CN1922328A - 从d-氨基酸制备l-氨基酸的方法 - Google Patents
从d-氨基酸制备l-氨基酸的方法 Download PDFInfo
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- CN1922328A CN1922328A CNA2005800052537A CN200580005253A CN1922328A CN 1922328 A CN1922328 A CN 1922328A CN A2005800052537 A CNA2005800052537 A CN A2005800052537A CN 200580005253 A CN200580005253 A CN 200580005253A CN 1922328 A CN1922328 A CN 1922328A
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Abstract
本发明涉及重组微生物,其与起始微生物相比具有更高浓度或活性的D-氨基酸氧化酶、L-氨基酸脱氢酶、NADH共底物再生酶的一种,及如果需要还有过氧化氢酶,本发明还涉及一种通过使用这些微生物从D-氨基酸制备L-氨基酸的方法。
Description
本发明涉及重组微生物,其与起始微生物相比具有更高浓度或活性的D-氨基酸氧化酶、L-氨基酸脱氢酶、NADH共底物再生酶中的一种、并且如果需要还具有过氧化氢酶,本发明还涉及通过使用这些微生物从D-氨基酸制备L-氨基酸的方法。
目前许多天然氨基酸是通过用遗传优化的细菌进行发酵而以对映异构体纯的形式而制备(de Graaf AA,Eggeling L,Sahm H 2001.Metabolic Engineering for L-Lysine Production by Corynebacteriumglutamicum.Adv.Biochem.Eng.Biotechnol.73:9-29;Sahm H,Eggeling L,Eikmanns B,Kramer R.1996,Construction of L-Lysine-,L-Threonine-,and L-Isoleucin Overproducing Strains of CorynebacteriumGlutamicum.Ann N Y Acad Sci 782:25-39)。
诚然,不是所有的蛋白质氨基酸(proteinogenic amino acids)并且仅极少数非天然氨基酸和D-氨基酸可以这种方式制备。由于对映异构体纯的氨基酸的化学合成非常昂贵,因此已经开发了一系列酶学方法,其中一些方法已经可以达到每年生产数吨的规模。
所述方法的范围包括从借助于酰基转移酶、酰胺酶、酯酶、海因酶(hydantoinase)、氨基酸氧化酶和蛋白酶的动态外消旋物裂解到利用裂合酶、氨基转移酶和脱氢酶的对映体选择性合成(Schmid A.et al.(2002)“The Use of Enzymes in the Chemical Industry in Europe”,Curr Opin Biotechnol 13(4):359-366)。
除了对映体选择性合成法之外,动态外消旋物裂解(dynamischkinetische Racematspaltungen)也特别有效,其中不希望的对映异构体被原位外消旋化。在动态外消旋物裂解中,通过组合D-或L-氨基酸氧化酶与非选择性化学还原初始亚氨基酸为主要氨基酸,也可以达到100%得率。然而,还原剂如NaBH4必须以至少25倍当量过量应用,这样导致这种方法非常昂贵(Enright et al.,“Stereoinversion ofBeta-and Gamma-Substituted Alpha Amino Acids Using a Chemo-Enzymatic Oxidation-Reduction Procedure”,Chemical Communications(2003),(20),2636-2637.)
