CN101511996A - 酶促还原炔衍生物的方法 - Google Patents
酶促还原炔衍生物的方法 Download PDFInfo
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- CN101511996A CN101511996A CNA2007800320370A CN200780032037A CN101511996A CN 101511996 A CN101511996 A CN 101511996A CN A2007800320370 A CNA2007800320370 A CN A2007800320370A CN 200780032037 A CN200780032037 A CN 200780032037A CN 101511996 A CN101511996 A CN 101511996A
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- described method
- enzyme
- aforementioned arbitrary
- reductase enzyme
- alkyl
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Abstract
本发明涉及通过在特定还原酶的存在下反应,酶促还原式(1)的炔衍生物的方法,其中R1表示H、C1-C6烷基、C2-C6链烯基或任选取代的碳环或杂环芳族或非芳族基团,R2表示H、C1-C6烷基或C2-C6链烯基。
Description
发明领域
本发明涉及酶促还原炔衍生物的方法。
背景技术
至今还未公开过不需要ATP的酶促还原炔衍生物的方法。
Takeshita,M.等人在Journal of Molecular Catalysis B:Enzymatic 5(1998)245-248中描述了用S-9大鼠肝级分和用面包酵母进行苯基-C4衍生物的不对称生物转化的实验。根据此文,与对照相比,使用大鼠肝级分,4-苯基-3-丁炔-2-酮没有显著转化为相应的丁烯-2-酮。使用酵母全细胞的实验产出的丁烯-2-酮的量也不显著(3%产率)。另一方面,作为主要组分形成相应的S-丁-2-醇(31%)、相应的丁-2-酮(8%)和相应的S-丁炔-2-醇(5%)。由于实验是用酵母全细胞进行的,故不可能给这些生物转化分派特定的酶活性。而且,实验必须在细胞辅因子例如ATP的存在下进行。
本发明的目的在于提供不需要ATP的酶促还原炔衍生物的方法。
发明简述
通过用还原酶OYE1、2和3及其功能等同物还原通式(1)的炔衍生物已实现了上述目的。
附图说明
附图显示
图1:在NADPH(左)或NADH(右)、EDTA和异丙醇存在下,使用OYE1-3,20mM 4-苯基-3-丁炔-2-酮在pH6.8,30℃,1h后的生物转化。所述酶在大肠杆菌(E.coli)TG10+中过量表达;TG10+自身用作对照;
图2:使用的酶OYE1、2和3的氨基酸序列;
图3:pAgro4的质粒图;
图4:pHSG575’的质粒图;
图5:pDHE1658 OYE1的质粒图;
图6:pDHE1658 OYE2的质粒图;和
图7:pDHE1658 OYE3的质粒图。
发明详述
本发明涉及由通式(1)的α,β-不饱和炔酮衍生物酶促制备通式(2)的烯酮衍生物的方法
其中
R1为H、C1-C6烷基、C2-C6链烯基或任选取代的碳环或杂环芳族或非芳族环,和
R2为H、C1-C6烷基或C2-C6链烯基,
所述方法通过如下方式进行:在还原酶的存在下还原式(1)的化合物,所述还原酶
(i)包含多肽序列SEQ ID NO:1、2、3、5、7或9的至少其中之一或
