp Met Met Asp Glu
145 150 155 160
Tyr Ser Arg Met Asp Glu Phe Asn Ser Pro Gly Phe Ile Thr Gly Lys
165 170 175
Pro Leu Val Leu Gly Gly Ser Lys Gly Arg Asp Arg Ala Thr Ala Glu
180 185 190
Gly Val Thr Ile Val Ile Gln Glu Ala Ala Lys Lys Arg Asn Ile Asp
195 200 205
Ile Lys Gly Ala Arg Val Val Ile Gln Gly Phe Gly Asn Ala Gly Ser
210 215 220
Phe Leu Ala Lys Phe Met Ser Asp Leu Gly Ala Lys Val Ile Gly Ile
225 230 235 240
Ser Asp Ala Tyr Gly Ala Leu His Asp Pro Asn Gly Leu Asp Ile Asp
245 250 255
Tyr Leu Leu Asp Arg Arg Asp Ser Phe Gly Thr Val Thr Thr Leu Phe
260 265 270
Glu Asn Thr Ile Thr Asn Gln Glu Leu Leu Glu Leu Asp Cys Asp Ile
275 280 285
Leu Val Pro Ala Ala Ile Glu Asn Gln Ile Thr Ala Glu Asn Ala His
290 295 300
Asn Ile Lys Ala Thr Ile Val Val Glu Ala Ala Asn Gly Pro Thr Thr
305 310 315 320
Ser Glu Ala Thr Lys Ile Leu Thr Glu Arg Gly Ile Leu Leu Val Pro
325 330 335
Asp Val Leu Ala Ser Ala Gly Gly Val Thr Val Ser Tyr Phe Glu Trp
340 345 350
Val Gln Asn Asn Met Gly Tyr Tyr Trp Glu Glu Glu Glu Val Gln Glu
355 360 365
Lys Leu Tyr Lys Lys Met Val Asp Ser Phe Glu Ala Val Tyr Thr Thr
370 375 380
Ala Thr Thr Arg Asn Ile Asp Met Arg Leu Ala Ala Tyr Met Val Gly
385 390 395 400
Val Arg Arg Thr Ala Glu Ala Ser Arg Phe Arg Gly Trp Val
405 410
<210> 3
<211> 6
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Gly Gly Gly Lys Gly Gly
1 5
<210> 4
<211> 4
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Val Val Thr Gly
1
<210> 5
<211> 4
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Phe Val Thr Gly
1
<210> 6
<211> 4
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Val Leu Thr Gly
1
<210> 7
<211> 4
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Val Phe Thr Gly
1
<210> 8
<211> 4
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Phe Ile Thr Gly
1
<210> 9
<211> 4
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Phe Phe Thr Gly
1
<210> 10
<211> 4
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Val Val Phe Gly
1
<210> 11
<211> 5
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Phe Val Phe Thr Gly
1 5
<210> 12
<211> 4
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Val Leu Phe Gly
1
<210> 13
<211> 4
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Val Phe Phe Gly
1
<210> 14
<211> 4
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Phe Leu Phe Gly
1
<210> 15
<211> 4
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Phe Phe Phe Gly
1
<210> 16
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for any kind of Amino acid residue.
<220>
<221> UNSURE
<222> (5)..(5)
<223> The 'Xaa' at location 2 stands for any kind of Amino acid residue.
<220>
<221> UNSURE
<222> (6)..(6)
<223> The 'Xaa' at location 2 stands for any kind of Amino acid residue.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (5)..(5)
<223> The 'Xaa' at location 5 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (6)..(6)
<223> The 'Xaa' at location 6 stands for Gln, Arg, Pro, or Leu.
<400> 16
Gly Xaa Arg Val Xaa Xaa Gly
1 5
<210> 17
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (5)..(5)
<223> n stands for a, c, g or t.
<220>
<221> misc_feature
<222> (6)..(6)
<223> n stands for a, c, g or t.
<220>
<221> misc_feature
<222> (7)..(7)
<223> k stands for g or t.
<400> 17
accgnnkccg gatgttttta ccaatgc 27
<210> 18
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (6)..(6)
<223> m stands for a or c.
<220>
<221> misc_feature
<222> (7)..(7)
<223> n stands for a, c, g or t.
<220>
<221> misc_feature
<222> (8)..(8)
<223> n stands for a, c, g or t.
<400> 18
tccggmnncg gtatatcttt ggtcggc 27
<210> 19
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (5)..(5)
<223> n stands for a, c, g or t.
<220>
<221> misc_feature
<222> (6)..(6)
<223> n stands for a, c, g or t.
<220>
<221> misc_feature
<222> (7)..(7)
<223> k stands for g or t.