本领域通常已知氨基酸脱氢酶对α-酮酸的氨基化。然而无可否认所得离析物与例如相应的外消旋氨基酸相比更加昂贵许多倍。
然而,通过联合氨基酸氧化酶与氨基酸脱氢酶,可以从氨基酸原位获得相应的酮酸。当这两种酶具有相反的对映体选择性时,D-氨基酸可以完全转变为L-氨基酸或者L一氨基酸完全转变为D-氨基酸。从外消旋物开始,从中可以产生对映异构体纯的化合物。
在此NADH共底物必须使用酶再生,因为其太昂贵以至于不能以化学计量的量应用。从其底物中释放二氧化碳并因此使得反应不可逆的酶如甲酸脱氢酶和苹果酸脱氢酶(脱羧化)是特别适用的(Hanson,R.L.et al.“Enzymatic Synthesis of L-6-Hydroxynorisoleucine”,Bioorganic & Medicinal Chemistry(1999),7(10),2247-2252,及Nakajima N.et al.,“Enzymatic Conversion of Racemic Methionine to theL-Enantiomer”,Journal of the Chemical Society,ChemicalCommunications(1990),(13),947-8)。
然而为了在无细胞***中迅速并完全地转化,必须存在过氧化氢酶,因为在由氨基酸氧化酶催化的氧化步骤中,产生过氧化氢,导致酮酸脱羧化及所述酶失活(Trost,E.-M.;Fischer,L.,“Minimization of By-Product Formation during D-Amino Acid OxidaseCatalyzed Racemate Resolution of D/L-Amino Acids”,Journal ofMolecular Catalysis B:Enzymatic(2002),19-20 189-195)。外消旋的氨基酸转化为对映异构体纯的氨基酸在这个***中可以达到>99%ee和>95%的得率。
然而这种方法昂贵,因为必须单独制备和分离4种不同的酶。
本发明的目的是提供一种方法,该方法可以避免昂贵地分离和提供将D-氨基酸转化为L-氨基酸所需的酶。
本发明的主题优选是重组微生物的静态细胞,其与起始微生物例如野生型微生物相比具有更高浓度或活性的D-氨基酸氧化酶、氨基酸脱氢酶、NADH共底物再生酶及如果需要还有过氧化氢酶。根据要被转化的底物和所述酶的底物范围组合氧化酶和脱氢酶。由此起始生物体当然不必须含有所述酶。
甲酸脱氢酶、苹果酸脱氢酶(德国专利102 40 603)或者醇脱氢酶可以用作NADH共底物的再生酶。
这种酶的编码多核苷酸的来源通常不限于重组微生物的菌株或者菌种。
转化宿主生物体的基因可以不用考虑来源而选择。
基因的来源可以是微生物、霉菌或者酵母,尤其对于D-AAO是原玻璃蝇节杆菌(Arthrobacter protophormiae)或者变异三角酵母(trigonopsis variabilis),对于LeuDH是腊状芽孢杆菌(bacilluscereus)。
存在稳定表达***的微生物优选用作宿主生物体,例如芽孢杆菌属、多种霉菌、葡萄球菌属或者链霉菌属,特别是大肠杆菌(E.coli)。
特别合适的氨基酸脱氢酶是来自芽孢杆菌属的L-亮氨酸脱氢酶(EP 792 933),来自红球菌(rhodococcus)属的谷氨酸脱氢酶、L-苯丙氨酸脱氢酶,来自高温放线菌属(thermoactinomyces)或者芽孢杆菌菌株的L-丙氨酸脱氢酶。
根据要被还原氨化的酮酸选择所述酶。
本发明的D-氨基酸氧化酶或者其编码多核苷酸均源自纤细红酵母(rhototorula gracillis)(美国专利6,187,574)、变异三角酵母(Long-Liu Lin et al.,Enzyme and Microbial Technol.27(2000),482-491)、念珠菌属(Candida)菌种、粗糙链孢霉(neurospora crassa)、verlicullium luteoralbo及不同的镰刀霉菌种,D-氨基酸氧化酶源自原玻璃蝇节杆菌(欧洲专利1 375 649)。
关于不同D-AAO的底物的综述可见于Gabler等(EnzymeMicrobiol.Technol.27(8):605-611(2000))所述。在欧洲专利897 006A1中,描述了来自红冬孢属(rhodosporidium)的D-AAO及其基因。根据要被转化的底物,选择D-氨基酸氧化酶和氨基酸脱氢酶的编码基因,并且用它们转化指定的宿主株。
例如,对于DL-甲硫氨酸或者DL-亮氨酸的去外消旋化,优选来自原玻璃蝇节杆菌的D-氨基酸氧化酶(欧洲专利1 375 649A)和来自腊状芽孢杆菌的亮氨酸脱氢酶的编码基因在大肠杆菌中是过表达的。
下表示出了可用于本发明的基因和属于它们的酶。
表1:D-氨基酸氧化酶
登记号 | ||