(ii)具有功能上等同的多肽序列,该序列与SEQ ID NO:1、2、3、5、7或9的序列同一性至少为80%。
本发明优选提供特别是E构型的式2化合物
其中R1和R2具有上述含义。特别地多于50%,特别地多于60、70或80%,但优选多于90%,例如95到99%,特别是约100%的式2化合物为E构型形式。
原则上,既可以用经纯化的或经富集的酶本身实施本发明方法,也可以用天然或重组表达此酶的微生物、用源于其的细胞匀浆物、或如果微生物分泌酶到环境中的话,使用培养物上清液来实施本发明方法。然而特别地,本发明上下文中,“酶促反应”包含在基本上无ATP的环境中的反应,即,优选地使用不再包含任何低分子量细胞成分例如特别是ATP的无细胞酶制品进行的反应,如使用纯的或富集的酶或适宜的蛋白质级分的反应。
除非另有说明,以下术语的含义为:
-C1-C6烷基特别是甲基、乙基、丙基、丁基、戊基或己基,及一次或多次分支的相应类似物,如异丙基、异丁基、仲丁基、叔丁基、异戊基或新戊基,特别优选所述的C1-C4烷基基团;
-C2-C6链烯基特别是具有2到6个碳原子的上述烷基基团的单不饱和类似物,特别优选相应的C2-C4链烯基基团。
-碳环和杂环芳族或非芳族环特别是任选稠合的、具有3到12个碳原子和任选地1到4个杂原子如N、S和O,特别是N或O的环。可以提及的实例为环丙基、环丁基、环戊基、环己基、环庚基、其单或多不饱和类似物如环丁烯基、环戊烯基、环己烯基、环庚烯基、环己二烯基、环庚二烯基;苯基和萘基;和5到7元饱和或不饱和的杂环基,其具有1到4个选自O、N和S的杂原子,其中该杂环可以任选地与其它杂环或碳环稠合。应特别提到来自以下的杂环基:吡咯烷、四氢呋喃、哌啶、吗啉、吡咯、呋喃、噻吩、吡唑、咪唑、噁唑、噻唑、吡啶、吡喃、嘧啶、哒嗪、吡嗪、苯并呋喃、吲哚和喹啉。该环状基团以及上述烷基和链烯基基团均可以任选地被取代一次或多次,例如1、2或3次。作为适宜的取代基的实例应提及:卤素,特别是F、Cl、Br;-OH、-SH、-NO2、-NH3、-SO3H、C1-C4烷基和C2-C4链烯基、C1-C4烷氧基;和羟基-C1-C4烷基;其中烷基和链烯基定义如上,烷氧基来自如上定义的相应烷基。
本发明的方法特别可以用通式(1)的炔进行,其中R1为分支或不分支形式的C1-C4烷基、分支或不分支形式的C2-C6链烯基或任选取代的苯基。
对于本发明的方法,特别适宜的底物为这样的通式(1)的炔,其中R1为任选取代的苯基,R2为CH3。
根据本发明使用的还原酶在一些情况下有时不仅还原相对于羰基官能团的α,β位的三键,还还原羰基官能团本身,从而形成相应的醇。而烯同样可以进一步部分还原为相应的烷。
适合本发明方法的还原酶为所有能够在依赖NAD(P)H且优选不依赖ATP的反应中将4-苯基-3-丁炔-2-酮还原为E-4-苯基-3-丁烯-2-酮的酶。此反应在下文中也称为模型反应。
4-苯基-3-丁炔-2-酮 E-4-苯基-3-丁烯-2-酮
而且,适合本发明方法的还原酶(有时也称为enoate还原酶)可以具有SEQ ID NO:1、2、3、5、7或9所示的多肽序列,或具有这样的多肽序列,该多肽序列与SEQ ID NO:1、2、3、5、7或9的序列同一性为至少80%,例如至少90%,或至少95%,及特别是至少97%、98%或99%。
具有SEQ ID NO:1的多肽名为OYE1,来自卡尔酵母(Saccharomycescarlsbergensis)(Genbank Q02899)。
具有SEQ ID NO:2的多肽由OYE2基因编码,其来自面包酵母(酿酒酵母(Saccharomyces cerevisiae)基因座位YHR179W)(GenbankQ03558)。
具有SEQ ID NO:3的多肽由OYE3基因编码,其来自面包酵母(酿酒酵母基因座位YPL171C)(Genbank P41816)。
