Screening and characterization of a novel esterase from a metagenomic library

被引:81
作者
Kim, YJ [1 ]
Choi, GS [1 ]
Kim, SB [1 ]
Yoon, GS [1 ]
Kim, YS [1 ]
Ryu, YW [1 ]
机构
[1] Ajou Univ, Coll Engn, Dept Mol Sci & Technol, Suwon 443749, South Korea
基金
新加坡国家研究基金会;
关键词
metagenome; esterase; functional screening; p-nitrophenyl acetate; enantioselectivity;
D O I
10.1016/j.pep.2005.06.008
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Metagenomes from various environmental soils were screened using alpha-naphthyl acetate and Fast Blue RR for a novel esterhydrolyzing enzyme on Escherichia coli. Stepwise fragmentations and subcloning of the initial insert DNA (30-40 kb) using restriction enzymes selected to exclude already known esterases with subsequent screenings resulted in a positive clone with a 2.5-kb DNA fragment. The cloned sequence included an open reading frame consisting of 1089 bp, designated as est25, encoding a protein of 363 amino acids with a molecular mass of about 38.3 kDa. Amino acid sequence analysis revealed only moderate identity (<= 48%) to the known esterases/lipases in the databases containing the conserved sequence motifs of esterases/lipases, such as HGGG (residues 124127), GxSxG (residues 199-203), and the putative catalytic triad composed of Sei-201, Asp303, and His333. Est25 was functionally overexpressed in a soluble form in E. coli with optimal activity at pH 7.0 and 25 degrees C. The purified Est25 exhibited hydrolyzing activity toward p-nitrophenyl (NP)-fatty acyl esters with short-length acyl chains (<= C-6) With the highest activity toward p-NP-acetate (K-m = 1.0 mM and V-max = 63.7 U/ing), but not with chain lengths >= C-g, demonstrating that Est25 is an esterase originated most likely from a mesophilic microorganism in soils. Est25 efficiently hydrolyzed (R,S)-ketoprofen ethyl ester with K-m of 16.4 mM and V-max of 59.1 U/mg with slight enantioselectivity toward (R)-ketoprofeii ethyl ester. This study demonstrates that functional screening combined with the sequential uses of restriction enzymes to exclude already known enzymes is a useful approach for isolating novel enzymes from a metagenome. (c) 2005 Elsevier Inc. All rights reserved.
引用
收藏
页码:315 / 323
页数:9
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