CN110184246B - 谷氨酸脱氢酶突变体及其应用 - Google Patents
谷氨酸脱氢酶突变体及其应用 Download PDFInfo
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- CN110184246B CN110184246B CN201910403331.2A CN201910403331A CN110184246B CN 110184246 B CN110184246 B CN 110184246B CN 201910403331 A CN201910403331 A CN 201910403331A CN 110184246 B CN110184246 B CN 110184246B
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- glufosinate
- glutamate dehydrogenase
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- ammonium
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Abstract
本发明公开了谷氨酸脱氢酶突变体及其应用,该突变体为下列之一:将SEQ ID NO.1所示氨基酸序列的第402位赖氨酸突变为苯丙氨酸或天冬氨酸;第406位异亮氨酸突变为苯丙氨酸或苏氨酸;第121位苏氨酸和123位亮氨酸进行组合突变;第379位丙氨酸和383位亮氨酸进行组合突变。本发明通过定向进化与半理性设计相结合的分子改造方法,显著提高了源于Pseudomonas putida的谷氨酸脱氢酶对2‑羰基‑4‑(羟基甲基膦酰基)丁酸(PPO)的催化活力;解决了还原胺化法制备L‑草铵膦工艺中谷氨酸脱氢酶酶活低的问题。
Description
技术领域
本发明涉及酶工程技术领域,尤其涉及谷氨酸脱氢酶突变体及其在催化2-羰基-4-(羟基甲基膦酰基)丁酸制备L-草铵膦中的应用。
技术背景
草铵膦(Phosphinothricin,又名Glufosinate)是一种含磷氨基酸类灭生性除草剂,化学名称为2-氨基-4-(羟基甲基膦酰基)丁酸,是全球第三大灭生性除草剂及第二大转基因作物除草剂。草铵膦具有杀草谱广、低毒、活性高、部分传导性和环境相容性好等特点,被广泛应用于非耕地、免耕地、农田作物、水生田等杂草防除。草铵膦的两种构型中,只有L-型具有除草活性(Herbicidal compositions[P].Patent application US4265654 A,1981),而且在土壤中易于降解,对环境的危害较小。
目前,市场销售的草铵膦都是外消旋体混合物,若能够以L-构形的纯光学异构体形式使用,草铵膦施用量将降低50%,可显著提高经济性、减轻环境压力。因此,光学纯L-草铵膦制备工艺的开发具有非常重要的意义。
谷氨酸脱氢酶(Glutamate dehydrogenase,EC 1.4.1.2–1.4.1.4)来源丰富且性质多样,在手性L-氨基酸合成领域具有非常大的应用潜力(L-glutamate dehydrogenases:Distribution,properties and mechanism[J].Comparative Biochemistry&PhysiologyB Comparative Biochemistry,1993,106(4):767-792.)。利用谷氨酸脱氢酶催化2-羰基-4-(羟基甲基膦酰基)丁酸(PPO)不对称还原胺化制备L-草铵膦,具有立体选择性严格、理论产率能够达到100%、产物易于分离提纯等显著优势。
