CN112831532A - 一种酶促合成d-亮氨酸的方法 - Google Patents
一种酶促合成d-亮氨酸的方法 Download PDFInfo
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- C12P13/00—Preparation of nitrogen-containing organic compounds
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- C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
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- C12Y101/99—Oxidoreductases acting on the CH-OH group of donors (1.1) with other acceptors (1.1.99)
- C12Y101/9901—Glucose dehydrogenase (acceptor) (1.1.99.10)
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- C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
- C12Y104/99—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with other acceptors (1.4.99)
- C12Y104/99001—D-Amino-acid dehydrogenase (1.4.99.1)
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Abstract
本发明公开了一种酶促合成D‑亮氨酸的方法,其以α‑酮异己酸为底物,用D‑氨基酸脱氢酶催化底物发生脱氢反应和氨基转移反应,得到D‑亮氨酸,产物ee值超过99%,工业化应用前景广阔。
Description
技术领域
本发明属于酶催化技术领域,具体地说,涉及一种酶促合成D-亮氨酸的方法以及用于该方法的D-氨基酸脱氢酶突变体。
背景技术
D-亮氨酸是一种非天然氨基酸,又名D-2-氨基-4-甲基戊酸,CAS号为328-38-1,是一种白色鳞片状粉末。其为一种支链氨基酸,可作为营养补充剂使用,有助于训练后肌肉的恢复,控制血糖,并给身体组织提供能量,增强免疫力。D-亮氨酸在医药、化工领域也有较多应用。
D-亮氨酸可通过发酵法(CN110330441A)获得;或者通过手性拆分DL-亮氨酸外消旋体方法(CN103981248A)获得,两类方法各有利弊,它们共同的特点是成本较高。发酵法生产周期长且发酵后的产物提取和纯化步骤繁琐,手性拆分不论是化学拆分法、酶法或诱导结晶法都必须用价格较高的亮氨酸为原料,在经济上不可行。
发明内容
为了降低D-亮氨酸的生产成本,本发明开发了一种通过合成法制备D-亮氨酸的新途径,该酶催化反应的路线如下所示。
发明人对于能够催化α-酮异己酸生成D-亮氨酸的氨基酸脱氢酶进行了广泛筛选,筛选出一种立体选择性高、且酶活力相对较高的氨基酸脱氢酶,其为热球状尿素芽孢杆菌(Ureibacillus thermosphaericus)来源的NADP+辅因子依赖的D型氨基酸脱氢酶(简称utDAADH)(SEQ ID NO:1)。进一步地,为了提高其酶活力,还通过基因工程对该野生酶进行改造,构建能高效催化α-酮异己酸反应的突变体,从而用于合成高光学纯度的D-亮氨酸。具体地,本发明包括如下技术内容。
一种酶促合成D-亮氨酸的方法,其以α-酮异己酸为底物,用D-氨基酸脱氢酶催化底物发生脱氢反应和氨基转移反应,得到D-亮氨酸。
