title: A novel carbonyl reductase with anti-Prelog stereospecificity from Acetobacter sp. CCTCC M209061: purification and characterization.
abstract: A novel carbonyl reductase (AcCR) catalyzing the asymmetric reduction of ketones to enantiopure alcohols with anti-Prelog stereoselectivity was found in Acetobacter sp. CCTCC M209061 and enriched 27.5-fold with an overall yield of 0.4% by purification. The enzyme showed a homotetrameric structure with an apparent molecular mass of 104 kDa and each subunit of 27 kDa. The gene sequence of AcCR was cloned and sequenced, and a 762 bp gene fragment was obtained. Either NAD(H) or NADP(H) can be used as coenzyme. For the reduction of 4'-chloroacetophenone, the Km value for NADH was around 25-fold greater than that for NADPH (0.66 mM vs 0.026 mM), showing that AcCR preferred NADPH over NADH. However, when NADH was used as cofactor, the response of AcCR activity to increasing concentration of 4'-chloroacetophenone was clearly sigmoidal with a Hill coefficient of 3.1, suggesting that the enzyme might possess four substrate-binding sites cooperating with each other The Vmax value for NADH-linked reduction was higher than that for NADPH-linked reduction (0.21 mM/min vs 0.17 mM/min). For the oxidation of isopropanol, the similar enzymological properties of AcCR were found using NAD+ or NADP+ as cofactor. Furthermore, a broad range of ketones such as aryl ketones, α-ketoesters and aliphatic ketones could be enantioselectively reduced into the corresponding chiral alcohols by this enzyme with high activity.
authors.affiliation: Lab of Applied Biocatalysis, College of Light Industry and Food Sciences, South China University of Technology, Guangzhou, Guangdong, China.
authors.forename: Xiao-Hong
authors.initials: XH
authors.lastname: Chen
authors.affiliation: Lab of Applied Biocatalysis, College of Light Industry and Food Sciences, South China University of Technology, Guangzhou, Guangdong, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Wei
authors.affiliation: State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou, Guangdong, China.
authors.forename: Xiao-Ting
authors.initials: XT
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou, Guangdong, China.
authors.forename: Min-Hua
authors.initials: MH
authors.lastname: Zong
authors.affiliation: Lab of Applied Biocatalysis, College of Light Industry and Food Sciences, South China University of Technology, Guangzhou, Guangdong, China; State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou, Guangdong, China.
authors.forename: Wen-Yong
authors.initials: WY
authors.lastname: Lou
mesh_terms: D019840:2-Propanol / Q000737:chemistry / Q000378:metabolism; D000091:Acetobacter / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000438:Alcohols / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D055162:Biocatalysis / Q000187:drug effects; D018533:Biomass; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007659:Ketones / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D012689:Sequence Homology, Nucleic Acid; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D000438:Alcohols; D001426:Bacterial Proteins; D007659:Ketones; D008670:Metals; D009243:NAD; D009249:NADP; D000429:Alcohol Oxidoreductases; D019840:2-Propanol
references.citation: Bioresour Technol. 2012 Jan;103(1):343-50
references.pmid: 22055107
references.citation: J Biol Chem. 1981 Feb 10;256(3):1206-13
references.pmid: 7005231
references.citation: J Biotechnol. 2013 May 10;165(1):52-62
references.pmid: 23471075
references.citation: J Biosci Bioeng. 2007 Feb;103(2):174-8
references.pmid: 17368401
references.citation: Adv Biochem Eng Biotechnol. 2010;120:195-242
references.pmid: 20182929
references.citation: Enzyme Microb Technol. 2011 May 6;48(6-7):472-9
references.pmid: 22113019
references.citation: Org Biomol Chem. 2011 Jun 7;9(11):4070-8
references.pmid: 21505708
references.citation: Bioresour Technol. 2013 Jun;137:111-5
references.pmid: 23587814
references.citation: Biotechnol Adv. 2012 Nov-Dec;30(6):1279-88
references.pmid: 22079798
references.citation: Bioprocess Biosyst Eng. 2011 Mar;34(3):315-22
references.pmid: 20960011
references.citation: Plant Physiol. 1983 Aug;72(4):959-63
references.pmid: 16663146
references.citation: Biosci Biotechnol Biochem. 2011;75(6):1055-60
references.pmid: 21670533
references.citation: Chem Soc Rev. 2012 Feb 21;41(4):1437-51
references.pmid: 22033698
references.citation: Bioresour Technol. 2011 Jan;102(2):1733-9
references.pmid: 20933386
references.citation: Nature. 1970 Aug 15;227(5259):680-5
references.pmid: 5432063
references.citation: Bioresour Technol. 2009 Dec;100(23):5560-5
references.pmid: 19564106
references.citation: Structure. 1996 Jan 15;4(1):33-45
references.pmid: 8805511
references.citation: BMC Biotechnol. 2011 Nov 20;11:110
references.pmid: 22099947
references.citation: Bioresour Technol. 2011 Jan;102(2):483-9
references.pmid: 20833539
references.citation: J Biol Chem. 1993 May 25;268(15):11002-7
references.pmid: 8388380
references.citation: Bioresour Technol. 2010 Feb;101(3):1054-9
references.pmid: 19767205
references.citation: Bioresour Technol. 2011 Jul;102(14):7023-8
references.pmid: 21570826