在公布号为CN106978453A的发明专利申请文献中,我们将来源于Pseudomonasputida的谷氨酸脱氢酶(PpGluDH,NCBI登录号:NP_742836.1)基因进行了克隆并在大肠杆菌中过量表达。该重组谷氨酸脱氢酶能够还原胺化2-羰基-4-(羟基甲基膦酰基)丁酸生成光学纯度较高的L-草铵膦。但是,从工业化应用的角度来讲,PpGluDH的催化活力较低,这会带来催化剂制备成本过高、后处理困难等一系列问题。因此,通过分子改造提高PpGluDH对PPO的催化活力是实现该L-草铵膦还原胺化合成工艺工业化应用的关键。
发明内容
本发明针对来源于Pseudomonas putida的谷氨酸脱氢酶对非天然底物2-羰基-4-(羟基甲基膦酰基)丁酸催化活力低的问题,提供了多个酶活显著提高的谷氨酸脱氢酶突变体及其在L-草铵膦合成中的应用。
具体技术方案如下:
本发明提供了一类谷氨酸脱氢酶突变体,所述谷氨酸脱氢酶突变体为下列之一:
(1)将SEQ ID NO.1所示氨基酸序列的第402位赖氨酸突变为苯丙氨酸或天冬氨酸;
(2)将SEQ ID NO.1所示氨基酸序列的第406位异亮氨酸突变为苯丙氨酸或苏氨酸;
(3)将SEQ ID NO.1所示氨基酸序列的第121位苏氨酸和123位亮氨酸分别突变为丝氨酸和组氨酸,或天冬酰胺和酪氨酸,或缬氨酸和酪氨酸,或丝氨酸和丝氨酸,或天冬酰胺和苯丙氨酸,或缬氨酸和苯丙氨酸,或异亮氨酸和酪氨酸;
(4)将SEQ ID NO.1所示氨基酸序列的第379位丙氨酸和383位亮氨酸分别突变为异亮氨酸和酪氨酸,或异亮氨酸和半胱氨酸,或丝氨酸和酪氨酸,或缬氨酸和丝氨酸,或苯丙氨酸和半胱氨酸,或酪氨酸和半胱氨酸,或半胱氨酸和半胱氨酸。
本发明利用易错PCR技术对来源于Pseudomonas putida的谷氨酸脱氢酶基因(Genebank ID:1044501)引入随机突变,构建随机突变文库,随后利用高通量筛选方法对该突变库进行筛选,获得的阳性突变株再利用HPLC法对其酶活进行复筛。复筛获得的对PPO催化活力有所提高的突变株通过测序分析其具体的突变情况。
基于上述定向进化获得阳性突变株的突变情况,设计并构建了三个突变文库,包含402位的饱和突变库、121位与123位的组合突变库、379位与383位的组合突变库。通过高通量筛选技术对这三个突变库进行筛选,获得的阳性突变株再利用HPLC法对其酶活进行复筛,复筛获得的对PPO催化活力有所提高的突变株通过测序分析其具体的突变情况。
本发明还提供了所述谷氨酸脱氢酶突变体的编码基因。
本发明还提供了包含所述谷氨酸脱氢酶突变体编码基因的表达载体与工程菌。
本发明还提供了所述谷氨酸脱氢酶突变体在催化2-羰基-4-(羟基甲基膦酰基)丁酸或其盐制备L-草铵膦中的应用。
本发明还提供了一种催化2-羰基-4-(羟基甲基膦酰基)丁酸或其盐制备L-草铵膦的方法,包括:
(1)制备表达谷氨酸脱氢酶突变体的基因工程菌,所述谷氨酸脱氢酶突变体如权利要求1所述;
(2)培养所述基因工程菌,制备酶液;
(3)将所述酶液加入含有底物2-羰基-4-(羟基甲基膦酰基)丁酸、氨基供体及还原型辅酶的混合体系中,进行还原胺化反应,制得L-草铵膦。
步骤(2)中,所述的酶液为基因工程菌的静息细胞悬液或者破胞粗酶液;当然,将该粗酶液进行纯化获得的纯酶也适用本发明所述的L-草铵膦制备方法。
作为优选,步骤(3)中,所述还原胺化反应的温度为15~50℃,反应液的pH值为6~10。
步骤(3)中,所述还原型辅酶为还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)。
作为优选,步骤(3)的反应体系中,还包括辅酶再生系统;所述辅酶再生系统为:以葡萄糖脱氢酶为辅酶再生酶、以葡萄糖为辅酶再生底物的辅酶再生系统。
更优选,步骤(3)中的反应体系中,所述葡萄糖脱氢酶基因(BsGDH-2M)来源于Bacillus subtilis,并通过分子改造提高其稳定性,其编码的氨基酸序列为SEQ ID NO.2
进一步地,步骤(3)中,所述氨基供体为硫酸铵。
与现有技术相比,本发明具有以下有益效果:
本发明通过定向进化与半理性设计相结合的分子改造方法,显著提高了源于Pseudomonas putida的谷氨酸脱氢酶对2-羰基-4-(羟基甲基膦酰基)丁酸(PPO)的催化活力;解决了还原胺化法制备L-草铵膦工艺中谷氨酸脱氢酶酶活低的问题;本发明谷氨酸脱氢酶突变体对PPO的催化活力显著提高,单位发酵液的酶活最高达11.21U/mL,为野生型的102倍;本发明谷氨酸脱氢酶突变体在L-草铵膦的制备中,表现出良好的催化效率,底物转化率>99%,L-草铵膦浓度最高达80.6g/L,ee值>99%,具有非常大的工业应用前景;本发明成功的解决了氨化还原反应制备L-草铵膦过程中的生物催化剂活力低的关键问题,为实现该工艺的工业化应用奠定了基础。
附图说明
图1为实施案例1中PpGluDH的野生型及突变体的活力测定结果。
图2为原料2-羰基-4-(羟基甲基膦酰基)丁酸(简称PPO)的质谱图。
图3为原料2-羰基-4-(羟基甲基膦酰基)丁酸(简称PPO)的核磁图。
其中,A图为PPO的1H NMR谱图;B图为PPO的13C NMR谱图。
图4为底物2-羰基-4-(羟基甲基膦酰基)丁酸的标样高效液相检测图谱(非手性分析,5mM);其中,底物PPO的保留时间为9.7min。
图5为外消旋草胺膦标样柱前衍生化高效液相图谱(手性分析,2mM);
其中,保留时间:L-草铵膦为6.3min,D-草铵膦为7.2min。
图6为实施案例4中反应液(反应结束后)柱前衍生化高效液相检测图谱(手性分析)。
具体实施方式
以下结合具体实施例,对本发明做进一步描述。应理解,以下实施例仅用于说明本发明而非用于限制本发明的范围。
本发明中的实验方法如无特别说明均为常规方法,基因克隆操作具体可参见J.萨姆布鲁克等编的《分子克隆实验指南》。
上游基因工程操作所用试剂:本发明实施例1中用于易错PCR的DNA聚合酶(EasyTaq DNA Polymerase)购自北京全式金生物;本发明实施例2中用于DNA聚合酶(Max DNA Polymerase)和Dpn I购自TaKaRa,宝生物工程(大连)有限公司;重组克隆试剂盒(ClonExpress II One Step Cloning Kit)购自南京诺唯赞生物科技有限公司;质粒提取试剂盒购自Axygen杭州有限公司;E.coli BL21(DE3)、质粒等购自Novagen公司;引物合成与基因测序工作由杭州擎科梓熙生物技术有限公司完成。以上试剂使用方法参考商品说明书。
本发明所涉及带有谷氨酸脱氢酶基因的重组大肠杆菌,由本实验室构建与保藏。所用载体为pET-28a(+),所用宿主为大肠杆菌BL21(DE3)。
下游催化工艺所用试剂:2-羰基-4-(羟基甲基膦酰基)丁酸(简称PPO)为人工合成,其质谱图与核磁图如附图2,3所示;DL-草铵膦标准品购自Sigma-Aldrich公司;NADP+与NADPH购于邦泰生物工程(深圳)有限公司;其他常用试剂购自国药集团化学试剂有限公司。在本申请文本中所使用的氨基酸三字母或单字母表达方式,采用IUPAC规定的氨基酸代码(Eur.J.Biochem.,138:9-37,1984)。
谷氨酸脱氢酶酶活标准检测体系:适量的酶液、100mM底物、10mM NADPH、500mMNH4 +((NH4)2SO4),总体系为400μL,反应介质为pH 7.5 0.1M磷酸盐缓冲液。35℃反应10min,反应结束后加入40uL 5M NaOH终止反应。样品中生成的L-草铵膦浓度通过柱前衍生化HPLC进行定量。
酶活单位(U)的定义:在标准的反应条件下,每分钟生成1μmol L-草铵膦所需要的酶量。
本发明通过高效液相色谱(HPLC)分析反应液中底物的浓度,监测反应的进行。HPLC分析方法为:色谱柱型号:QS-C18,5μm,4.6mm×250mm。流动相:50mM(NH4)2HPO4,加入1%的10%四丁基氢氧化铵水溶液,用50%磷酸(质量分数)调pH至3.6,加入8%乙腈。检测波长:205nm。流速:1.0mL/min。柱温:40℃。底物出峰情况见附图4。