gi2140775gi1616634gi27806895EP 897 006US 6,187,574 | 原玻璃蝇节杆菌变异三角酵母牛(bos taurus)红冬孢属纤细红酵母 | D-氨基酸氧化酶D-氨基酸氧化酶D-天冬氨酸氧化酶D-氨基酸氧化酶D-氨基酸氧化酶 |
表2:L-氨基酸脱氢酶
登记号 | ||
gi6741938gi625925---gi16080244gi118533 | 腊状芽孢杆菌红球菌属人(homo sapiens)枯草芽孢杆菌(bacillus subtilis)牛 | L-亮氨酸脱氢酶L-苯丙氨酸脱氢酶L-赖氨酸脱氢酶L-丙氨酸脱氢酶谷氨酸脱氢酶 |
然后对获得的DNA序列可以使用已知的算法或者序列分析程序例如Staden(Nucleic Acids Research 14,217-232(1986))、Marck(Nucleic Acids Research 16,1829-1836(1988))的程序或者Butler的GCG程序(Methods of Biochemical Analysis 39,74-97(1998))进行研究。
本领域技术人员可获知借助于聚合酶链反应(PCR)扩增DNA序列的指导,见Gait:Oligonucleotide Synthesis:A Practical Approach,(IRL Press,Oxford,UK,1984)和Newton and Graham:PCR(SpektrumAkademischer Verlag,Heidelberg,Germany 1994)所述。
本发明的另一个目的是提供在选择的宿主株中可自主复制的载体,其通常彼此相容并含有编码本发明需要的酶的至少一个基因。
DNA载体可以通过已知的转化技术导入真核或原核细胞中。
优选的载体是含有编码两种酶、特别例如苹果酸脱氢酶和氨基酸脱氢酶或者苹果酸脱氢酶和D-氨基酸氧化酶的编码核苷酸序列的载体。
载体上的编码核苷酸序列的组合对于氨基酸脱氢酶和D-氨基酸氧化酶也是有益的。
在这个***中仍缺少的酶的核苷酸序列位于另一个载体上。
通常,在低拷贝数的载体中克隆具有良好表达能力的基因,使用具有较高拷贝数和/或强启动子的载体克隆具有较弱表达能力的基因。将宿主细胞以这样的方式用这些载体转化,所述方式即与起始生物体相比,转化后的宿主细胞含有所需用于将D-氨基酸转化为L-氨基酸的三种或者如果需要四种酶的编码核苷酸序列的各至少一个额外拷贝。
通过这种方式可以成功地过表达所述核苷酸序列。
为了实现过表达,可以增加相应基因的拷贝数,或者可以突变位于结构基因上游的启动子和调节区域或者核糖体结合位点。
在结构基因上游形成的表达盒以相似方式发挥作用。通过诱导型启动子,还可以在发酵生产氨基酸期间增加表达。通过延长m-RNA的寿命可以同样改良表达。另外,酶活性也可以通过防止酶蛋白的降解而加强。所述基因或基因构建体可以以不同拷贝数存在于质粒中,或者可以整合进并在染色体中扩增。或者,所述被研究的基因的过表达可另外通过改变培养基的组成和培养程序而实现。
本领域技术人员在如下文献中可发现关于这方面及其它方面的指导:Martin等(Bio/Technology 5,137-146(1987))、Guerrero等(Gene 138,35-41(1994))、Tsuchiya和Morinaga(Bio/Technology 6,428-430(1988))、Eikmanns等(Gene 102,93-98(1991))、欧洲专利472869、美国专利4,601,893、Schwarzer和Pühler(Bio/technology 9,84-87(1991)、Reinscheid等(Applied and Environmental Microbiology60,126-132(1994))、LaBarre等(Joumal of Bacteriology 175,1001-1007(1993))、专利申请WO 96/15246、Malumbres等(Gene 134,15-24(1993))、未审查的日本申请JP-A-10-229891、Jensen和Hammer(Biotechnology and Bioengineering 58,191-195(1998))、Makrides(Microbiological Reviews 60:512-538(1996))及已知的遗传学和分子生物学教科书。
过表达导致相应酶的胞内活性或者浓度增加。
这种增加与未转化的微生物(起始微生物)的酶的浓度或者活性相比通常增加至少10-500%、或者100-500%、直至最大1000或者2000%。
本发明还涉及一种通过使用对映体选择性酶合成途径从D-氨基酸中制备L-氨基酸的方法,所述方法特征在于其利用一种重组微生物,该重组微生物与转化起始微生物相比具有更高浓度或活性的D-氨基酸氧化酶、L-氨基酸脱氢酶、NADH共底物再生酶及如果需要还有过氧化氢酶,将其与含有D-氨基酸的溶液反应并分离所得L-氨基酸。
