CN116555206A - 一种d-氨基酸氧化酶及其在制备l-草铵膦或其中间体中的应用 - Google Patents
一种d-氨基酸氧化酶及其在制备l-草铵膦或其中间体中的应用 Download PDFInfo
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- CN116555206A CN116555206A CN202210114692.7A CN202210114692A CN116555206A CN 116555206 A CN116555206 A CN 116555206A CN 202210114692 A CN202210114692 A CN 202210114692A CN 116555206 A CN116555206 A CN 116555206A
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- amino acid
- acid oxidase
- glufosinate
- salt
- oxidase
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Abstract
本发明公开了一种D‑氨基酸氧化酶及其在制备L‑草铵膦或其中间体中的应用。一种D‑氨基酸氧化酶,其氨基酸序列与SEQ ID NO:1相比包含选自以下一个或多个的氨基酸残基差异:K29G/H/I/N/Q/W/Y/C/L;V42C/D/E/H/Y;E195N/Y/Q;C234L;V326W;并具有不低于如SEQ ID NO:1的氨基酸序列所示的D‑氨基酸氧化酶的活性和/或热稳定性。本发明提供了一种热稳定性更高的D‑氨基酸氧化酶,在提高酶活性的同时,也扩大了酶的使用温度范围,且在较低温度下使用,能够延长酶的使用寿命;在较高温度下使用,可提高酶的催化效率。
Description
技术领域
本发明属于生物技术领域,具体涉及一种D-氨基酸氧化酶及其在制备L-草铵膦或其中间体中的应用。
背景技术
草铵膦与草甘膦、百草枯并称世界三大除草剂,都具有非选择性。其中,由于百草枯极强的毒性,近年来在多个国家严被格限制或禁止使用;草甘膦的应用广泛,但我国A级绿色食品已将其从准用名单中去除。草铵膦具有活性高、除草谱广、环境相容性好等特点,并且其发挥除草活性的速度优于草甘膦,也可在杂草对草甘膦产生抗性的地区使用,因此具有很大的市场开发潜力和广阔的应用前景。
2020年,全球草铵膦的市场规模达到10.5亿美元,近6年全球市场复合增长率为6.3%,属于增长最快的非选择性除草剂。因此开发或优化L-草铵膦生产工艺对于提高经济效益、降低使用成本、减轻环境压力具有重要意义。
商业化应用的草铵膦是外消旋体的混合物,有D型和L型两种异构体,但只有L型具有活性,且在土壤中易分解、毒性小。L-草铵膦的生产主要有两类方法:化学法和生物法。化学法生产草铵膦主要是Strecker工艺和热裂解-ACA工艺。其中Srecker工艺流程复杂,涉及原料易燃易爆,且产品的收率较低。热裂解-ACA工艺复杂,且甲基二氯化磷(MDP)是其前端关键中间体,也是该工艺的技术壁垒。生物法反应条件温和、生产流程简单,且产物易于分离,但其产量相对较低,成本较高,目前还未实现产业化。
CN105603015A中发明了以2-氧代-4-(羟基(甲基)膦酰基)丁酸(PPO)及其盐为底物,丙氨酸为氨基供体,利用离体的转氨酶或体外表达转氨酶的细胞催化底物,与丙氨酸发生转氨反应,得到L-草铵膦;CN106978453B中发明了一种利用氨基酸脱氢酶制备L-草铵膦的方法,该方法以2-羰基-4-(羟基甲基膦酰基)丁酸或其盐为底物进行转胺化反应,获得L-草铵膦。此方法原料转化率较高,但底物合成工艺复杂,成本较高。