L-草铵膦的手性分析及浓度分析通过柱前衍生化高效液相色谱进行,具体的分析方法为:
(2)衍生化试剂:分别称取0.03g邻苯二甲醛与0.1g N-乙酰-L-半胱氨酸,用400uL乙醇助溶,再加入4mL 0.2mol/L硼酸缓冲液(pH 9.8),振荡使其充分溶解,4℃冰箱保存备用(不超过4天)。
(3)衍生化反应与测定:取100μL样品加入150μL衍生化试剂,混匀后至于25℃保温5min。加入1mL纯水进行稀释,过膜,进样20uL进行分析。
D-草铵膦与L-草铵膦的出峰情况,见附图5。
实施例1基于易错PCR的Pseudomonas putida来源谷氨酸脱氢酶(PpGluDH)定向进化
步骤一:PpGluDH重组菌的活化及质粒提取
将带有pET-28a(+)-PpGluDH重组质粒的大肠杆菌工程菌使用LB培养基进行活化与培养。
LB培养基的具体配方为:蛋白胨10g/L,酵母粉5g/L,NaCl 10g/L,用去离子水溶解后定容,121℃灭菌20min,待用。固体培养基为LB培养基加入2%的琼脂。
将保存有PpGluDH重组菌的甘油管划线至含有LB固体培养基(50μg/mL卡那霉素)的平皿上,37℃静置培养12h。从平皿上挑取单菌落接入含50μg/mL卡那霉素的5mL LB培养基中,37℃、200rpm培养12h。在获得培养液后,根据质粒提取试剂盒的操作说明书进行质粒的提取。
步骤二:随机突变库的构建
以步骤一中提取的pET-28a(+)-PpGluDH质粒为模版,通过易错PCR(Error-pronePCR)向PpGluDH基因引入随机突变,所用引物及PCR反应体系分别见表1、表2。
表1易错PCR所用引物
a注:在上、下游引物中分别加入限制性内切酶切位点,如下划线所示,具体限制性内切酶见引物序列括号内
表2 PCR扩增体系
易错PCR扩增条件:
1)预变性94℃3min;
2)变性94℃30s,退火58℃30s,延伸72℃2min(此阶段循环35次);
3)后延伸72℃8min;
4)4℃保存。
PCR扩增结束后,扩增产物用1.0%琼脂糖凝胶电泳来检测,目标条带用DNA回收纯化试剂盒进行纯化回收。回收后的PCR扩增产物及表达载体pET-28a(+)分别用相应的限制性内切酶在37℃双酶切3h。酶切体系如下表所示:
表3酶切体系
酶切完成后用DNA纯化试剂盒对酶切产物进行纯化回收。用T4 DNA连接酶将酶切后的PCR扩增产物连接至具有相对应切口的表达载体pET-28a(+)上,连接体系如下表所示:
表4酶接体系
将酶连产物转化至E.coli BL21(DE3)感受态细胞中,涂平板,置于37℃倒置培养至长出合适大小的单菌落。
步骤三:随机突变库的高通量筛选-初筛
在灭菌的96深孔板中加入200μL的LB培养基(50μg/mL的卡那霉素),使用灭菌枪头挑取单菌落至96深孔板。然后将深孔板置于37℃,200rpm培养8h,称之为一级板。在另外的灭菌的96孔板中加入400μL的LB培养基(50μg/mL的卡那霉素)作为二级板,在一级板中吸取50μL菌液至二级板,一级板加入20%的甘油放置于-80℃冰箱长期保藏。然后将二级板置于37℃进行震荡培养3h,加入终浓度为0.5mM的IPTG诱导,然后将二级板置于18℃,200rpm培养18h。
二级板在4000rpm,4℃下离心20min收集菌体,然后置于-80℃进行冷冻3h以上。将二级板从-80℃取出,室温下放置0.5h解冻,之后在每孔加入300μL溶菌液(10mM pH 7.5磷酸盐缓冲液,750mg/L溶菌酶,10mg/L DNaseⅠ),震荡使细胞悬浮,并置于摇床37℃,200rpm孵育1h。孵育结束后,4000rpm,4℃离心20min,取上清进行酶活测定。