苹果酸酶的过表达对于作为共底物再生酶是有益的,其中在含有完整细胞催化剂和要被转化的D-氨基酸的缓冲水溶液中,同时含有与要被转化的D-氨基酸的数量相比至少等摩尔比率的、优选1.5-6倍摩尔比率的L-苹果酸盐或者L-苹果酸。
如果需要,过氧化氢酶被过表达作为分解过氧化物的酶。来自不同生物体的过氧化氢酶均是合适的,例如来自大肠杆菌的酶(过氧化氢酶HPII(羟基过氧化物酶II)登记号:gi115722)。
D-氨基酸的转化优选使用静态细胞进行。静态细胞应理解为是存活的、但在一定条件下不增殖的细胞。
文中所述氨基酸是指天然存在的以及合成的α-氨基酸,例如Beyer-Walter,有机化学教科书(Lehrbuch der organischen Chemie),S.Hirzel Publishers,Stuttgart,22nd edition,1991,p.822及其后所述的那些。
优选采用的是选自如下一组的D和L氨基酸的混合物、其外消旋物或者纯D-对映异构体:赖氨酸、精氨酸、苯丙氨酸、缬氨酸、鸟氨酸、亮氨酸、组氨酸、正亮氨酸、酪氨酸、丙氨酸、谷氨酸和头孢菌素,特别是甲硫氨酸。
某些酶特别适合转化不同的D-氨基酸(见Gabler et al.,2000)。
例如,来自原玻璃蝇节杆菌的D-AAO例如特别适于转化碱性和疏水性氨基酸。
本领域已知合适的酶和核苷酸序列。
在本发明的方法中,不需要分离各个酶。另一个优势是本发明应用的细胞(总细胞催化剂)在反应后可易于分离。
在根据现有技术使用分离的酶将D-甲硫氨酸转化为L-甲硫氨酸的方法中,另一方面必须加入过氧化氢酶(Nakajima et al.,1990)。
本发明使用的微生物也是本发明的一个目的,其可以连续培养或者以分批方式(分批培养)或者补料分批或者重复补料分批培养方式不连续培养。
已知的培养方法在Chmiel(Biofahrensstechnik 1.Einführung indie Biofahrenstechnik,Gustav Fischer Verlag,Stuttgart 1991)或者Storhas(Bioreaktoren und periphere Einrichtungen,Vieweg Verlag,Braunschweig/Wiesbaden,1994)的教科书中描述。
所用培养基必须以适当方式符合各个菌株的需要。关于不同微生物的培养基的描述见于美国细菌学会(American Society forBacteriology)的“Manual of Methods for General Bacteriology”手册(Washington D.C.,USA 1981)。
可使用的碳源包括糖及碳水化合物,例如葡萄糖、蔗糖、乳糖、果糖、麦芽糖、糖蜜、淀粉和纤维素,油和脂肪如豆油、葵花油、落花生油和椰子油,脂肪酸如棕榈酸、硬脂酸和亚油酸,醇如甘油和乙醇,及有机酸如乙酸。这些物质可单独或混合使用。
可使用的氮源包括含氮有机化合物如胨、酵母提取物、肉膏、麦芽汁、玉米浆、大豆粉和尿素,或无机化物如硫酸铵、氯化铵、磷酸铵、碳酸铵和硝酸铵。
可使用的磷源包括磷酸、磷酸二氢钾或磷酸氢二钾,或相应钠盐。培养基另外还必须含有为生长所需的金属盐如硫酸镁或硫酸铁。最后,除了上述物质之外,还加入必需生长激素如氨基酸和维生素。所述额外的添加物质可以一次添加的形式加入培养基或在培养期间以适当方式补料添加。
为了调节培养物的pH值,可以适当方式加入碱性化合物如氢氧化钠、氢氧化钾、氨或氨水,或酸性化合物如磷酸或硫酸。为了控制泡沫产生,可以利用抗泡沫剂例如脂肪酸聚乙二醇酯。为了保持质粒的稳定性,可以加入适当的选择性功能物质例如抗生素。为了保持有氧条件,可向培养基中充入氧气或含氧混合气,例如空气。培养温度通常在20℃和45℃之间,优选25℃-40℃。持续培养直至经过对数生长期。此目的通常在10~20小时范围内达到。随后优选地收获细胞、洗涤并导入缓冲液中形成pH6-9、特别是pH6.8-7.9的悬浮液。细胞浓度为1-6%,特别是1.5-4%(湿重)。它们通常使用物理或化学方法而透化,例如使用甲苯,如Wilms et al.,J.Biotechnol.,vol.86(2001),19-30所述,使得需代谢的D-氨基酸穿透细胞壁并可以泄漏出。
然后将该悬浮液与含有D-氨基酸和L-苹果酸盐或者L-苹果酸的溶液混合。在10-40℃、特别是在25-36℃,在pH6.8-8.9、优选pH7.5-8.5条件下发生转化。通过加热至70-100℃结束。
实施例1:构建质粒
为了证实反应原理,构建携带下述不同基因组合的一系列质粒:苹果酸酶(MAE)、亮氨酸-脱氢酶(LeuDH)、来自原玻璃蝇节杆菌的D-AAO(ApD-AAO)和来自变异三角酵母的D-AAO(TvD-AAO)。使用表3所列出的引物和质粒。