上述方法中,可以在反应体系中添加葡萄糖脱氢酶和辅酶NADPH。由于D-氨基酸脱氢酶是NADP+辅因子依赖的酶,反应体系中包含葡萄糖脱氢酶依赖的NADP+辅因子再生***,例如添加有葡萄糖脱氢酶和辅酶NADPH(即NADP+)。借助于NADP+辅因子再生***,可进一步降低D-亮氨酸的生产成本,在经济上是有利的。
优选地,反应体系中还可以添加有铵盐或者氨水作为氨供体。
在一种实施方式中,上述方法中使用的D-氨基酸脱氢酶是热球状尿素芽孢杆菌(Ureibacillus thermosphaericus)来源的D-氨基酸脱氢酶SEQ ID NO:1或者与其具有90%以上同源性、优选地95%以上同源性、优选地98%以上同源性、更优选地99%以上同源性的突变体。
MSKIRIGIVGYGNLGRGVEAAIQQNPDMELVAVFTRRDPKTVAVKSNVKVLHVDDAQSYKDEIDVMILCGGSATDLPEQGPYFAQYFNTIDSFDTHARIPDYFDAVNAAAEQSGKVAIISVGWDPGLFSLNRLLGEVVLPVGNTYTFWGKGVSQGHSDAIRRIQGVKNAVQYTIPIDEAVNRVRSGENPELSTREKHARECFVVLEEGADPAKVEHEIKTMPNYFDEYDTTVHFISEEELKQNHSGMPHGGFVIRSGKSDEGHKQIIEFSLNLESNPMFTSSALVAYARAAYRLSQNGDKGAKTVFDIPFGLLSPKSPEDLRKELL(SEQ ID NO:1)。
优选地,上述突变体的氨基酸序列为SEQ ID NO:3或者SEQ ID NO:4。但本发明的D-氨基酸脱氢酶突变体不限于此。其中,D-氨基酸脱氢酶突变体SEQ ID NO:3在专利文献CN202110108232.9中已报道过,研究发现其也可以催化α-酮异己酸反应生成D-亮氨酸。
MSKIRIGIVGYGNLGRGVEAAIQQNPDMELVAVFTRRDPKTVAVKSNVKVLHVDDAQSYKDEIDVMILCGGSATDLPEQGPYFAQYFNTIDSFATHARIPDYFDAVNAAAEQSGKVAIISVGWDPGLFSLNRLLGEVVLPVGNTYTFWGKGVSQGHSDAIRRIQGVKNAVQYTIPIDEAVNRVRSGENPELSTREKHAVECFVVLEEGADPAKVEHEIKTMPNYFDEYDTTVHFISEEELKQNHSGMPTGGFVIRSGKSDEGHKQIIEFSLNLESNPMFTSSALVAYARAAYRLSQNGDKGAKTVFDIPFGLLSPKSPEDLRKELL(SEQ ID NO:3)。
其为野生型D-氨基酸脱氢酶SEQ ID NO:1第94位的D替换为A、第199位的R替换为V、第249位的H替换为T的突变体。
另一个D-氨基酸脱氢酶突变体SEQ ID NO:4与SEQ ID NO:3的区别仅在于1第94位的D替换为L,其氨基酸序列为:
MSKIRIGIVGYGNLGRGVEAAIQQNPDMELVAVFTRRDPKTVAVKSNVKVLHVDDAQSYKDEIDVMILCGGSATDLPEQGPYFAQYFNTIDSFLTHARIPDYFDAVNAAAEQSGKVAIISVGWDPGLFSLNRLLGEVVLPVGNTYTFWGKGVSQGHSDAIRRIQGVKNAVQYTIPIDEAVNRVRSGENPELSTREKHAVECFVVLEEGADPAKVEHEIKTMPNYFDEYDTTVHFISEEELKQNHSGMPTGGFVIRSGKSDEGHKQIIEFSLNLESNPMFTSSALVAYARAAYRLSQNGDKGAKTVFDIPFGLLSPKSPEDLRKELL(SEQ ID NO:4)。
其为野生型D-氨基酸脱氢酶SEQ ID NO:1第94位的D替换为L、第199位的R替换为V、第249位的H替换为T的突变体。