SEQ ID NO:5、7和9所示的序列与SEQ ID NO:1、2和3相对应,与其差别仅在于额外的N端甲硫氨酸残基。
为本文中所述的目的,序列同一性用威斯康辛大学(University ofWisconsin)Genetics Computer Group(GCG)的“GAP”计算机程序确定,用10.3版本以及GCG推荐的标准参数。
这样的还原酶可以用本领域技术人员已知的靶向或随机诱变方法从SEQ ID NO:1、2、3、5、7或9开始获得。然而,备选地,也可以在微生物中寻找催化上述模型反应且氨基酸序列已经或通过诱变方法具有与SEQ ID NO:1、2、3、5、7或9的所需序列同一性的还原酶,所述微生物优选属于以下的属:Alishewanella、交替球菌属(Alterococcus)、Aquamonas、Aranicola、杀雄菌属(Arsenophonus)、Azotivirga、Brenneria、巴克纳氏菌属(Buchnera)(蚜虫第一代内共生体(P-endosymbionts))、布戴约维采菌属(Budvicia)、布丘氏菌属(Buttiauxella)、Candidatus Phlomobacter、西地西菌属(Cedecea)、柠檬酸杆菌属(Citrobacter)、Dickeya、爱德华菌属(Edwardsiella)、肠杆菌属(Enterobacter)、欧文氏菌属(Erwinia)、埃希氏菌属(Escherichia)、爱文菌属(Ewingella)、Grimontella、哈夫尼亚菌属(Hafnia)、克雷伯菌属(Klebsiella)、克吕沃尔菌属(Kluyvera)、勒克菌属(Leclercia)、勒米诺菌属(Leminorella)、米勒菌属(Moellerella)、摩根菌属(Morganella)、肥杆菌属(Obesumbacterium)、泛菌属(Pantoea)、果胶杆菌属(Pectobacterium)、发光杆菌属(Photorhabdus)、邻单胞菌属(Plesiomonas)、布拉格菌属(Pragia)、变形杆菌属(Proteus)、普罗威登斯菌属(Providencia)、拉恩菌属(Rahnella)、劳特菌属(Raoultella)、沙门氏菌属(Salmonella)、Samsonia、沙雷氏菌属(Serratia)、志贺氏菌属(Shigella)、Sodalis、塔特姆菌属(Tatumella)、特拉布斯氏菌属(Trabulsiella)、魏格沃菌(Wigglesworthia)、致病杆菌(Xenorhabdus)、耶尔森菌属(Yersinia)或约克菌属(Yokenella)。
还原酶可以以纯化或部分纯化形式、或以微生物本身的形式使用。从微生物中获得和纯化脱氢酶的方法为本领域技术人员所熟知。
优选在适宜的辅因子(也称为共底物)的存在下用还原酶进行对映选择性还原。通常用于还原酮的辅因子为NADH和/或NADPH。还原酶还可以以细胞体系的形式使用,该细胞体系固有地含有辅因子,或可以加入氧化还原介体(A.Schmidt,F.Hollmann和B.Bühler“Oxidation ofAlcohols”,于K.Drauz和H.Waldmann,Enzyme Catalysis in OrganicSynthesis 2002,卷III,991-1032,Wiley-VCH,Weinheim)。
此外优选在适宜的还原剂存在下用还原酶进行对映选择性还原,其中所述还原剂可以使在还原过程中被氧化的辅因子再生。适宜的还原剂的实例为糖,特别是己糖如葡萄糖、甘露糖、果糖,和/或可氧化的醇,特别是乙醇、丙醇或异丙醇,及甲酸、亚磷酸盐或氢分子。为氧化该还原剂及随之令辅酶再生,可以加入第二脱氢酶,例如当用葡萄糖作为还原剂时加入葡萄糖脱氢酶,或当用甲酸作为还原剂时加入甲酸脱氢酶。其可以以游离或固定化酶形式,或以游离或固定化细胞的形式使用。其制备可以单独进行或可以通过在(重组的)还原酶菌株中共表达来实现。