为了扩增ApD-AAO(Apdao;gi32140775)、TvD-AAO(TVdao;gi1616634)和苹果酸酶(mae;gi:1787752)的基因,来自原玻璃蝇节杆菌、变异三角酵母和大肠杆菌K12的基因组DNA用作模板。
亮氨酸脱氢酶(leudh;gi:6741938)的基因使用质粒pT1L(=pLeuB)扩增(Ansorge M.B.Kula M.R.(2000),InvestigatingExpression Systems for the Stable Large-Scale Production ofRecombinant L-leucine dehydrogenase from bacillus cereus inEscherichia coli.App1.Microbiol.Biotechnol.53:668-73)。
使用表3列出的合适引物,通过PCR***限制性切割位点以进行克隆。使用正向引物DAAOTvforNdeI,将内含子从Tvdao基因中去除。
在将Apdao基因克隆在pH1M中的过程中,通过PCR掺入Shine-Dalgarno序列和额外的限制性切割位点(EcoRV、PstI和NotI切割位点),其中使用反向引物ApNdeIrev(表1)。
表3:质粒和引物
名称 | 质粒/基因 | 引物 | |
pE1D | pET21a/Apdao | 欧洲专利1 375 649A | |
pHWG640 | pACYC-Derivativ/mae | Altenbucher et al.,Curr.Opin.Biotechnol.,2001,12(6),559-563 | |
pT1L(=pLeuB) | pTrc99a/leudh | Ansorge et al.,Appl.Microbiol.Biotechnol.,2000,53(6),668-673 | |
pAD1M | pACYC-Duet/mae | MEfNdeIMErEcoRV | 5′ggaattc catatggatattcaaaaaagagtgag 3′5′gcgc gatatctattagatggaggtacggcggta 3′ |
pAD2DM | pAD1M/Apdao | ApNcoIfor_2ApPstIrev | 5′catg ccatgggtcccacagcaccgttgag 3′5′ttt ctgcaggctagctggccggctcgccagcca 3′ |
pAD3LM | pAD1M/leudh | LeuDHfNcoI_2LeuDHrSacI | 5′cat gccatgggtacattagaaatcttcgaata 3′5′cg gagctctattagcgacggctaataatat 3′ |
pH1DM | pHWG640/Apdao | ApNdeIfor_2ApNdeIrev | 5′ggaattc catatgcccacagcaccgttgag 3′5′gaattc catatgttatatctccttcttgcggccgcctgcaggatatcctagctggccggctcgccagcca 3′ |
pE2D | pET21a/Tvdao | DAAOTvforNdeIDAAOTvrevBamHI | 5′ggaattc catatggctaaaatcgttgttattggtgccggtgttgccggtttaactacagctctt 3′5′cgg gatccctaaaggtttggacgagtaagagctctttc 3′ |
pJ1L | pJOE4036/leudh | LeuDHfSacILeuDHrSacI | 5′cg gagctcatgacattagaaatcttcgaata 3′5′cg gagctctattagcgacggctaataatat 3′ |
实施例2:构建表达菌株
通过合适的质粒组合,构建表4中列出的重组大肠杆菌菌株,其均含有氨基酸氧化酶、亮氨酸脱氢酶和苹果酸酶的基因。
选择来自Novagen的BL21(DE3)和来自Stratgene的JM109作为起始菌株。
表4:大肠杆菌菌株
No. | 大肠杆菌菌株 | 质粒1 | 质粒2 | 诱导剂 | 抗性 |
1 | BL21(DE3) | pAD3LM | pE1D | IPTG | ApR,CmR |
2 | BL21(DE3) | pAD3LM | pE2D | IPTG | ApR,CmR |
3 | BL21(DE3) | pAD2DM | pJ1L | ITPG+Rha | ApR,CmR |
4 | BL21(DE3) | pAD2DM | pT1L | IPTG | ApR,CmR |
5 | JM109 | pH2DM | pJ1L | Rha | ApR,CmR |
6 | BL21(DE3) | pH2DM | pJ1L | Rha | ApR,CmR |
7 | JM109 | pH2DM | pT1L | IPTG+Rha | ApR,CmR |