US9834802B2中发明了以D,L-草铵膦为原料,在D-氨基酸氧化酶突变体的催化作用下生成中间产物PPO,PPO在氨基酸转氨酶的催化作用下生成L-草铵膦。该方法反应得到的组合物含L-草铵膦、D-草铵膦、PPO,其中L-草铵膦含量达到80%以上,D-草铵膦的含量小于10%、PPO含量为小于20%。但是其酶活稳定性仍有待提高。
因此迫切需要寻找一种具有较高的酶活稳定性且D-草铵膦转化率高,L-草铵膦ee值高的D-氨基酸氧化酶。
发明内容
本发明所要解决的技术问题是为了克服现有技术中D-氨基酸氧化酶的酶活性不高、酶活稳定性差和L-草铵膦产率低等缺陷。为解决上述技术问题,本发明提供了一种D-氨基酸氧化酶及其在制备L-草铵膦或其中间体中的应用。热稳定性更高的D-氨基酸氧化酶,在提高酶活性的同时,也扩大了酶的使用温度范围,且在较低温度下使用,能够延长酶的使用寿命;在较高温度下使用,可提高酶的催化效率。热稳定性高的酶具有提高化学反应速率、提高产品质量、活性稳定、耐储藏等优点。
本发明的第一方面提供一种D-氨基酸氧化酶,其氨基酸序列与SEQ ID NO:1相比包含选自以下一个或多个的氨基酸残基差异:
K29G/H/I/N/Q/W/Y/C/L;V42C/D/E/H/Y;E195N/Y/Q;C234L;V326W;
并具有不低于如SEQ ID NO:1的氨基酸序列所示的D-氨基酸氧化酶的活性和/或热稳定性。
本发明中,氨基酸残基间的“/”代表该氨基酸残基对应的位点具有不同的氨基酸残基差异的选择。例如,E195N/Y/Q代表第195位的差异可以是N、Y或Q。
本发明的一些优选的实施例中,所述D-氨基酸氧化酶的氨基酸序列与SEQ ID NO:1相比包含选自以下两个或多个的氨基酸残基差异:V42Y、E195Y、V326W、C234L。
较佳地,本发明所述D-氨基酸氧化酶与SEQ ID NO:1相比包含C234L的氨基酸残基差异,并进一步包含V42Y、E195Y和V326W中的一个或两个的氨基酸残基差异。
更佳地,所述D-氨基酸氧化酶与SEQ ID NO:1相比具有选自以下任一组的氨基酸残基差异:
C234L和V42Y;
C234L和E195Y;
C234L和V326W;
C234L、K29C和V42Y;
C234L、K29G和V42Y;
C234L、K29L和V42Y;
C234L、V326W和V42Y;
C234L、V42Y和E195Y;
C234L、E195Y和V326W。
本发明的另一些优选的实施例中,所述D-氨基酸氧化酶与SEQ ID NO:1相比包含E195Y的氨基酸差异,并进一步包含V42Y或V326W的氨基酸残基差异;
较佳地,所述D-氨基酸氧化酶与SEQ ID NO:1相比具有选自以下任一组的氨基酸残基差异:E195Y和V42Y;
E195Y和V326W;
E195Y、K29Q和V42Y;
E195Y、K29W和V42Y;
E195Y、K29Y和V42Y。
本发明的另一些优选的实施例中,所述的D-氨基酸氧化酶与SEQ ID NO:1相比具有选自以下任一组的氨基酸残基差异:
K29G;K29H;K29I;K29N;K29Q;K29W;K29Y;V42C;V42D;V42E;V42H;V42P;V42Y;E195H;E195N;E195Y;E195Q;C234L;V326W。
本发明的第二方面提供一种分离的核酸,所述核酸编码本发明所述的D-氨基酸氧化酶。
本发明的第三方面提供一种重组表达载体,其包含如本发明所述的核酸。
本发明的第四方面提供一种转化体,其包含如本发明所述的核酸或如本发明所述的重组表达载体。
本发明所述的转化体的宿主细胞较佳地为埃希氏大肠杆菌(Escherichia coli),例如E.coli BL21(DE3)。