可选地,上述葡萄糖脱氢酶来源于蜡样芽胞杆菌(Bacillus cereus),氨基酸序列SEQ ID NO:6,编码基因可以是SEQ ID NO:7(GenBank序列号为AE016877.1),可通过大肠杆菌表达得到。但与本发明的D-氨基酸脱氢酶或其上述突变体配合使用的葡萄糖脱氢酶不限于此。
本发明的另一个方面提供了一种D-氨基酸脱氢酶突变体,其氨基酸序列为SEQ IDNO:4。
该D-氨基酸脱氢酶突变体可以通过基因工程菌的发酵表达而获得。当D-氨基酸脱氢酶突变体SEQ ID NO:4的表达宿主菌是大肠杆菌时,其编码基因的核苷酸序列可以是SEQID NO:5。
该编码基因可以克隆在合适的载体上,然后将载体转化入大肠杆菌感受态细胞中,得到表达D-氨基酸脱氢酶突变体SEQ ID NO:4的转化子。优选该载体是PET系列质粒,比如载体是pET24a或pET28a等,但不限于此。
通过基因工程菌的发酵,可获得D-氨基酸脱氢酶突变体SEQ ID NO:4。例如,在微生物发酵后,菌体用缓冲液重悬,超声破碎,离心,收集上清过柱层析,洗脱目的蛋白,即得纯化的D-氨基酸脱氢酶突变体。
本发明的另一个方面提供了上述D-氨基酸脱氢酶突变体SEQ ID NO:4或者SEQ IDNO:3在制备D-亮氨酸中的应用。
本发明提供了一种制备高光学纯度D-亮氨酸的新思路,采用D-氨基酸脱氢酶比如SEQ ID NO:4,以100mM的α-酮异己酸为底物,可以在8个小时内,达到转化率95%,产物D-亮氨酸的ee值超过99%,显示出较好的工业化开发和应用前景。
具体实施方式
在探索酶法合成D-型氨基酸比如D-叔亮氨酸、D-亮氨酸等时,筛选出野生型D-氨基酸脱氢酶,其来源于微生物热球状尿素芽孢杆菌(Ureibacillus thermosphaericus),GenBank序列号为BAK86217.1,氨基酸序列为SEQ ID NO:1。当其在大肠杆菌中表达时,其编码基因可以是SEQ ID NO:2。
实验发现其催化活力偏低,难以满足工业化生产D-叔亮氨酸和D-亮氨酸等的要求。于是通过生物信息学技术对该野生型酶SEQ ID NO:1进行分析,判断其氨基酸序列中一些位点在酶的结构和功能方面起到关键作用,然后通过定点饱和突变等技术对其进行改造,重点在于对催化底物发生脱氢反应和氨基转移反应的活性位点氨基酸(包括94位天冬氨酸位点、199位精氨酸位点、249位组氨酸)进行替换,以期获得酶活力提高的突变体。通过对众多突变体的筛选,筛选出(D94A、R199V、H249T)的突变体SEQ ID NO:3和(D94L、R199V、H249T)的突变体SEQ ID NO:4等一系列酶活力提高的突变体。
研究发现,这些突变体对于底物的选择性较强,比如突变体SEQ ID NO:3可高效地催化三甲基丙酮酸反应生成D-叔亮氨酸,如同专利文献CN202110108232.9报道的那样,本文中以引用方式将其部分内容引入本文中。但当底物换成α-酮异己酸时,其催化活力明显下降。而另一个氨基酸仅有一个位点之差(第94位丙氨酸A与亮氨酸L的区别)的突变体SEQID NO:4却对底物α-酮异己酸表现出较高的酶活力和高度的立体选择性,两种突变体对于底物选择性产生如此差异的原因有待于进一步分析研究。
在本发明中,术语“野生(型)”、“野生酶”、“野生型酶”表示相同的意义,都是指D型氨基酸脱氢酶的野生序列SEQ ID NO:1。相对应地,D-氨基酸脱氢酶突变体SEQ ID NO:3和SEQ ID NO:4也可以简称为“突变体”或者“突变酶”。
本发明的D-氨基酸脱氢酶突变体SEQ ID NO:4的氨基酸数量只有326个,且结构明确,因此本领域技术人员很容易获得其编码基因、包含这些基因的表达盒和质粒、以及包含该质粒的转化体。这些基因、表达盒、质粒、转化体可以通过本领域技术人员所熟知的基因工程构建方式获得。
基于催化功能相同考虑,本文中将野生型D-氨基酸脱氢酶SEQ ID NO:1、其突变体SEQ ID NO:3和SEQ ID NO:4统称为D-氨基酸脱氢酶。
与本发明的D-氨基酸脱氢酶配合使用的葡萄糖脱氢酶也可以通过大肠杆菌表达。