配制筛选用测活混合溶液:pH 7.5磷酸盐缓冲液(0.1M),2mM NADPH,20mM底物PPO,1M NH4 +。向新的96孔板(反应板)各孔中加入200μL测活混合液,并置于37℃保温15min。吸取200μL酶液加入反应板中开始反应,分别于20min,40min和60min取样100uL加入到已经预先加好100uL pH 7.5磷酸缓冲液(0.1M)的酶标板中,用酶标仪测定340nm处吸光值。吸光值越低代表其催化活力越高,挑选吸光值较对照(野生型)显著降低的突变株作为候选菌株进行复筛。
步骤四:复筛
利用HPLC对初筛表现出酶活显著提高的突变体进行复筛。将一级板上相应的突变株经平皿划线活化后,挑单菌落接种至含50μg/mL卡那霉素的5mL LB液体培养基中,37℃震荡培养12h。按2%的接种量转接至50mL同样含50μg/mL卡那霉素的LB液体培养基中,37℃震荡培养至OD600达到0.6~0.8左右时,加入IPTG至其终浓度为0.5mM,18℃下诱导培养16h。培养结束后,将培养液12000g 4℃离心10min,弃上清,收集菌体。将培养结束后收集到的菌体,用50mM pH 7.5的磷酸盐缓冲液洗涤菌体两次后,重悬于pH 7.5的磷酸盐缓冲液中,400W功率超声破碎30次,每次超声持续3s,间歇7s。将此细胞破碎液12000g4℃离心10min去除沉淀,得到的上清为粗酶液。
按照标准酶活检测体系对各突变株对PPO的粗酶液酶活进行测定。最终获得13株酶活具有显著提高的突变体,其中包含8个单点突变株、4个两点突变株及1个三点突变株,单位体积发酵液酶活为0.167-0.897U/mL,是原始谷氨酸脱氢酶的1.5-8.2倍,具体见附图1。通过回复突变分析了两点及三点突变株中起正向突变作用的突变,确定这些突变株中起正向作用的为V29A、L354P、I406T这三个突变。对PpGluDH定向进化获得的阳性突变进行总结,如表5所示。
表5通过定向进化获得的阳性突变总结
注:a)突变株相对于PpGluDH野生型(0.11U/mL)的酶活提高倍数
实施例2聚焦饱和突变库的构建及筛选
分析实施例1结果,对PpGluDH的121、123、379、383及402位氨基酸残基进行饱和突变。设计引物(表6),以pET-28a(+)-PpGluDH质粒为模版,以K402X-F、T121X/L123X-F或A379X/L383X-F与Aid-R为引物对进行PCR获得线性化短片段,再以K402X-R、T121X/L123X-R或A379X/L383X-R与Aid-F为引物对进行PCR获得线性化长片段。参考重组克隆试剂盒(ClonExpress II One Step Cloning Kit)使用说明书进行后续的DpnⅠ消化、胶回收、重组及转化操作,构建了402位的饱和突变库(K402X)、121位与123位的组合突变库(T121X/L123X)、379位与383位的组合突变库(A379X/L383X)3个突变库。
表6聚焦饱和突变库构建所用引物
通过实施例1所述高通量筛选方法对这3个突变库进行初筛,再通过HPLC法进行复筛。最终从402位的饱和突变库(K402X)中筛选获得两株对PPO酶活显著提高的突变株:K402F与K402D;从121位与123位的组合突变库(T121X/L123X)中筛选出7株对PPO酶活显著提高的突变株:T121S/L123H、T121N/L123Y、T121V/L123Y、T121S/L123S、T121N/L123F、T121V/L123F、T121I/L123Y;从379位与383位的组合突变库(A379X/L383X)中筛选出7株对PPO酶活显著提高的突变株:A379I/L383Y、A379I/L383C、A379S/L383Y、A379V/L383S、A379F/L383C、A379Y/L383C、A379C/L383C。具体的突变及酶活变化情况见表7。
表7突变库的筛选结果
注:a)突变株相对于PpGluDH野生型(0.11U/mL)的酶活提高倍数