本发明的第五方面提供一种制备如本发明所述的D-氨基酸氧化酶的方法,所述方法包括在适于表达所述D-氨基酸氧化酶的条件下培养如本发明所述的转化体。
本发明的第六方面提供一种2-氧代-4-(羟基甲基氧膦基)丁酸或其盐的制备方法,所述制备方法包括以下步骤:在如本发明所述的D-氨基酸氧化酶的存在下,将底物进行氧化反应,即得所述的2-氧代-4-(羟基甲基氧膦基)丁酸或其盐。
所述底物较佳地为D-草铵膦或其盐,所述D-草铵膦或其盐可单独存在,或与L-草铵膦或其盐共同存在,例如所述底物以消旋草铵膦或其盐的形式存在。
所述的氧化反应较佳地在通气的条件下进行;所述通气较佳地为通入空气或氧气;所述通气的速率较佳地为0.5VVM-1.5VVM例如1VVM。
所述的氧化反应较佳地在过氧化氢酶的存在下进行。
所述D-氨基酸氧化酶较佳地以D-氨基酸氧化酶的菌体、粗酶、纯酶或固定化酶的形式存在。
所述底物的浓度较佳地为0.1~0.5mol/L;较佳地为0.17mol/L;
所述的氧化反应的反应体系的pH较佳地为7-9,例如8。
所述的氧化反应的反应体系的温度较佳地为20-50℃,例如25℃。
更佳地,所述D-氨基酸氧化酶的菌体与所述底物的质量比为1:(0.5-3),例如1:1。
进一步更佳地,所述过氧化氢酶与所述D-氨基酸氧化酶的菌体的质量比为1:(20-60);例如1:40。
所述氧化反应的反应体系较佳地包括缓冲液,所述缓冲液优选为磷酸缓冲液,例如磷酸二氢铵和磷酸氢二铵、磷酸氢二钠和磷酸二氢钠。
所述缓冲液用于调控反应体系的pH,较佳地,所述缓冲液的pH为8.0。
本发明的第七方面提供一种L-草铵膦或其盐的制备方法,其包括如下步骤:
(1)通过如本发明所述的制备方法获得2-氧代-4-(羟基甲基氧膦基)丁酸或其盐;
(2)在谷氨酸脱氢酶、无机氨基供体和还原型辅酶的存在下,将步骤(1)获得的2-氧代-4-(羟基甲基氧膦基)丁酸或其盐进行氨化反应,即得所述L-草铵膦或其盐。
在本发明所述的L-草铵膦或其盐的制备方法中,所述还原型辅酶较佳地为NADPH或NADH。
在本发明所述的L-草铵膦或其盐的制备方法中,所述的无机氨基供体较佳地为氨气、硫酸铵、氯化铵、磷酸氢二铵、乙酸铵、甲酸铵和碳酸氢铵中的一种或多种,所述氨气的使用形式较佳地为氨水。
在本发明所述的L-草铵膦或其盐的制备方法中,所述氨化反应的反应体系的pH较佳地为7-10,更佳地为8.4-8.6。
在本发明所述的L-草铵膦或其盐的制备方法中,所述氨化反应的反应温度较佳地为28-35℃,更佳地为30-33℃。
在本发明所述的L-草铵膦或其盐的制备方法中,所述谷氨酸脱氢酶的氨基酸序列较佳地如CN201910434350.1中的突变体1-4。
在本发明所述的L-草铵膦或其盐的制备方法中,所述谷氨酸脱氢酶与所述底物D-草铵膦的质量比较佳地为1:(0.5-3),例如1:1.25。
在本发明所述的L-草铵膦或其盐的制备方法中,所述底物D-草铵膦与所述无机氨基供体的摩尔比较佳地为1:(1~1.5),例如1:1。
本发明所述的L-草铵膦或其盐的制备方法较佳地还包括以下步骤:在脱氢酶以及氢供体的存在下,将氧化型辅酶进行还原反应,得到所述的还原型辅酶即可。
所述的脱氢酶较佳地为葡萄糖脱氢酶、醇脱氢酶或甲酸脱氢酶;
所述的氢供体较佳地为葡萄糖、异丙醇或甲酸盐。
更佳地,当所述的脱氢酶为醇脱氢酶时,所述的氢供体为异丙醇;当所述的脱氢酶为葡萄糖脱氢酶时,所述的氢供体为葡萄糖;当所述的脱氢酶为甲酸脱氢酶时,所述的氢供体为甲酸盐。
本发明所述的L-草铵膦或其盐的制备方法可以分步骤进行,例如先进行氧化反应制备获得2-氧代-4-(羟基甲基氧膦基)丁酸或其盐,再进行氨化反应制备获得L-草铵膦或其盐;也可以通过“一锅法”的方法进行,例如将所有原料混合后制备获得L-草铵膦或其盐。