本发明方法的一个优选实施方案是用酶系统再生辅因子,该酶系统中使用第二脱氢酶,特别优选葡萄糖脱氢酶。
还可以方便地加入其它促进还原的添加物例如金属盐或螯合剂,例如EDTA。
根据本发明使用的还原酶可以以游离或固定化形式使用。固定化酶指固定到惰性载体上的酶。适合的载体材料和在其上固定化的酶在EP-A-1149849、EP-A-1069 183和DE-A 10019377及其中引述的文献中公开。这些出版物在这方面的公开全部地通过引用并入本文。适合的载体材料包括例如粘土、粘土材料如高岭石、硅藻土、珍珠岩、二氧化硅、氧化铝、碳酸钠、碳酸钙、纤维素粉、阴离子交换材料、合成聚合物如聚苯乙烯、丙烯酸树脂、酚醛树脂、聚氨基甲酸酯和聚烯烃如聚乙烯和聚丙烯。载体材料通常以细碎的颗粒形式用于制备与载体结合的酶,优选为多孔形式。载体材料的颗粒大小通常不大于5mm,特别是不大于2mm(分级曲线)。当以全细胞催化剂形式使用脱氢酶时,可以相似地选择游离或固定化形式。载体材料的实例为藻酸钙和角叉菜胶。酶和细胞也均可以直接用戊二醛进行交联(交联以得到CLEA)。相应的和其它固定化方法在例如J.Lalonde和A.Margolin“Immobilization of Enzyme”于K.Drauz和H.Waldmann,Enzyme Catalysis in Organic Synthesis 2002,卷III,991-1032,Wiley-VCH,Weinheim中有所描述。
反应可以在水性或非水性反应介质中或在双相系统或(微)乳剂中进行。水性反应介质优选为缓冲溶液,其pH通常为4到8,优选为5到8。除了水,水性溶剂还可以包含至少一种醇,例如乙醇或异丙醇、或二甲亚砜。
非水性反应介质指基于液体反应介质的总重量,反应介质中包含的水按重量计少于1%,优选按重量计少于0.5%。反应特别可以在有机溶剂中进行。
适合的有机溶剂为例如脂族烃,优选具有5到8个碳原子,如戊烷、环戊烷、己烷、环己烷、庚烷、辛烷或环辛烷,卤代脂族烃,优选具有1或2个碳原子,如二氯甲烷、氯仿、四氯甲烷、二氯乙烷或四氯乙烷,芳族烃如苯、甲苯、二甲苯、氯苯或二氯苯,脂肪族无环和环状醚或醇,优选具有4到8个碳原子,如乙醇、异丙醇、二乙醚、甲基叔丁醚、乙基叔丁醚、二丙醚、二异丙醚、二丁醚、四氢呋喃,或酯如乙酸乙酯或乙酸正丁酯,或酮如甲基异丁基酮或二噁烷,或其混合物。特别优选使用上述醚,尤其是四氢呋喃。
例如,使用还原酶的还原可以在水性有机反应介质或水性反应介质中进行,该水性有机反应介质例如为以任何比例混合的水/异丙醇,例如1∶99到99:1或10:90到90:10。
底物(1)在酶促还原中优选使用的浓度为0.1g/l到500g/l,特别优选1g/l到50g/l,可以连续或间断进料。
酶促还原通常在低于所使用的还原酶的失活温度但高于-10℃的反应温度下进行。特别优选在0到100℃的范围内,特别是15到60℃,尤其是20到40℃,例如于约30℃。
例如,一种可行的操作方案是例如通过搅拌或振荡将底物(1)与还原酶、溶剂、及辅酶(如果适当的话)、用于再生辅酶的第二脱氢酶和/或其它还原剂(如果适当的话)充分混合。但是,还可以将还原酶固定在反应器例如柱中,令包含底物和,如果适当的话,辅酶和/或共底物的混合物通过反应器。为此目的,可以令混合物循环通过反应器直至实现期望的转化。
在此情况下,在相对于羰基官能团的α,β位的三键被还原为双键,有时羰基官能团本身也被还原为醇官能团。基于混合物中存在的底物,通常实施还原直至发生至少70%,特别优选至少85%,特别是至少95%的转化。也可以利用常规方法如气相色谱或高压液相色谱,跟踪反应进程,即,随后的双键还原。