8 | BL21(DE3) | pH2DM | pT1L | IPTG+Rha | ApR,CmR |
菌株的名称(1-8)在如下实施例中相应保留。
实施例3:胞内酶活性的核实
将相应于表4的菌株在标准条件下在pH7.5的Luria-Bertani(LB)培养基中培养(angezogen)。
根据转化的质粒,将100μg/ml的氨苄青霉素和/或34μg/ml的氯霉素加入培养基中。
为了进行酶表达,将重组大肠杆菌菌株在需氧条件下在具有20ml培养基的100ml振动瓶中培养。将细胞在37℃以200rpm在振动旋转机中保温,并当OD550为大约0.5时用100μM IPTG和/或0.2%鼠李糖诱导。在这种诱导之后,将菌株在30℃进一步保温。
测定的酶活性示于图2。
白色:D-AAO;灰色:MAE;MAE;黑色:LeuDH。
菌株6示出鼠李糖启动子的有效性。
实施例4:与完整重组菌株的生物转化反应
(A):D,L-甲硫氨酸
作为去外消旋化的实例,将甲硫氨酸的外消旋物用作底物并用相应于表2的完整的重组大肠杆菌细胞菌株1转化,所述重组大肠杆菌细胞菌株1同时含有pAD3LM质粒(携带亮氨酸脱氢酶和苹果酸酶的基因)和pE1D(携带原玻璃蝇节杆菌的D-氨基酸氧化酶的基因)。
通过HPLC,L-met的催化合成可以通过D-met的氧化酶催化分解及通过还原性氨基化而实现。相继利用的是:25mM D,L-met,100mM L-苹果酸盐,0.7M NH4Cl,50mM tris,10mM MgCl2,终pH为8.0。将细胞悬浮在50mM TEA/HCl pH 7.6缓冲液中,并与10μl/ml甲苯混合,将此悬浮液在30℃搅拌30分钟,然后静置进行反应。反应中细胞浓度在1ml终体积中为3.3%(每单位体积湿重)。在Thermomixer 5436(Eppendorf公司)中以1000rpm摇动进行转化。通过在95℃加热5分钟结束反应,通过HPLC分析上清(klare _berstand)。
为了确定D-和L-met的浓度,在稀释后将样品衍生化。为此加入20μl在100mM硼酸钠缓冲液(pH 10.4)中的260mM异丁酸-L-半胱氨酸和170mM邻苯二甲酰二醛的溶液。HPLC分离条件可公开获得(L.Krieg et al.(2002)Screening for amidases:Isolation andCharacterization of a Novel D-amidase from variovorax paradoxus.Adv.Synth.Catal.,344(9):965-73)。
D-met的分解和L-met的形成概括示于图3。获得的对映异构体纯的L-met产物的终浓度为25mM。
(B)D-亮氨酸
以与A部分所述相似的方式,将25mM D,L-亮氨酸溶液与同样的微生物开始反应。反应本身及HPLC分析相应于已经描述的实施例。
D-leu的分解和L-leu的形成一起示于图4。获得的对映异构体纯的L-leu产物的终浓度为25mM。
Claims (20)
1.重组微生物,其与起始微生物相比具有更高浓度或活性的D-氨基酸氧化酶、L-氨基酸脱氢酶、NADH共底物NADH再生酶之一,如果需要则还具有过氧化氢酶。
2.权利要求1的微生物,其包含静态细胞。
3.权利要求1或2的微生物,其具有更高浓度或活性的甲酸脱氢酶。
4.权利要求1或2的微生物,其具有更高浓度或活性的苹果酸脱氢酶。
5.权利要求1或2的微生物,其具有更高浓度或活性的醇脱氢酶。
6.权利要求1-5任一项的微生物,其具有更高浓度或活性的L-亮氨酸脱氢酶。
7.权利要求1-5任一项的微生物,其具有更高浓度或活性的L-氨基酸脱氢酶。
8.权利要求1-4任一项的微生物,其中编码所述酶的多个基因源自其它物种微生物、霉菌或者酵母。
9.权利要求1-8任一项的微生物,其中D-氨基酸氧化酶、L-氨基酸脱氢酶、NADH辅酶再生酶及如果需要还有过氧化氢酶的胞内活性与起始微生物相反是增高的,其中这些酶的编码核苷酸序列的拷贝数被增加或者使用强启动子或者组合这两种措施。
10.载体,其中一或多个编码核苷酸序列编码D-氨基酸氧化酶、氨基酸脱氢酶或者编码一种NADH共底物再生酶。
11.权利要求10的载体,其含有D-氨基酸脱氢酶的编码核苷酸序列和苹果酸脱氢酶的编码核苷酸序列。
12.权利要求10的载体,其含有氨基酸脱氢酶的编码核苷酸序列和苹果酸脱氢酶的编码核苷酸序列。
13.权利要求10的载体,其含有氨基酸脱氢酶的编码核苷酸序列及D-氨基酸氧化酶的编码核苷酸序列。
14.以权利要求10-13任一项的载体转化的微生物,所述转化的微生物与起始微生物相比含有各个编码所述酶的编码核苷酸序列的至少一个额外拷贝。
15.通过使用对映体选择性酶合成途径从D-氨基酸中生产L-氨基酸的方法,其特征在于将重组微生物与含有D-氨基酸的溶液反应,并分离所得L-氨基酸,所述重组微生物与起始微生物(例如野生型)相比具有更高浓度或活性的氨基酸氧化酶、氨基酸脱氢酶、NADH共底物再生酶及如果需要还具有过氧化氢酶。