本发明的第八方面提供一种如本发明所述的D-氨基酸氧化酶在制备L-草铵膦或其盐、2-氧代-4-(羟基甲基氧膦基)丁酸或其盐中的应用。
本发明提及的DAAO酶与D-氨基酸氧化酶可互换地使用。
本发明中D-氨基酸氧化酶作用底物的量都以D-草铵膦或其盐来计算。
在符合本领域常识的基础上,上述各优选条件,可任意组合,即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于:提供了一种热稳定性更高的D-氨基酸氧化酶,在提高酶活性的同时,也扩大了酶的使用温度范围,且在较低温度下使用,能够延长酶的使用寿命;在较高温度下使用,可提高酶的催化效率。
具体实施方式
下面通过实施例的方式进一步说明本发明,但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。
产物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:
(1)色谱条件:Agilent ZORBAX Eclipse plus C18,3.5μm,150*4.6mm。流动相A:0.1%TFA+H2O,流动相B:0.1%TFA+CAN。检测波长:340nm,流速:1.0mL/min,柱温:30℃。
(2)衍生化试剂:Marfey试剂
(3)衍生反应:称取50mg供试品于25ml容量瓶中,加15ml稀释液(纯水:乙腈=50:50)并超声5min,再加纯化水稀释至刻度,混合均匀。移取上述溶液1mL于5mL容量瓶中,加入1mL的Marfey’s reagent溶液和0.1mL碳酸氢钠(1M)溶液,盖上盖子于50℃烘箱中避光加热1小时,反应结束后再加入0.1mL盐酸溶液,混合均匀。
移取1mL上述混合溶液,再加入4mL的稀释液,混合均匀即可倒入进样瓶。进样10μL进行分析。
PPO通过离子对色谱分析,具体的分析方法为:
色谱条件:ΜLtimate AQ-C18,5μm,4.6*250mm;流动相:0.05mol/L磷酸氢二铵PH=3.6:10%四丁基氢氧化铵水溶液:乙腈=91:1:8;检测波长:205nm;流速:1.0mL/min;柱温:25℃。
样品:5mg/mL H2O溶液。进样10μL进行分析。
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加J.萨姆布鲁克等编的《分子克隆实验指南》。
本发明中的氨基酸简写符号如无特殊说明均为本领域常规,具体简写符号对应的氨基酸如表1所示。
表1
所述氨基酸对应的密码子也为本领域常规,具体氨基酸与密码子的对应关系如表2所示。
表2
pET28a质粒和bugbuster protein extraction reagent购买自Novagen公司;NdeI酶、HindIII酶购买自Thermo Fisher公司;E.coli BL21感受态细胞购买自北京鼎国昌盛生物技术有限责任公司;过氧化氢酶购买自山东丰泰生物科技有限公司。
实施例1 D-氨基酸氧化酶(DAAO)的制备
DAAO酶工程菌来源于专利CN111019916B所公开的含seq.79的工程菌株,氨基酸序列如SEQ ID NO:1所示,核苷酸序列如SEQ ID NO:12所示。
LB液体培养基组成: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。
将上述DAAO酶工程菌经平皿划线活化后,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%接种量转接至150mL同样含50μg/mL卡那霉素的新鲜LB液体培养基中,37℃震荡至OD600达到0.8左右时,降温至30℃,加入IPTG至其终浓度为0.5mM,诱导培养16h,培养结束后,将培养液10000rpm离心10min,弃上清液,收集菌体,置于-20℃冰箱中保存,待用。