在本发明上下文中,具体公开的酶的“功能等同物”或类似物为这样的多肽,其与该具体公开的酶不同,但仍具有期望的生物活性,例如底物特异性。因此,“功能等同物”指例如这样的酶,其催化模型反应,并且具有包含SEQ ID NO:1、2或3所示氨基酸序列之一的酶的至少20%,优选50%,特别优选75%,非常特别优选90%活性。另外,功能等同物还优选在pH4到10之间是稳定的,最好其最适pH在pH5和8之间,最适温度在20℃到80℃的范围内。
根据本发明,“功能等同物”还特别指突变体,其在上述氨基酸序列的至少一个序列位置上具有与具体公开的氨基酸不同的氨基酸,但仍具有一种上述的生物活性。因此“功能等同物”包含这样的突变体,其可以由一个或多个氨基酸的添加、替代、缺失和/或倒位得到,所述的修饰可以发生在任何序列位置上,只要能得到具有本发明性质谱的突变体即可。功能等同物还尤其存在于突变体和未修饰的多肽在反应模式上性质相一致的情况下,即,例如以不同速率转化相同底物的情况。
适宜的氨基酸替代的实例可见下表:
原始残基 替代实例
Ala Ser
Arg Lys
Asn Gln;His
Asp Glu
Cys Ser
Gln Asn
Glu Asp
Gly Pro
His Asn;Gln
Ile Leu;Val
Leu Ile;Val
Lys Arg;Gln;Glu
Met Leu;Ile
Phe Met;Leu;Tyr
Ser Thr
Thr Ser
Trp Tyr
Tyr Trp;Phe
Val Ile;Leu
以上意义的“功能等同物”也可以是所述多肽的“前体”和“功能衍生物”。
在此上下文中,“前体”为多肽的天然或合成的前体,其具有或没有期望的生物活性。
本发明多肽的“功能衍生物”同样可以借助于已知技术在氨基酸侧链官能团上或在其N或C末端制备。这样的衍生物包括例如羧酸基团的脂族酯,羧酸基团的酰胺(其可以通过与氨或与伯胺或仲胺反应得到);通过与酰基反应制备的游离氨基的N-酰基衍生物;或通过与酰基反应制备的游离羟基的O-酰基衍生物。
在可能发生蛋白质糖基化的情况下,本发明的“功能等同物”包括上述类型的蛋白质的去糖基化或糖基化形式,以及可以通过改变糖基化模式得到的修饰形式。
当然,“功能等同物”还包括可以从其它生物获得的多肽和天然存在的变体。例如,可以通过序列比较确立同源序列区域的范围,并基于本发明的特定需要确定等同的酶。
“功能等同物”同样还包括本发明多肽的片段,优选单个结构域或序列基元,其具有例如期望的生物功能。
此外,“功能等同物”可以为融合蛋白,其包含上述多肽序列之一或由其衍生的功能等同物、和功能与之不同的、作N或C端功能性连接(即融合蛋白的各部分之间相互的功能损害可忽略)的、至少一个其它异源序列。这样的异源序列的非限制性例子为例如信号肽或酶。
可以通过筛选突变体例如截短突变体的组合文库,鉴定本发明蛋白质的同系物。例如,蛋白质变体的多样化文库(variegated library)可以通过在核酸水平进行组合诱变而产生,例如通过对合成的寡核苷酸的混合物进行酶促连接而产生。有大量方法可以用于从简并寡核苷酸序列制备潜在同系物的文库。简并基因序列的化学合成可以在DNA自动合成仪中进行,然后可以将合成的基因连接入适合的表达载体。使用一组简并基因使得可以在一个混合物中提供编码期望的一组潜在蛋白质序列的所有序列。合成简并寡核苷酸的方法为本领域技术人员已知(例如Narang,S.A.(1983)Tetrahedron 39:3;Itakura等人(1984)Annu.Rev.Biochem.53:323;Itakura等人,(1984)Science 198:1056;Ike等人(1983)Nucleic AcidsRes.11:477).