本发明采用D-氨基酸脱氢酶和葡萄糖脱氢酶偶联反应方式,以α-酮异己酸、葡萄糖为底物,NADP+为辅因子,一锅法制备D-亮氨酸。其中葡萄糖是葡萄糖脱氢酶的底物,反应时,葡萄糖脱氢酶催化葡萄糖氧化,同时将NADP+还原为NADPH。
当作为生物催化剂用于生产D-亮氨酸时,本发明催化合成中使用的D-氨基酸脱氢酶和葡萄糖脱氢酶可以呈现酶的形式或者表达微生物发酵菌体的形式。所述酶的形式包括游离酶、固定化酶,包括纯化酶、粗酶、发酵液、载体固定的酶等。
以下结合具体实施例对本发明做进一步详细说明。应理解,以下实施例仅用于说明本发明而非用于限定本发明的范围。
本文中涉及到多种物质的添加量、含量及浓度,其中所述的百分含量,除特别说明外,皆指质量百分含量。
实施例
材料和方法
实施例中的全基因合成、引物合成及测序皆由苏州金唯智生物科技有限公司完成。
实施例中的分子生物学实验包括质粒构建、酶切、连接、感受态细胞制备、转化、培养基配制等等,主要参照《分子克隆实验指南》(第三版),J.萨姆布鲁克,D.W.拉塞尔(美)编著,黄培堂等译,科学出版社,北京,2002)进行。必要时可以通过简单试验确定具体实验条件。
PCR扩增实验根据质粒或DNA模板供应商提供的反应条件或试剂盒说明书进行。必要时可以通过简单试验予以调整。
LB培养基:10g/L胰蛋白胨,5g/L酵母提取物,10g/L氯化钠,pH7.2。(LB固体培养基另加20g/L琼脂粉。)
TB培养基:24g/L酵母提取物、12g/L胰蛋白胨、16.43g/L K2HPO4.3H2O、2.31g/LKH2PO4、5g/L甘油,pH7.0-7.5。(TB固体培养基另加20g/L琼脂粉。)
为描述方便起见,在实施例中有时将某一种酶蛋白编号、其基因编号和其表达菌株编号混用/套用,本领域技术人员容易理解它们在不同的环境指代不同的生物体含义。
按照专利文献CN202110108232.9中实施例1-3报道的方法,分别构建野生型D-氨基酸脱氢酶SEQ ID NO:1、葡萄糖脱氢酶SEQ ID NO:6、D-氨基酸脱氢酶(D94A、R199V、H249T)突变体SEQ ID NO:3的表达菌株。
实施例1野生型D-氨基酸脱氢酶表达菌株的构建
对于热球状尿素芽孢杆菌(Ureibacillus thermosphaericus)来源的野生型D-氨基酸脱氢酶SEQ ID NO:1,进行密码子优化,全基因合成编码基因序列SEQ ID NO:2,并在基因两端设计限制性内切酶位点Nde I和XhoI,亚克隆到载体pET24a(Novagen)的相应位点,获得重组质粒pET24a-utDAADH。将重组质粒pET24a-utDAADH转化表达宿主大肠杆菌BL21(DE3),得到表达野生酶的重组大肠杆菌utDAADH。
实施例2葡萄糖脱氢酶表达菌株的构建
对于蜡样芽胞杆菌(Bacillus cereus)来源的葡萄糖脱氢酶SEQ ID NO:6,全基因合成编码基因序列SEQ ID NO:7,并在基因两端设计限制性内切酶位点Nde I和XhoI,亚克隆到载体pET24a(Novagen)的相应位点,获得重组质粒pET24a-bcGDH。将重组质粒pET24a-bcGDH转化表达宿主大肠杆菌BL21(DE3),得到表达葡萄糖脱氢酶的重组大肠杆菌bcGDH。
实施例3构建(D94A、R199V、H249T)突变菌种
按照专利文献CN202110108232.9实施例3报道的方法,构建(D94A、R199V、H249T)突变体SEQ ID NO:3表达菌株,包括如下步骤:
3.1以utDAADH菌株的质粒pET24a-utDAADH质粒为模板,针对野生型D-氨基酸脱氢SEQ ID NO:1中第94位、199位、249位三个位点,通过基因定点突变技术将其改造成D94A、R199V、H249T,然后按照实施例1中的方法,构建包含这三种突变的突变体表达菌株utDAADH-M。构建过程中所用的引物如下:utDAADH-94F:5’-CAACACCATCGACTCTTTCGCCACCCACGCTCGTATC-3’,utDAADH-199F:5’-CTACCCGTGAAAAACACGCTGTTGAATGCTTCGTTGTTC-3’,utDAADH-199R:5’-GAACAACGAAGCATTCAACAGCGTGTTTTTCACGGGTAG-3’,utDAADH-249R:5’-GATAACGAAGCCGCCGGTGGGCATACCAGAGTG-3’。
