不对称还原大位阻二芳香基甲酮的羰基还原酶的基因克隆及性质分析
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摘要
由于二芳香基甲酮的位阻大及羰基两侧的取代基差异性较小,对其进行不对称还原是生物催化中具有挑战性的难题之一。文中通过对毕赤酵母GS115基因组序列的分析,发现了一个潜在羰基还原酶基因pascr。将该基因克隆、表达在大肠杆菌Rosetta2(DE3)中,通过Ni-NTA对重组蛋白进行了分离纯化,并对酶的性质进行了研究。PasCR专一性利用NADPH作为辅酶,其最适反应pH为6.5;最适反应温度为35℃;凝胶层析实验结合SDS-PAGE分析表明PasCR在溶液中以二聚体形式存在。PasCR能够不对称还原位阻较大的二芳香基甲酮类化合物,如4-甲基二苯甲酮、4-氯二苯甲酮、2-氯二苯甲酮等,对4-甲基二苯甲酮的还原产物的ee值达到了85%。
Asymmetric reduction of bulky diaryl ketones is still one of the challenging tasks in biocatalysis.By genomic data mining,a putative carbonyl reductase gene pascr was found in Pichia pastoris GS115.pascr was cloned and over-expressed in Escherichia coli Rosseta2(DE3).The recombinant enzyme was purified to homogeneity by Ni-NTA column and its catalytic properties were studied.PasCR strictly used NADPH as cofactor,gel filtration and SDS-PAGE analysis suggested that the native form of PasCR was a dimmer.PasCR exhibited the highest activity at 35 °C in phosphate buffer at pH 6.5.The enzyme catalyzed the reduction of some bulky diaryl ketones,such as 4-methylbenzophenone,2-methylbenzophenone and 4-chlorobenzophenone,especially for 4-methylbenzophenone,the product S-alcohol was obtained with 85% ee.
Asymmetric reduction of bulky diaryl ketones is still one of the challenging tasks in biocatalysis.By genomic data mining,a putative carbonyl reductase gene pascr was found in Pichia pastoris GS115.pascr was cloned and over-expressed in Escherichia coli Rosseta2(DE3).The recombinant enzyme was purified to homogeneity by Ni-NTA column and its catalytic properties were studied.PasCR strictly used NADPH as cofactor,gel filtration and SDS-PAGE analysis suggested that the native form of PasCR was a dimmer.PasCR exhibited the highest activity at 35 °C in phosphate buffer at pH 6.5.The enzyme catalyzed the reduction of some bulky diaryl ketones,such as 4-methylbenzophenone,2-methylbenzophenone and 4-chlorobenzophenone,especially for 4-methylbenzophenone,the product S-alcohol was obtained with 85% ee.
引文
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[3]Truppo MD,Pollard D,Devine P.Enzyme-catalyzed enantioselective diaryl ketone reductions.Org Lett,2007,9(2):335 338.
[4]Welch CJ,Grau B,Moore J,et al.Use of chiralHPLC-MS for rapid evaluation of theyeast-mediated enantioselective bioreduction of adiaryl ketone.J Org Chem,2001,66(20):6836 6837.
[5]Corey EJ,Helal CJ.Reduction of carbonylcompounds with chiral oxazaborolidine catalysts:Anew paradigm for enantioselective catalysis and apowerful new synthetic method.Angew Chem IntEd,1998,37(15):1986 2012.
[6]Yang Y,Zhu DM,Piegat TJ,et al.Enzymaticketone reduction:mapping the substrate profile of ashort-chain alcohol dehydrogenase(YMR226c)from Saccharomyces cerevisiae.Tetrahedron:Asymmetry,2007,18(15):1799 1803.
[7]Moore JC,Pollard DJ,Kosjek B,et al.Advances inthe enzymatic reduction of ketones.Acc Chem Res,2007,40(12):1412 1419.
[8]Zhu DM,Wu QQ.Engineering theenantioselectivity of biocatalysts.Chin J Biotech,2009,25(12):1770 1778(in Chinese).朱敦明,吴洽庆.生物催化剂立体选择性的基因工程改造.生物工程学报,2009,25(12):1770 1778.
[9]Li H,Zhu DM,Hua L,et al.Enantioselectivereduction of diaryl ketones catalyzed by a carbonylreductase from sporobolomyces salmonicolor andits mutant enzymes.Adv Synth Catal,2009,351(4):583 588.
[10]Zhu DM,Mukherjee C,Biehl ER,et al.Discoveryof a mandelonitrile hydrolase from Bradyrhizobiumjaponicum USDA110 by rational genome mining.JBiotech,2007,129(4):645 650.
[11]Yu HM,Luo H,Shi Y,et al.Application ofbioinformatics in researches of industrialbiocatalysis.Chin J Biotech,2004,20(3):325 330(in Chinese).于慧敏,罗晖,史悦,等.生物信息学在工业生物催化研究中的应用.生物工程学报,2004,20(3):325330.
[12]De Schutter K,Lin YC,Tiels P,et al.Genome sequence of the recombinant protein production host Pichia pastoris.Nat Biotech,2009,27(6):561566.
[13]Bain P A,Yoo M,Clarke T,et al.Multiple forms of mouse3beta-hydroxysteroid dehydrogenase/delta5-delta4isomerase and differential expression in gonads,adrenal glands,liver,and kidneys of both sexes.Proc Nat Acad Sci USA,1991,88(20):88708874.
[14]Kataoka M,Hoshino-Hasegawa A,Thiwthong R,et al.Gene cloning of an NADPH-dependent menadione reductase from Candida macedoniensis,and its application to chiral alcohol production.Enzyme Microb Technol,2006,38(7):944951.
[15]Kita K,Fukura T,Nakase KI,et al.Cloning,overexpression,and mutagenesis of the Sporobolomyces salmonicolor AKU4429gene encoding a new aldehyde reductase,which catalyzes the stereoselective reduction of ethyl 4-chloro-3-oxobutanoate to ethyl(S)-4-chloro-3-hydroxybutanoate.Appl Environ Microbiol,1999,65(12):52075211.
[16]Costello CA,Payson RA,Menke MA,et al.Purification,characterization,cDNA cloning and expression of a novel ketoreductase from Zygosaccharomyces rouxii.Eur J Biochem,2000,267(17):54935501.
[17]Choi YH,Choi HJ,Kim D,et al.Asymmetric synthesis of(S)-3-chloro-1-phenyl-1-propanol using Saccharomyces cerevisiae reductase with high enantioselectivity.Appl Microbiol Biotechnol,2010,87(1):185193.
[18]Joernvall H,Persson B,Krook M,et al.Short-chain dehydrogenases/reductases(SDR).Biochemistry,1995,34(18):60036013.
[19]Takemoto M,Achiwa K.The synthesis of optically active pyridyl alcohols from the corresponding racemates by Catharanthus roseus cell cultures.Tetrahedron:Asymmet,1995,6(12):29252928.
[20]Salvi NA,Chattopadhyay S.Studies on Rhizopus arrhizus mediated enantioselective reduction of arylalkanones.Tetrahedron,2001,57(14):28332839.
[21]Takemoto M,Tanaka K.Synthesis of optically activeα-phenylpyridylmethanols by Camellia sinensis cell culture.J Mol Catal B:Enzym,2001,15(4/6):173176.
[22]Takemoto M,Achiwa K.Synthesis of optically activeα-phenylpyridylmethanols with Baker's yeast.Chem Pharm Bull,1994,42(4):802805.