对于筛选通过点突变或截短制备的组合文库的基因产物,和在cDNA文库中筛选具有选定性质的基因产物,有若干技术为本领域已知。可以改造这些技术以适于如下基因文库的快速筛选,该基因文库通过组合诱变本发明的同系物而产生。筛选大的基因文库的最常用高通量筛选技术包括:将基因文库克隆到可复制的表达载体中,用得到的载体文库转化适合的细胞,在一定条件下表达组合基因,在该条件下,对期望的活性的检测有助于分离编码该检测到的产物的基因的载体。递推式系综诱变(Recursiveensemble mutagenesis)(REM)是增加文库中功能性突变体频率的技术,可以与筛选试验进行组合使用以鉴定同系物(Arkin和Yourvan(1992)PNAS 89:7811-7815;Delgrave等人(1993)Potein Engineering 6(3):327-331)。
本发明还涉及核酸序列(单链和双链DNA和RNA序列,例如cDNA和mRNA),其编码本发明的具有还原酶活性的酶。优选编码例如SEQ IDNO:1、2或3所示的氨基酸序列或其特征性的部分序列的核酸序列。
本文提及的所有核酸序列都可以用本质上已知的方式从核苷酸构件通过化学合成制备,例如对双螺旋的各重叠互补核酸构件进行片段缩合。寡核苷酸的化学合成可以例如用已知方式通过亚磷酰胺法进行(Voet,Voet,第二版,Wiley Press New York,896-897页)。添加合成的寡核苷酸、用DNA聚合酶的Klenow片段补平缺口和连接反应,以及一般的克隆方法均在Sambrook等人(1989),分子克隆实验指南(Molecular克隆:Alaboratory manual),冷泉港实验室出版社(Cold Spring HarborLaboratory Press)中有描述。
用于实施本发明的酶促还原方法的其它实施方案:
根据本发明使用的还原酶可以以游离或固定化酶的形式用于本发明的方法。
本发明的方法中,pH最好保持在pH4和12之间,优选在pH4.5和9之间,特别优选在pH5和8之间。
对于本发明的方法,可以使用包含编码还原酶的核酸、核酸构建体或载体的生长细胞。还可以使用静止细胞或破裂的细胞。破裂的细胞指例如经用例如溶剂处理而导致可通透的细胞,或经酶处理、机械处理(例如弗氏压碎器或超声)或任何其它方式而分裂的细胞。这样得到的粗提物有利地适用于本发明的方法。本发明方法还可以使用纯化的或部分纯化的酶。固定化的微生物或酶同样适合,可以有利地用于反应中。
本发明的方法可以分批、半分批或连续进行。
本发明方法可以有利地在生物反应器中,按照例如在生物技术(biotechnology),卷3,第2版,Rehm等人编(1993,特别是第II章)中所描述的进行。
下列实施例旨在对本发明进行举例说明,而不是限制性的。在此方面参考附图进行。
实验部分
实施例1:制备表达还原酶的大肠杆菌TG10+转化体和相应的转化构建体
除非另有说明,在本发明上下文中进行的克隆步骤可以如在Sambrook等人(1989)上述引文中所述的那样进行,克隆步骤为例如限制性切割、琼脂糖凝胶电泳、纯化DNA片段、将核酸转移至硝酸纤维素膜和尼龙膜、连接DNA片段、微生物转化、微生物培养、噬菌体复制和重组DNA的序列分析。
重组大肠杆菌株TG10+(OYE)构建如下:
大肠杆菌TG10来自大肠杆菌TG1(Stratagene)。TG10是四环素抗性和鼠李糖营养缺陷型菌株。通过向TG10引入下列质粒得到TG10+:
a)pAgro4(陪伴分子+链霉素抗性)(参见图3)和
b)pHSG575(陪伴分子+氯霉素抗性)(参见图4)。
pAgro4是具有来自大肠杆菌的groELS陪伴蛋白基因的pZ载体的衍生物。PZ则是pACYC质粒的衍生物。pAgro4在例如Nucleic Acids Res.,1997,25,1203,Mol.Microbiol.,2001,40,397中有描述。
pHSG575是具有来自大肠杆菌的lacIq阻抑蛋白基因的pSC101的衍生物,在例如Gene,1987,61,63,Mol.Microbiol.,2001,40,397中有描述。
TG10+(OYE)是由进一步向TG10+引入如下质粒而得到的:
pDHE1650(鼠李糖慢启动子(slow rhamnose promoter)+氨苄青霉素抗性+一个oye基因)。