将培养结束后收集到的菌体,用50mM pH 8.0磷酸缓冲液洗涤两次,之后重悬于50mL pH 8.0的磷酸缓冲液中,均质破碎,破碎液离心去除沉淀,得到含重组DAAO酶的粗酶液。
实施例2 D-氨基酸氧化酶(DAAO)突变体的构建
一、工程菌活化和质粒提取
将实施例1所述活化的DAAO酶工程菌接入含有5mL LB培养基的试管中,37℃、200rpm培养8-12h。在获得培养菌体后,根据生工质粒提取试剂盒的操作说明书进行质粒提取。所得质粒直接用于点突变,或者置于-80℃冰箱长期保存。
二、突变体文库(K29位、V42位点、E195位点、C234位点、V326位点)的构建
基因突变采用全质粒PCR的方法,获得突变基因。
以上述步骤所提取质粒为模板,针对突变的D-氨基酸氧化酶序列的K29位、V42位点、E195位点、C234位点、V326位点进行突变的突变体文库构建设计PCR的引物序列,获得目标突变体的基因。引物序列具体如表3所示:
表3
其中,N代表A、G、C、T中任何一种核苷酸,M代表A或C,K代表G或T;其根据所述位点需突变成的氨基酸的编码核苷酸来选择,如K29-正向引物中的NNK可以代表AAG(赖氨酸)、AAT(天冬氨酸)、AGG(精氨酸)或AGT(丝氨酸)等。
PCR扩增体系为:
表4
试剂 | 用量(μL) |
2×的PCR buffer(含高保真酶) | 25 |
正向引物(10μM) | 1 |
反向引物(10μM) | 1 |
模板 | 1 |
去离子水 | 22 |
PCR扩增程序如下:
表5
加入DpnI酶对PCR产物进行消化,37℃,2h。反应完成转化至E.coli BL21感受态细胞,涂布在含有50μg/mL卡纳霉素的LB培养基,37℃培养过夜,收菌,得到包含突变体文库的转化子。
三、组合突变
将筛出的有益突变,通过overlap PCR的方式,实现多种不同的突变组合,形成新的突变转化子。
实施例3高通量突变体文库初筛
将实施例2中所得转化子接种96孔板培养,加IPTG至其终浓度为0.5mM,30℃过夜诱导。之后收菌,加bugbuster protein extraction reagent裂解,离心取上清液得DAAO突变体酶液。
热稳定性筛选方法:
将上述上清酶液置于60℃水浴锅,热处理20min。再使用“酶活检测分析”,筛选阳性克隆。
酶活检测分析:取100μL pH为8.0的100mM底物(D,L-草铵膦,购自上海阿拉丁生化科技股份有限公司),再加50μL的显色液(含60mg/mL的TBHBA(3-羟基-2,4,6-三溴苯甲酸)和100mg/mL的4-AAP(4-氨基安替比林))和25μL HRP(辣根过氧化物酶,0.1mg/mL),最后加25μL的上述DAAO突变体酶液,得酶标板200μL反应体系,在30℃,pH 8.0的条件下对其进行分析。分别在0min和20min记录510nm处的吸光度,取差值,以野生型为参照系,筛选阳性克隆子。筛选出酶活性与Enz.01相当或更高,且热稳定增强的阳性克隆子且见表6。
表6
实施例4热稳定性提高的突变体复筛
突变体复筛的酶活检测方法:
5mL反应体系中,加入1mL 500mM的D,L-草铵膦(铵盐),0.25mL热处理(60℃,20min)后的DAAO突变体粗酶液,1.25mL辣根过氧化物酶(HRP),2.5mL显色染料溶液(含60mg/mL的TBHBA和100mg/mL的4-AAP),反应介质为pH为8.0的磷酸氢二钠-磷酸二氢钠缓冲液,于30℃摇床震荡反应,每隔2min,取反应液扫描510nm下的吸光值,做吸光度和时间(min)的酶反应动力学曲线,根据曲线斜率计算酶活。