实施例3谷氨酸脱氢酶野生型(PpGluDH)、葡萄糖脱氢酶双酶耦联制备L-草铵膦
菌体的培养及粗酶液制备:将保藏有野生型PpGluDH和葡萄糖脱氢酶(BsGDH-2M,SEQ ID NO.2)的工程菌的甘油管经平皿划线活化后,挑单菌落接种至含50μg/mL卡那霉素的50mL LB液体培养基中,37℃震荡培养12h。按2%的接种量转接至1L同样含50μg/ml Kan的新鲜LB液体培养基中,37℃震荡培养至OD600达到0.6左右时,加入IPTG至其终浓度为0.5mM,18℃下诱导培养16h。培养结束后,将培养液12000g 4℃离心10min,收集菌体,超声破胞制备粗酶液。
反应体系为30mL,含有500mM底物PPO,600mM葡萄糖,250mM(NH4)2SO4与0.5mM NADP+。谷氨酸脱氢酶菌体(干重)浓度为1.25g/L,葡萄糖脱氢酶菌体(干重)浓度为1.25g/L。通过水浴控制反应温度为35℃,反应过程通过滴加氨水控制pH为7.5。反应12h后利用非手性HPLC检测PPO的残余浓度,同时利用柱前衍生化高效液相色谱检测L-草铵膦的生成量与ee值。
反应结束数据如下:PPO剩余373mM,转化率25.4%。L-草铵膦的生成浓度为20.5g/L,ee值>99%。
实施例4谷氨酸脱氢酶突变株(PpGluDH-T121N/L123Y),葡萄糖脱氢酶双酶耦联制备L-草铵膦
按实施例3中同样的方法培养表达谷氨酸脱氢酶(PpGluDH-T121N/L123Y)和葡萄糖脱氢酶(BsGDH-2M,SEQ ID NO.2)的工程菌,离心收集细胞,并超声破胞制备粗酶液。
反应体系为30mL,含有500mM底物PPO,600mM葡萄糖,250mM(NH4)2SO4与0.5mM NADP+。谷氨酸脱氢酶菌体(干重)浓度为1.25g/L,葡萄糖脱氢酶菌体(干重)浓度为1.25g/L。通过水浴控制反应温度为35℃,反应过程通过滴加氨水控制pH为7.5。反应2.5h后利用非手性HPLC检测PPO的残余浓度,同时利用柱前衍生化高效液相色谱检测L-草铵膦的生成量与ee值。
反应结束数据如下:PPO剩余2.4mM,转化率99.5%。L-草铵膦的生成浓度为80.6g/L,ee值>99%(附图6)。
实施例5谷氨酸脱氢酶突变株(PpGluDH-A379C/L383C),葡萄糖脱氢酶双酶耦联制备L-草铵膦
按实施例3中同样的方法培养表达谷氨酸脱氢酶(PpGluDH-A379C/L383C)和葡萄糖脱氢酶(BsGDH-2M,SEQ ID NO.2)的工程菌,离心收集细胞,并超声破胞制备粗酶液。
反应体系为30mL,含有500mM底物PPO,600mM葡萄糖,250mM(NH4)2SO4与0.5mM NADP+。谷氨酸脱氢酶菌体(干重)浓度为1.25g/L,葡萄糖脱氢酶菌体(干重)浓度为1.25g/L。通过水浴控制反应温度为35℃,反应过程通过滴加氨水控制pH为7.5。反应5h后利用非手性HPLC检测PPO的残余浓度,同时利用柱前衍生化高效液相色谱检测L-草铵膦的生成量与ee值。
反应结束数据如下:PPO剩余3.4mM,转化率99.3%。L-草铵膦的生成浓度为79.4g/L,ee值>99%。
实施例6谷氨酸脱氢酶突变株(PpGluDH-I406F),葡萄糖脱氢酶双酶耦联制备L-草铵膦
按实施例3中同样的方法培养表达谷氨酸脱氢酶(PpGluDH-I406F)和葡萄糖脱氢酶(BsGDH-2M,SEQ ID NO.2)的工程菌,离心收集细胞,并超声破胞制备粗酶液。
反应体系为30mL,含有500mM底物PPO,600mM葡萄糖,250mM(NH4)2SO4与0.5mM NADP+。谷氨酸脱氢酶菌体(干重)浓度为1.25g/L,葡萄糖脱氢酶菌体(干重)浓度为1.25g/L。通过水浴控制反应温度为35℃,反应过程通过滴加氨水控制pH为7.5。反应10h后利用非手性HPLC检测PPO的残余浓度,同时利用柱前衍生化高效液相色谱检测L-草铵膦的生成量与ee值。
