title: Thermophilic esterase from Thermomyces lanuginosus: molecular cloning, functional expression and biochemical characterization.
abstract: A novel esterase encoding gene, tle, was cloned from the thermophilic fungus Thermomyces lanuginosus DSM 10635. The tle had an open reading frame of 945bp encoding TLE of 314 amino acids with a theoretical molecular mass of 34.5kDa. The putative catalytic triad of TLE was consisted of Ser151, His279, and Asp249. TLE was heterologously expressed in Escherichia coli in biologically active form and purified to homogeneity. Several biochemical properties of TLE were studied: Among the tested p-nitrophenol esters, TLE showed the highest hydrolytic activity with p-nitrophenyl butyrate (C4) and exhibited the maximum activity at 60°C and pH 8.5. The enzyme was stable at temperatures below 60°C and retained 53% of the maximum activity after treatment at 70°C for 60min. Esterase activity was notably enhanced by addition of Ca(2+) and Ba(2+), respectively. Furthermore, TLE showed high enantioselectivity (E=95) in the kinetic resolution of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE), which produce a valuable chiral intermediate-(3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid for Pregabalin. These unique properties of the esterase indicate that TLE is a potential candidate for industrial application.
authors.affiliation: Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310014, PR China; Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou 310014, PR China.
authors.forename: Xiao-Jun
authors.initials: XJ
authors.lastname: Li
authors.affiliation: Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310014, PR China; Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou 310014, PR China.
authors.forename: Ren-Chao
authors.initials: RC
authors.lastname: Zheng
authors.affiliation: Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310014, PR China; Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou 310014, PR China.
authors.forename: Zhe-Ming
authors.initials: ZM
authors.lastname: Wu
authors.affiliation: Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310014, PR China; Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou 310014, PR China.
authors.forename: Xu
authors.initials: X
authors.lastname: Ding
authors.affiliation: Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310014, PR China; Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou 310014, PR China. Electronic address: zhengyg@zjut.edu.cn.
authors.forename: Yu-Guo
authors.initials: YG
authors.lastname: Zheng
mesh_terms: D000595:Amino Acid Sequence; D001464:Barium / Q000737:chemistry; D001483:Base Sequence; D002087:Butyrates / Q000378:metabolism*; D002118:Calcium / Q000737:chemistry; D002208:Caproates / Q000737:chemistry; D020134:Catalytic Domain / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D004950:Esterases / Q000235:genetics*; D032641:Eurotiales / Q000201:enzymology*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
chemical_list: D002087:Butyrates; D002208:Caproates; D011994:Recombinant Proteins; D001464:Barium; C033592:4-nitrophenyl butyrate; C481311:5-methylhexanoic acid; D004950:Esterases; D002118:Calcium
keywords: Characterization; Cloning; Enantioselectivity; Esterase; Thermomyces lanuginosus