单位酶活的定义:在特定反应条件(30℃)下,每分钟生成1μmol H2O2所需要的酶量,酶活单位是U。
根据以上酶活测定方法,检测热处理前和热处理后酶活,计算热稳定性相对Enz.1提高的倍数。结果见下表7。*表示热稳定性提高1~1.2倍(不含1.2),**表示热稳定性提高1.2~2倍(不含2),***表示热稳定性提高2倍及以上。热稳定性提高倍数计算方法:突变体热处理后酶活与处理前酶活比/Enz.01热处理后酶活与处理前酶活比。
表7
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如上表所述,说明多数单点突变酶和组合突变酶热稳定的提高效果显著,Enz.27、Enz.29、Enz.32效果更佳。
实施例5 L-草铵膦的制备
本发明涉及的反应路线如下所示:
根据来源于短乳杆菌(Lactobacillus brevis KB290)(GenBank登录号为BAN05992.1)的Cyclopentanol dehydrogenase基因序列,全合成醇脱氢酶基因。
LB液体培养基组成: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。
醇脱氢酶基因连pET28a,酶切位点NdeI&HindIII,将重组载体转化至宿主E.coliBL21(DE3)感受态细胞,得到含有醇脱氢酶基因的工程菌株。将含有醇脱氢酶基因的工程菌在经平皿划线活化后,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%接种量转接至50ml同样含50μg/mL卡那霉素的新鲜LB液体培养基中,37℃震荡至OD600达到0.8左右时,加入IPTG至其终浓度为0.5mM,18℃诱导培养16h。培养结束后,将培养液10000rpm离心10min,弃上清液,收集菌体,置于-20℃超低温冰箱中保存,待用。
酶液制备:实施例4所筛选出的酶活性高的菌体(酶编号为Enz.27、Enz.29、Enz.32)置于pH 7.0的50mmol/L磷酸铵缓冲液中均质,菌体与缓冲液比例为1:5(g:mL),均质后加絮凝剂絮凝离心得上清。
向10L夹套反应釜中加入4600g水,投入0.28g磷酸二氢铵和5.57g磷酸氢二铵搅拌溶清后向反应釜内投入400g D,L-草铵膦(铵盐),搅拌溶解,氨水调节溶液pH 7.9-8.1,向反应釜内投入5g过氧化氢酶(酶活80万μ/g),投入1000mL(200g菌体)突变体DAAO酶液(Enz.27、Enz.29或Enz.32),空气鼓气,通气量控制在每分钟1反应体积,控制温度25℃,反应20h。
控制温度为30-33℃,加入0.4g NADP+、78g异丙醇、50g氯化铵,氨水调节pH 8.4-8.6,加入0.35g乙醇脱氢酶(ADH)菌体。待温度、pH正常,向釜内投入160g谷氨酸脱氢酶(即本公司CN201910434350.1中突变体1-4)菌体,开始反应。反应6h,检测PPO残留量≤2%。
表8
反应结果如表8所示;Enz.27、Enz.29和Enz.32反应24h后的ee值均在98%以上,显著优于Enz.1。
SEQUENCE LISTING
<110> 弈柯莱生物科技(上海)股份有限公司
<120> 一种D-氨基酸氧化酶及其在制备L-草铵膦或其中间体中的应用
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aaagaagctg gtccgcgtca agctaaatgg gaggaagcaa ccttcaaaca gtgggtggac 240
ttcgttccgc aaggtctggc aatgtggctg aaaggtaccc gccgctttgc agaaaccgaa 300