pDHE1650是pJOE2702的衍生物,其中“老黄酶”的基因已克隆在pJOE2702的NdeI和PstI或HindIII切割位点之间。pJOE2702则制备如下:从大肠杆菌JM109扩增rhaB序列,克隆进入pBR322衍生物pBTAC1的SphI或EcoRI切割位点。用合成寡核苷酸得到pET11a的核糖体结合位点和NdeI切割位点,置入pBTAC1的BamHI/EcoRI切割位点之间。该质粒在例如Methods Enzymol.,1992,216,457,Mol.Microbiol.,1996,21,1037中有描述。
以上引用的文献通过引用并入本文。
克隆了下列老黄酶(oye)基因:
a)oye1:卡尔酵母(Genbank Q02899)
b)oye2:酿酒酵母(Genbank Q03558)
c)oye3:酿酒酵母(Genbank P41816)。
由此得到下列质粒:
pDHE1650 OYE1(参见图5)
pDHE1650 OYE2(参见图6)
pDHE1650 OYE3(参见图7)
实施例2:生物转化实验
a)酶的生产
重组菌株在含100μg/ml氨苄青霉素、100μg/ml链霉素和20μg/ml氯霉素的LB培养基(10g/l胰蛋白胨,10g/l NaCl和5g/l酵母提取物)中培养。用0.1mM IPTG诱导两种陪伴分子的蛋白质过量表达,用0.5g/l L-鼠李糖诱导OYE的蛋白质过量表达。培养物在37℃以120rpm振荡22小时。收获的细胞储存在-20℃。
a)生物转化
生物转化在1ml的反应体积中进行,在磁性搅拌下30℃进行1小时。反应混合物的起始浓度如下:10g干生物质/l,10%异丙醇[体积/体积],20mM4-苯基-3-丁炔-2-酮,5mM EDTA,2mM NADP+,1U/ml来自嗜酸热原体(Thermoplasma acidophilum)的葡萄糖脱氢酶,50mM D-葡萄糖,50mM MES缓冲液(用KOH调节至pH6.8)。对于用NADH进行的生物转化,用15mM NADH替换NADPH再生系统(NADP+,葡萄糖脱氢酶和葡萄糖)。
b)实验评估
反应混合物用氯仿提取,提取物用毛细管气相色谱(GC)(使用具有Supelco BPX5毛细管柱(25m×0.32mm内径,固定相的膜厚度为0.5μm)的Varian Star 3400 GC和FID检测器)检测。产物的身份通过与样品标准物的共洗脱和GC/MS数据进行确认。该GC/MS使用Restek RTX-5MS毛细管柱(30m×0.25mm内径,固定相的膜厚度为0.25μm),在惠普(Hewlett-Packard)5890,II系列气相色谱仪上进行,该色谱仪与惠普5972TID质谱仪连接。用安捷伦科技有限公司(Agilent Technologies)的MSDChemStation软件分析层析谱和m/z比。而且,使用Wiley6文库基于片段化模式筛选数据库以鉴定未知化合物。用Bruker 300MHz GYRO NMR测定形成的4-苯基-3-丁烯-2-酮产物的顺式和反式构型。用1D-WINNMR软件分析数据,将得到的谱图与纯度99%的反式-4-苯基-3-丁烯-2-酮标准物进行比较。
附图1中概括了发现的实验结果,用初始生产率(mM/h)表示。观察到令人惊讶的E-4-苯基-3-丁烯-2-酮的特异性形成。
序列表
<110>巴斯夫欧洲公司(BASF Aktiengesellschaft)
<120>酶还原炔衍生物的方法
<130>M/47256
<160>9
<170>PatentIn version 3.3
<210>1
<211>399
<212>PRT
<213>卡尔酵母
<400>1
<210>2
<211>399
<212>PRT
<213>酿酒酵母
<400>2
<210>3
<211>399
<212>PRT
<213>酿酒酵母
<400>3
<210>4
<211>5490
<212>DNA
<213>人工
<220>
<223>编码OYE1的质粒
<220>
<221>CDS
<222>(4)..(1203)
<400>4
<210>5
<211>400
<212>PRT
<213>人工
<220>
<223>合成的构建体
<400>5
<210>6
<211>5491
<212>DNA
<213>人工
<220>