16.权利要求15的方法,其特征在于应用权利要求1-9和14的微生物。
17.权利要求15或16的方法,其特征在于选择苹果酸脱氢酶作为NADH再生共底物,并且含有D-氨基酸的溶液形成L-苹果酸盐或者L-苹果酸。
18.权利要求15-17任一项的方法,其特征在于微生物的静态细胞被分解。
19.权利要求15-18任一项的方法,其特征在于所用细胞的细胞壁通过化学或者物理措施而变得可通透,用以从溶液中吸收D-氨基酸。
20.权利要求15-19任一项的方法,其特征在于应用选自如下一组的D-氨基酸或者外消旋DL-氨基酸:赖氨酸、精氨酸、苯丙氨酸、缬氨酸、鸟氨酸、亮氨酸、组氨酸、正亮氨酸、酪氨酸、丙氨酸、谷氨酸、头孢菌素、特别是甲硫氨酸。
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Cited By (5)
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CN102174632A (zh) * | 2009-05-19 | 2011-09-07 | 重庆邮电大学 | 一种非天然l-氨基酸的生物催化去消旋化制备方法 |
WO2015055127A1 (zh) * | 2013-10-17 | 2015-04-23 | 苏州同力生物医药有限公司 | 一种左旋吡喹酮的制备方法 |
CN106520651A (zh) * | 2016-11-08 | 2017-03-22 | 江南大学 | 一种利用酶法转化生产l‑正缬氨酸的方法 |
CN107502647A (zh) * | 2017-09-15 | 2017-12-22 | 浙江大学 | 一种生物酶法去消旋化制备l‑草铵膦的方法 |
WO2019128387A1 (zh) * | 2017-12-28 | 2019-07-04 | 苏州同力生物医药有限公司 | 一种酶法拆分制备(s)-1,2,3,4-四氢异喹啉-1-甲酸及其衍生物的方法 |
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JPWO2006132145A1 (ja) * | 2005-06-09 | 2009-01-08 | ダイセル化学工業株式会社 | L−アミノ酸の製造方法 |
JP5291934B2 (ja) | 2005-08-02 | 2013-09-18 | 株式会社カネカ | D−アミノ酸オキシダーゼ、およびl−アミノ酸、2−オキソ酸、又は環状イミンの製造方法。 |
EP2046949B1 (en) * | 2006-07-19 | 2014-01-15 | Ajinomoto Co., Inc. | A method for producing an l-amino acid using a bacterium of the enterobacteriaceae family |
JP5005293B2 (ja) * | 2006-08-22 | 2012-08-22 | 株式会社カネカ | D−アミノ酸オキシダーゼ、およびl−アミノ酸、2−オキソ酸、又は環状イミンの製造方法。 |
JPWO2008047656A1 (ja) * | 2006-10-12 | 2010-02-25 | 株式会社カネカ | L−アミノ酸の製造方法 |
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US9267116B2 (en) | 2008-12-09 | 2016-02-23 | Kaneka Corporation | Amino acid dehydrogenase, and process for producing L-amino acid, 2-oxo acid or D-amino acid |
US9080192B2 (en) | 2010-02-10 | 2015-07-14 | Codexis, Inc. | Processes using amino acid dehydrogenases and ketoreductase-based cofactor regenerating system |
SG11201608272RA (en) * | 2014-04-30 | 2016-11-29 | Evonik Degussa Gmbh | Method for producing l-amino acids using an alkaliphilic bacteria |
US10364448B2 (en) | 2014-07-11 | 2019-07-30 | Sumitomo Chemical Company, Limited | Method for producing L-α-amino acid compound |