以pET24a-utDAADH质粒为模板,以utDAADH-94F和utDAADH-199R为引物对,直接进行P1片段扩增,以2utDAADH-199F和utDAADH-249R为引物对进行P2片段扩增,再以utDAADH-94F和utDAADH-249R为引物对,通过P1和P2片段的over-lapping PCR扩增出大片段P,然后以大片段P为引物进行MegaPrimer PCR,构建定点突变体表达质粒。
P1和P2片段50μL PCR反应体系:10ng质粒模板,10pmol的引物对,1×KOD plusbuffer,0.2mM dNTP,1.5mM MgSO4,5个单位的KOD-plus DNA聚合酶。
P1和P2片段PCR反应条件:95℃1min;98℃10s,57℃30s,68℃1min/kbp,30个循环;68℃10min。
PCR结束后,P1片段PCR产物大约353bp,P2片段PCR产物大约188bp,P1和P2 PCR产物分别切胶回收。
以切胶回收后的片段P1和片段P2为模板,以utDAADH-94F和utDAADH-249R为引物进行over-lapping PCR,获得长度大约为502bp的片段P,切胶回收。
Over-lapping PCR反应体系:5μl P1片段,5μl P2片段,10pmol的引物对,1×KODplus buffer,0.2mM dNTP,1.5mM MgSO4,5个单位的KOD-plus DNA聚合酶。
Over-lapping PCR反应条件:95℃3min;98℃10s,60℃30s,68℃1min/kbp,25个循环;68℃10min。
以片段P作为大引物,pET24a-utDAADH质粒为模板,用KOD-plus DNA聚合酶做MegaPrimer PCR,其反应体系:250ng P片段,10ng质粒模板,1×KOD plus buffer,0.2mMdNTP,1.5mM MgSO4,5个单位的KOD-plus DNA聚合酶。
MegaPrimer PCR反应条件:94℃5min;98℃10s,60℃30s,68℃2min/kbp,25个循环;68℃10min。
DpnI消化质粒模板,化学转化大肠杆菌E.coli BL21(DE3)感受态细胞,对阳性克隆菌株进行试管培养,抽提质粒,质粒测序确定突变菌株utDAADH-M构建成功。该菌株可表达突变酶SEQ ID NO:3。
实施例4构建(D94L、R199V、H249T)突变菌种
与实施例3类似地,构建另一种(D94L、R199V、H249T)突变体SEQ ID NO:4表达菌株,包括如下步骤:
以utDAADH菌株的质粒pET24a-utDAADH质粒为模板,对94位、199位、249位三个位点,通过定点突变技术改造成D94L,R199V,H249T,构建包含这三种突变的突变菌株utDAADH-M437。构建过程中所用的引物如下:utDAADH-94LF:5’-CAACACCATCGACTCTTTCCTGACCCACGCTCGTATC-3’utDAADH-199F:5’-CTACCCGTGAAAAACACGCTGTTGAATGCTTCGTTGTTC-3’utDAADH-199R:5’-GAACAACGAAGCATTCAACAGCGTGTTTTTCACGGGTAG-3’utDAADH-249R:5’-GATAACGAAGCCGCCGGTGGGCATACCAGAGTG-3’