<223>编码OYE2的质粒
<220>
<221>CDS
<222>(4)..(1203)
<400>6
<210>7
<211>400
<212>PRT
<213>人工
<220>
<223>合成的构建体
<400>7
<210>8
<211>5474
<212>DNA
<213>人工
<220>
<223>编码OYE3的质粒
<220>
<221>CDS
<222>(4)..(1203)
<400>8
<210>9
<211>400
<212>PRT
<213>人工
<220>
<223>合成的构建体
<400>9
Claims (11)
1.由通式(1)的α,β-不饱和炔酮衍生物酶促制备通式(2)的烯酮衍生物的方法,
其中
R1为H、C1-C6烷基、C2-C6链烯基或任选取代的碳环或杂环芳族或非芳族环,和
R2为H、C1-C6烷基或C2-C6链烯基,
所述方法通过如下方式进行:在还原酶的存在下还原式(1)的化合物,所述还原酶
(i)包含多肽序列SEQ ID NO:1、2、3、5、7或9中的至少一个,或
(ii)具有功能上等同的多肽序列,该多肽序列与SEQ ID NO:1、2、3、5、7或9的序列同一性至少为80%。
2.权利要求1所述的方法,其中所述还原为ATP不依赖性的,使用NADPH或NADH作为辅因子进行。
3.权利要求2所述的方法,其中使用的辅因子通过酶再生。
4.权利要求3所述的方法,其中辅因子通过葡萄糖脱氢酶再生。
5.前述任一权利要求所述的方法,其中还原在水性、水性-醇性或醇性反应介质中进行。
6.前述任一权利要求所述的方法,其中还原酶为固定化形式。
7.前述任一权利要求所述的方法,其中酶选自来自卡尔酵母(Genbank Q02899)、酿酒酵母(Genbank Q03558)和酿酒酵母(GenbankP41816)的还原酶。
8.前述任一权利要求所述的方法,其中使用R1为任选取代的芳基且R2为C1-C6烷基的式(1)化合物进行反应。
9.前述任一权利要求所述的方法,其中得到式(2)的化合物的E异构体。
10.前述任一权利要求所述的方法,其中反应在0到45℃的温度和/或pH6到8进行
11.通过前述任一权利要求所述的方法制备的式(2)化合物作为中间体在活性成分的化学或酶促合成中的用途。
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EP06120008A EP1894999A1 (de) | 2006-09-01 | 2006-09-01 | Verfahren zur enzymatischen Reduktion von Alkinderivaten |
PCT/EP2007/059071 WO2008025831A1 (de) | 2006-09-01 | 2007-08-30 | Verfahren zur enzymatischen reduktion von alkinderivaten |
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EP2145904A1 (de) | 2008-07-18 | 2010-01-20 | Basf Se | Verfahren zur enzymkatalysierten Hydrolyse von Polyacrylsäureestern sowie dafür zu verwendende Esterasen |
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EP2061880B1 (de) | 2009-12-09 |
CN101511996B (zh) | 2015-08-05 |
EP1894999A1 (de) | 2008-03-05 |
ES2336724T3 (es) | 2010-04-15 |
US8227218B2 (en) | 2012-07-24 |
EP2061880A1 (de) | 2009-05-27 |
WO2008025831A1 (de) | 2008-03-06 |
ATE451452T1 (de) | 2009-12-15 |
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