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DE19606494A1 (de) | 1996-02-22 | 1997-08-28 | Degussa | Enzym mit LeuDH-Aktivität, dafür codierende Nukleotid-Sequenz und Verfahren zur Herstellung des Enzyms |
ES2109194B1 (es) * | 1996-04-22 | 1998-07-16 | Antibioticos Sa | Un procedimiento para producir la enzima d-aminoacido oxidasa de rhodotorula gracilis en celulas hospedantes. |
WO2003000909A2 (en) * | 2001-06-21 | 2003-01-03 | Diversa Corporation | Methods for the manufacture of pure single enantiomer compounds and for selecting enantioselective enzymes |
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DE10240603A1 (de) | 2002-09-03 | 2004-03-11 | Degussa Ag | Verwendung von Malat-Dehydrogenase zur NADH-Regenerierung |
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2004
- 2004-02-19 DE DE102004008445A patent/DE102004008445A1/de not_active Withdrawn
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2005
- 2005-01-27 CN CN201410044828.7A patent/CN103834607A/zh active Pending
- 2005-01-27 WO PCT/EP2005/000768 patent/WO2005090590A1/de active Application Filing
- 2005-01-27 BR BRPI0506795-2A patent/BRPI0506795A/pt not_active Application Discontinuation
- 2005-01-27 CN CNA2005800052537A patent/CN1922328A/zh active Pending
- 2005-01-27 EP EP05707022A patent/EP1716241A1/de not_active Withdrawn
- 2005-01-27 RU RU2006133266/13A patent/RU2006133266A/ru unknown
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CN107502647B (zh) * | 2017-09-15 | 2020-12-15 | 浙江大学 | 一种生物酶法去消旋化制备l-草铵膦的方法 |
WO2019128387A1 (zh) * | 2017-12-28 | 2019-07-04 | 苏州同力生物医药有限公司 | 一种酶法拆分制备(s)-1,2,3,4-四氢异喹啉-1-甲酸及其衍生物的方法 |
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DE102004008445A1 (de) | 2005-09-08 |
CN103834607A (zh) | 2014-06-04 |
US20060063238A1 (en) | 2006-03-23 |
BRPI0506795A (pt) | 2007-05-22 |
US7217544B2 (en) | 2007-05-15 |
RU2006133266A (ru) | 2008-03-27 |
WO2005090590A1 (de) | 2005-09-29 |
EP1716241A1 (de) | 2006-11-02 |
JP2007522810A (ja) | 2007-08-16 |
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