以pET24a-utDAADH质粒为模板,以utDAADH-94LF和utDAADH-199R为引物对,直接进行P3片段扩增,以2utDAADH-199F和utDAADH-249R为引物对进行P4片段扩增,再以utDAADH-94LF和utDAADH-249R为引物对,通过P3和P4片段的over-lapping PCR扩增出大片段P-1,然后以P-1大片段为引物进行MegaPrimer PCR,构建定点突变菌株utDAADH-M437。
P3和P4片段50μL PCR反应体系:10ng质粒模板,10pmol的引物对,1×KOD plusbuffer,0.2mM dNTP,1.5mM MgSO4,5个单位的KOD-plus DNA聚合酶。
P3和P4片段PCR反应条件:95℃1min;98℃10s,57℃30s,68℃1min/kbp;30个循环;68℃10min。
PCR结束后,P3片段PCR产物大约353bp,P4片段PCR产物大约188bp,P3和P4 PCR产物分别切胶回收。
以切胶回收后的片段P3和片段P4为模板,以utDAADH-94LF和utDAADH-249R为引物进行over-lapping PCR,获得长度大约为502bp的片段P-1,切胶回收。
Over-lapping PCR反应体系:5μL P3片段,5μL P4片段,10pmol的引物对,1×KODplus buffer,0.2mM dNTP,1.5mM MgSO4,5个单位的KOD-plus DNA聚合酶。
Over-lapping PCR反应条件:95℃3min;98℃10s,60℃30s,68℃1min/kbp;25个循环;68℃10min。
以片段P-1作为大引物,pET24a-utDAADH质粒为模板,用KOD-plus DNA聚合酶做MegaPrimer PCR,其反应体系:250ng P-1片段,10ng质粒模板,1×KOD plus buffer,0.2mMdNTP,1.5mM MgSO4,5个单位的KOD-plus DNA聚合酶。
MegaPrimer PCR反应条件:94℃5min;98℃10s,60℃30s,68℃2min/kbp,25个循环;68℃10min。DpnI消化质粒模板,化学转化大肠杆菌E.coli BL21(DE3)感受态细胞,对阳性克隆菌株进行试管培养,抽提质粒,质粒测序确定突变菌株utDAADH-M437构建成功。该菌株可表达突变酶SEQ ID NO:4。
实施例5酶活力比较
5.1摇瓶发酵
分别从utDAADH、utDAADH-M和utDAADH-M437的LB培养平板上挑取单菌落,分别接种至含有50μg/mL的硫酸卡那霉素LB液体培养基中,37℃,230rpm培养过夜。按照体积比1:100的比例,将过夜培养物分别转接至1L含有50μg/mL硫酸卡那霉素的TB培养基中,于37℃,230rpm培养至OD600=0.6-0.8时,加入终浓度0.1mM的IPTG,25℃,200rpm培养过夜。然后于4℃,8000rpm,离心10min,收集菌体。
5.2提取utDAADH、utDAADH-M和tDAADH-M437表达的D-氨基酸脱氢酶纯酶
菌体用50mL平衡缓冲液(20mM磷酸钾盐缓冲液,200mM NaCl,pH7.8)重悬,然后超声破碎,破碎后的菌体于4℃,12000rpm,离心20min,收集上清。上清以1mL/min的速率加入含10mL Ni-NAT基质的亲和层析柱中,然后用含有50mM咪唑的平衡缓冲液冲洗柱料,洗脱杂质。最后用含有500mM咪唑的平衡缓冲液冲洗脱目的蛋白,收集峰值洗脱液。
洗脱液经截留分子量为10kDa的超滤管进行脱盐处理,得纯酶。
5.3按照与步骤5.1和5.2类似的方法,制备得到葡萄糖脱氢酶SEQ ID NO:6纯酶。
5.4测定D-氨基酸脱氢酶的比活力
采用500μl反应体系:200mM甘氨酸-KOH缓冲液(pH 10.5),200mM氯化铵,20mMα-酮异己酸,5mM NADPH,50μl步骤5.2中的脱盐纯酶,450μl纯水,45℃水浴,反应20min,通过测定340nm下的吸光值变化判断活力的大小。
