Screening and characterization of a novel esterase from a metagenomic library.
Metagenomes from various environmental soils were screened using alpha-naphthyl acetate and Fast Blue RR for a novel ester-hydrolyzing 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 (< or = 48%) to the known esterases/lipases in the databases containing the conserved sequence motifs of esterases/lipases, such as HGGG (residues 124-127), GxSxG (residues 199-203), and the putative catalytic triad composed of Ser201, 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 (< or = C6) with the highest activity toward p-NP-acetate (Km=1.0 mM and Vmax = 63.7 U/mg), but not with chain lengths > or = C8, demonstrating that Est25 is an esterase originated most likely from a mesophilic microorganism in soils. Est25 efficiently hydrolyzed (R,S)-ketoprofen ethyl ester with Km of 16.4 mM and Vmax of 59.1 U/mg with slight enantioselectivity toward (R)-ketoprofen 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.
Kim
Yun-Jung
YJ
Department of Molecular Science and Technology, College of Engineering, Ajou University, San 5, Woncheon-dong, Yeongtong-gu, Suwon 443-749, Republic of Korea.
Choi
Gi-Sub
GS
Kim
Seung-Bum
SB
Yoon
Gee-Sun
GS
Kim
Yong-Sung
YS
Ryu
Yeon-Woo
YW
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D004950:Esterases / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism; D015698:Genomic Library; D006863:Hydrogen-Ion Concentration; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010802:Phylogeny; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D004950:Esterases