gccgatctgc tgggccattg gtataaggac atcgtgccga attaccgcca tctgaacccg 360
agcgattgtc cgccgggcgc aattggcgtg acctacgata ctttaagcgt taacgccccg 420
aagttctgtc agtatctgca gcgcgaagca cagaaactgg gcgtgacctt cgaacgccgt 480
ttagttacct ctttagagca gattgcagat ggcgccgatc tgatcgttaa tgcaaccggt 540
ctgggcgcca aaagcatcgc cggtgtggaa gatcaagaag tggaacctat ccgcggccaa 600
acagtgctga ttaagagcaa ctgtaaacgc ctgaccactg acagtagtga cccgaaaagc 660
ccggcctaca ttattccgcg cccgggtggt gaagtgatct gtggtggtac ctatttagtg 720
ggtaactatg atttaagcgt ggatccggcc accattccgc gcattttaaa acattgtctg 780
cgtttagacc cgagtattag caccgacggc acactggaag gcatcgaaat tttacgccat 840
aacgttggtc tgcgtcccgc tcgtcgtggt ggtccgcgtg tggaattaga acgcgtgagc 900
ttcccgctga agcgcggtca gagtctgtta gctttaggca ccgccaaagc agcagaaggt 960
aaagcaagcc gtaccgtgcc ggttgttcat gcctatggct tcagcagcgc cggttaccaa 1020
caaggttggg gcgcagcttt agaagttcgt gatctggtgg accaagctat tggtagcagc 1080
agtagtgcca gtagtggccg ttatttagcc aaactg 1116
Claims (14)
1.一种D-氨基酸氧化酶,其氨基酸序列与SEQ ID NO:1相比包含选自以下一个或多个的氨基酸残基差异:
K29G/H/I/N/Q/W/Y/C/L;V42C/D/E/H/Y;E195N/Y/Q;C234L;V326W;
并具有不低于如SEQ ID NO:1的氨基酸序列所示的D-氨基酸氧化酶的活性和/或热稳定性。
2.如权利要求1所述的D-氨基酸氧化酶,其特征在于,所述D-氨基酸氧化酶的氨基酸序列与SEQ ID NO:1相比包含选自以下两个或多个的氨基酸残基差异:V42Y、E195Y、V326W、C234L。
3.如权利要求2所述的D-氨基酸氧化酶,其特征在于,所述D-氨基酸氧化酶与SEQ IDNO:1相比包含C234L的氨基酸残基差异,并进一步包含V42Y、E195Y和V326W中的一个或两个的氨基酸残基差异;
较佳地,所述D-氨基酸氧化酶与SEQ ID NO:1相比具有选自以下任一组的氨基酸残基差异:
C234L和V42Y;
C234L和E195Y;
C234L和V326W;
C234L、K29C和V42Y;
C234L、K29G和V42Y;
C234L、K29L和V42Y;
C234L、V326W和V42Y;
C234L、V42Y和E195Y;
C234L、E195Y和V326W。
4.如权利要求2所述的D-氨基酸氧化酶,其特征在于,所述D-氨基酸氧化酶与SEQ IDNO:1相比包含E195Y的氨基酸差异,并进一步包含V42Y或V326W的氨基酸残基差异;