同时采用Thermo Scientific公司的BCA Protein Assay Kit试剂盒测定纯酶的蛋白浓度,从而获得纯酶的比活力。
测试结果表明,对于催化底物α-酮异己酸转化为D-亮氨酸的反应,突变体SEQ IDNO:4的单位酶活力相比野生型酶SEQ ID NO:1提高了211倍;突变体SEQ ID NO:3的单位酶活力相比野生型酶SEQ ID NO:1提高了55倍。
实施例6突变体SEQ ID NO:4用于合成D-亮氨酸
采用D-氨基酸脱氢酶SEQ ID NO:4和葡萄糖脱氢酶SEQ ID NO:6纯酶,按如下方法进行底物α-酮异己酸的催化反应。
50ml反应体系:甘氨酸-KOH缓冲液(20mM,pH 10)中,100mM的α-酮异己酸,0.3M葡萄糖,1mM辅酶NADP+,150mM氯化铵,0.1mg/ml葡萄糖脱氢酶,D-氨基酸脱氢酶纯酶10U/ml,用氨水校正反应体系pH保持10.0。于45℃反应4小时后,补加10U/ml D-氨基酸脱氢酶纯酶,继续反应至6-10小时,取样离心后,上清过0.22μm膜后,直接进行HPLC分析,最终确定反应8小时,底物转化率超过95%,产物ee值大于99%。
HPLC检测方法:安捷伦1260;Kromasil 100C18色谱柱(250×4mm,5μm);流动相A:10mM乙酸钠,pH 6.00;流动相B:85%乙腈水溶液;衍生剂:0.1372g邻苯二甲醛和0.0589gN-异丁酰-L-半胱氨酸,用0.1M硼酸缓冲液(pH 10.4)定容至10ml;进样量:5μl;柱温30℃;流速:1ml/min;检测波长:334nm。
综上所述,本发明筛选的D-氨基酸脱氢酶SEQ ID NO:1及其突变体SEQ ID NOs:3-4能够以α-酮异己酸为底物,催化合成D-亮氨酸,例如,采用100mM的底物浓度,可以在8个小时内,底物转化率超过95%,产物D-亮氨酸的ee值超过99%,具有工业化开发和应用潜力。
序列表
<110> 洛阳华荣生物技术有限公司
<120> 一种酶促合成D-亮氨酸的方法
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Claims (10)
1.一种酶促合成D-亮氨酸的方法,其特征在于,以α-酮异己酸为底物,用D-氨基酸脱氢酶催化底物发生脱氢反应和氨基转移反应,得到D-亮氨酸。
2.如权利要求1所述的方法,其特征在于,反应体系中添加有葡萄糖脱氢酶和辅酶NADPH。
3.如权利要求1所述的方法,其特征在于,反应体系中还添加有铵盐或者氨水作为氨供体。
4.如权利要求1所述的方法,其特征在于,所述D-氨基酸脱氢酶是热球状尿素芽孢杆菌来源的D-氨基酸脱氢酶SEQ ID NO:1或者与其具有90%以上同源性的突变体。
5.如权利要求4所述的方法,其特征在于,所述突变体的氨基酸序列为SEQ ID NO:3或者SEQ ID NO:4。
6.如权利要求2所述的方法,其特征在于,所述葡萄糖脱氢酶的氨基酸序列为SEQ IDNO:6。
7.一种D-氨基酸脱氢酶突变体,其特征在于,氨基酸序列为SEQ ID NO:4。
8.编码如权利要求7所述D-氨基酸脱氢酶突变体的基因。
9.如权利要求8所述的基因,其特征在于,核苷酸序列为SEQ ID NO:5。
10.如权利要求7中所述的D-氨基酸脱氢酶突变体SEQ ID NO:4在制备D-亮氨酸中的应用。
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CN112877307A (zh) * | 2021-01-27 | 2021-06-01 | 洛阳华荣生物技术有限公司 | 一种氨基酸脱氢酶突变体及其应用 |
CN112877307B (zh) * | 2021-01-27 | 2023-10-31 | 洛阳华荣生物技术有限公司 | 一种氨基酸脱氢酶突变体及其应用 |
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