<400> 19
tgggnnkaca gttagctact ttgagtgg 28
<210> 20
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (7)..(7)
<223> m stands for a or c.
<220>
<221> misc_feature
<222> (8)..(8)
<223> n stands for a, c, g or t.
<220>
<221> misc_feature
<222> (9)..(9)
<223> n stands for a, c, g or t.
<400> 20
aactgtmnnc ccacctgcgc ttgctaa 27
<210> 21
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (7)..(7)
<223> n stands for a, c, g or t.
<220>
<221> misc_feature
<222> (8)..(8)
<223> n stands for a, c, g or t.
<220>
<221> misc_feature
<222> (9)..(9)
<223> k stands for g or t.
<400> 21
aaaatgnnkg atagctttga agcagtata 29
<210> 22
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (7)..(7)
<223> m stands for a or c.
<220>
<221> misc_feature
<222> (8)..(8)
<223> n stands for a, c, g or t.
<220>
<221> misc_feature
<222> (9)..(9)
<223> n stands for a, c, g or t.
<400> 22
gctatcmnnc atttttttgt acagtttttc 30
<210> 23
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (8)..(8)
<223> n stands for a, c, g or t.
<220>
<221> misc_feature
<222> (9)..(9)
<223> n stands for a, c, g or t.
<220>
<221> misc_feature
<222> (10)..(10)
<223> k stands for g or t.
<400> 23
catgtggnnk accctgaagt gcgggatt 28
<210> 24
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (7)..(7)
<223> m stands for a or c.
<220>
<221> misc_feature
<222> (8)..(8)
<223> n stands for a, c, g or t.
<220>
<221> misc_feature
<222> (9)..(9)
<223> n stands for a, c, g or t.
<400> 24
cagggtmnnc cacatgctca gtgctttaa 29
Claims (9)
1.一种NADH偏好型草铵膦脱氢酶突变体,其特征在于,由来源于堆肥赖氨酸杆菌(Lysinibacillus composti)的野生型草铵膦脱氢酶突变所得,野生型草铵膦脱氢酶的氨基酸序列如SEQ ID No.2所示,所述NADH偏好型草铵膦脱氢酶突变体的突变位点选自以下一种:
(1)A144G-V375F-M91A;
(2)A144G-V345A-M91A;
(3)A144G。
2.编码如权利要求1所述NADH偏好型草铵膦脱氢酶突变体的基因。
3.一种基因工程菌,包括宿主细胞和转入宿主细胞的目的基因,其特征在于,所述目的基因包含如权利要求2所述的基因。
4.如权利要求3所述的基因工程,其特征在于,目的基因还包括葡萄糖脱氢酶的编码基因。
5.如权利要求4所述的基因工程,其特征在于,葡萄糖脱氢酶的编码基因序列对应的GenBank登录号为KM817194.1。
6.权利要求1所述NADH偏好型草铵膦脱氢酶突变体、权利要求2所述基因或权利要求3~5任一所述基因工程菌在制备L-草铵膦中的应用。
7.一种L-草铵膦的制备方法,其特征在于,以2-羰基-4-(羟基甲基膦酰基)-丁酸为底物,在无机氨基供体、辅酶再生循环系统及对应辅助底物存在的条件下,利用催化剂催化底物反应获得L-草铵膦;
所述催化剂为以下一种:
(1)权利要求1所述NADH偏好型草铵膦脱氢酶突变体;
(2)能够表达权利要求1所述NADH偏好型草铵膦脱氢酶突变体的基因工程菌或该基因工程菌经裂解得到的粗酶液。
8.如权利要求7所述的制备方法,其特征在于,所述辅酶再生循环系统使用葡萄糖脱氢酶、甲酸脱氢酶或醇脱氢酶偏好型的辅酶再生循环系统。
9.功能序列和结构模拟相结合的基因挖掘方法,其特征在于,包括以下步骤:
(1)分析NADH型谷氨酸脱氢酶应具备的特性序列,特征序列包括:
(1.1)蛋白质大小:候选蛋白质的长度为300-500个氨基酸,
(1.2)草铵膦脱氢酶的两段必要特性序列:第一段为GGGKGG,其中X位置表示任意一种氨基酸,同时第二段为VVTG、FVTG、VLTG、VFTG、FITG、FFTG、VVFG、FVFTG、VLFG、VFFG、FLFG、FFFG其中之一,
(1.3)NADH结合的特性序列:GXRVXXG;
(2)根据特征序列搜索基因库;
(3)对步骤(2)搜索获得的基因进行聚类分析和蛋白结构模拟;
(4)选择基因聚合度高且蛋白结构与已知草铵膦脱氢酶结构类似的基因作为候选基因。
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