较佳地,所述D-氨基酸氧化酶与SEQ ID NO:1相比具有选自以下任一组的氨基酸残基差异:
E195Y和V42Y;
E195Y和V326W;
E195Y、K29Q和V42Y;
E195Y、K29W和V42Y;
E195Y、K29Y和V42Y。
5.如权利要求1所述的D-氨基酸氧化酶,其特征在于,其氨基酸序列与SEQ ID NO:1相比具有选自以下任一组的氨基酸残基差异:
K29G;K29H;K29I;K29N;K29Q;K29W;K29Y;V42C;V42D;V42E;V42H;V42P;V42Y;E195H;E195N;E195Y;E195Q;C234L;V326W。
6.一种分离的核酸,其特征在于,所述核酸编码如权利要求1-5任一项所述的D-氨基酸氧化酶。
7.一种重组表达载体,其包含如权利要求6所述的核酸。
8.一种转化体,其包含如权利要求6所述的核酸或如权利要求7所述的重组表达载体;较佳地,所述转化体的宿主细胞为埃希氏大肠杆菌(Escherichia coli)例如E.coliBL21(DE3)。
9.一种制备如权利要求1-5任一项所述的D-氨基酸氧化酶的方法,其特征在于,所述方法包括在适于表达所述D-氨基酸氧化酶的条件下培养如权利要求8所述的转化体。
10.一种2-氧代-4-(羟基甲基氧膦基)丁酸或其盐的制备方法,其特征在于,所述制备方法包括以下步骤:在如权利要求1-5任一项所述的D-氨基酸氧化酶的存在下,将底物进行氧化反应,即得所述的2-氧代-4-(羟基甲基氧膦基)丁酸或其盐;
较佳地:
所述底物为D-草铵膦或其盐,所述D-草铵膦或其盐可单独存在,或与L-草铵膦或其盐共同存在;例如所述底物以消旋草铵膦或其盐的形式存在;
和/或,所述的氧化反应在通气的条件下进行;所述通气较佳地为通入空气或氧气;所述通气的速率较佳地为0.5VVM-1.5VVM例如1VVM;
和/或,所述的氧化反应在过氧化氢酶的存在下进行;
和/或,所述D-氨基酸氧化酶以D-氨基酸氧化酶的菌体、粗酶、纯酶或固定化酶的形式存在;
和/或,所述底物的浓度为0.1~0.5mol/L;较佳地为0.17mol/L;
和/或,所述的氧化反应的反应体系的pH为7-9,较佳地为8;
和/或,所述的氧化反应的反应体系的温度为20-50℃,较佳地为25℃;
更佳地,所述D-氨基酸氧化酶的菌体与所述底物的质量比为1:(0.5-3),例如1:1;和/或,所述过氧化氢酶与所述D-氨基酸氧化酶的菌体的质量比为1:(20-60);例如1:40。
11.如权利要求10所述的制备方法,其特征在于,所述氧化反应的反应体系包括缓冲液,所述缓冲液优选为磷酸缓冲液,例如磷酸二氢铵和磷酸氢二铵、磷酸氢二钠和磷酸二氢钠。
12.一种L-草铵膦或其盐的制备方法,其包括如下步骤:
(1)通过如权利要求10或11所述的制备方法获得2-氧代-4-(羟基甲基氧膦基)丁酸或其盐;
(2)在谷氨酸脱氢酶、无机氨基供体和还原型辅酶的存在下,将步骤(1)获得的2-氧代-4-(羟基甲基氧膦基)丁酸或其盐进行氨化反应,即得所述L-草铵膦或其盐。
13.如权利要求12所述的制备方法,其特征在于,
所述还原型辅酶为NADPH或NADH;
和/或,所述的无机氨基供体为氨气、硫酸铵、氯化铵、磷酸氢二铵、乙酸铵、甲酸铵和碳酸氢铵中的一种或多种,所述氨气的使用形式较佳地为氨水;
和/或,所述氨化反应的反应体系的pH为7-10,较佳地为8.4-8.6;
和/或,所述氨化反应的反应温度为28-35℃,较佳地为30-33℃。
14.一种如权利要求1-5任一项所述的D-氨基酸氧化酶在制备L-草铵膦或其盐、2-氧代-4-(羟基甲基氧膦基)丁酸或其盐中的应用。
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