反应结束数据如下:PPO剩余4.6mM,转化率99.1%。L-草铵膦的生成浓度为78.7g/L,ee值>99%。
实施例7谷氨酸脱氢酶突变株(PpGluDH-K402F),葡萄糖脱氢酶双酶耦联制备L-草铵膦
按实施例3中同样的方法培养表达谷氨酸脱氢酶(PpGluDH-K402F)和葡萄糖脱氢酶(BsGDH-2M,SEQ ID NO.2)的工程菌,离心收集细胞,并超声破胞制备粗酶液。
反应体系为30mL,含有500mM底物PPO,600mM葡萄糖,250mM(NH4)2SO4与0.5mM NADP+。谷氨酸脱氢酶菌体(干重)浓度为1.25g/L,葡萄糖脱氢酶菌体(干重)浓度为1.25g/L。通过水浴控制反应温度为35℃,反应过程通过滴加氨水控制pH为7.5。反应12h后利用非手性HPLC检测PPO的残余浓度,同时利用柱前衍生化高效液相色谱检测L-草铵膦的生成量与ee值。
反应结束数据如下:PPO剩余37.6mM,转化率92.5%。L-草铵膦的生成浓度为72.8g/L,ee值>99%。
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Claims (10)
1.谷氨酸脱氢酶突变体,其特征在于,所述谷氨酸脱氢酶突变体为下列之一:
(1)将SEQ ID NO.1所示氨基酸序列的第402位赖氨酸突变为苯丙氨酸或天冬氨酸;
(2)将SEQ ID NO.1所示氨基酸序列的第406位异亮氨酸突变为苯丙氨酸或苏氨酸;
(3)将SEQ ID NO.1所示氨基酸序列的第121位苏氨酸和123位亮氨酸分别突变为丝氨酸和组氨酸,或天冬酰胺和酪氨酸,或缬氨酸和酪氨酸,或丝氨酸和丝氨酸,或天冬酰胺和苯丙氨酸,或缬氨酸和苯丙氨酸,或异亮氨酸和酪氨酸;
(4)将SEQ ID NO.1所示氨基酸序列的第379位丙氨酸和383位亮氨酸分别突变为异亮氨酸和酪氨酸,或异亮氨酸和半胱氨酸,或丝氨酸和酪氨酸,或缬氨酸和丝氨酸,或苯丙氨酸和半胱氨酸,或酪氨酸和半胱氨酸,或半胱氨酸和半胱氨酸。
2.如权利要求1所述的谷氨酸脱氢酶突变体的编码基因。
3.包含权利要求2所述编码基因的表达载体与工程菌。
4.如权利要求1所述的谷氨酸脱氢酶突变体在催化2-羰基-4-(羟基甲基膦酰基)丁酸或其盐制备L-草铵膦中的应用。
5.一种催化2-羰基-4-(羟基甲基膦酰基)丁酸或其盐制备L-草铵膦的方法,其特征在于,包括:
(1)制备表达谷氨酸脱氢酶突变体的基因工程菌,所述谷氨酸脱氢酶突变体如权利要求1所述;
(2)培养所述基因工程菌,制备酶液;
(3)将所述酶液加入含有底物2-羰基-4-(羟基甲基膦酰基)丁酸、氨基供体及还原型辅酶的体系中,进行还原胺化反应,制得L-草铵膦。
6.如权利要求5所述的催化2-羰基-4-(羟基甲基膦酰基)丁酸或其盐制备L-草铵膦的方法,其特征在于,步骤(3)中,所述还原胺化反应的温度为15~50℃,反应液的pH值为6~10。
7.如权利要求5所述的催化2-羰基-4-(羟基甲基膦酰基)丁酸或其盐制备L-草铵膦的方法,其特征在于,步骤(3)中,所述还原型辅酶为NADPH。
8.如权利要求5所述的催化2-羰基-4-(羟基甲基膦酰基)丁酸或其盐制备L-草铵膦的方法,其特征在于,步骤(3)的反应体系中,还包括辅酶再生系统;所述辅酶再生系统为:以葡萄糖脱氢酶为辅酶再生酶、以葡萄糖为辅酶再生底物的辅酶再生系统。
9.如权利要求8所述的催化2-羰基-4-(羟基甲基膦酰基)丁酸或其盐制备L-草铵膦的方法,其特征在于,所述葡萄糖脱氢酶的氨基酸序列如SEQ ID NO.2所示。
10.如权利要求5所述的催化2-羰基-4-(羟基甲基膦酰基)丁酸或其盐制备L-草铵膦的方法,其特征在于,步骤(3)中,所述氨基供体为硫酸铵。
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