Characterization of a Novel Alkaline Family VIII Esterase with S-Enantiomer Preference from a Compost Metagenomic Library.
A novel esterase gene, est7K, was isolated from a compost metagenomic library. The gene encoded a protein of 411 amino acids and the molecular mass of the Est7K was estimated to be 44,969 Da with no signal peptide. Est7K showed the highest identity of 57% to EstA3, which is an esterase from a drinking water metagenome, when compared with the enzymes with reported properties. Est7K had three motifs, SMTK, YSV, and WGG, which correspond to the typical motifs of family VIII esterases, SxxK, Yxx, and WGG, respectively. Est7K did not have the GxSxG motif in most lipolytic enzymes. Three additional motifs, LxxxPGxxW, PLGMxDTxF, and GGxG, were found to be conserved in family VIII enzymes. The results of the phylogenetic analysis and the alignment study suggest that family VIII enzymes could be classified into two subfamilies, VIII.1 and VIII.2. The purified Est7K was optimally active at 40°C and pH 10.0. It was activated to exhibit a 2.1-fold higher activity by the presence of 30% methanol. It preferred short-length p-nitrophenyl esters, particularly p-nitrophenyl butyrate, and efficiently hydrolyzed glyceryl tributyrate. It did not hydrolyze β-lactamase substrates, tertiary alcohol esters, glyceryl trioleate, fish oil, and olive oil. Est7K preferred an Senantiomer, such as (S)-methyl-3-hydroxy-2-methylpropionate, as the substrate. The tolerance to methanol and the substrate specificity may provide potential advantage in the use of the enzyme in pharmaceutical and other biotechnological processes.
Department of Pharmacy, and Research Institute of Life Pharmaceutical Sciences, Sunchon National University, Suncheon 57922, Republic of Korea.
Hyun Woo
HW
Lee
Department of Pharmacy, and Research Institute of Life Pharmaceutical Sciences, Sunchon National University, Suncheon 57922, Republic of Korea.
Won Kyeong
WK
Jung
Department of Agricultural Chemistry, Sunchon National University, Suncheon 57922, Republic of Korea.
Yong Ho
YH
Kim
Department of Chemistry, Sookmyung Women's University, Seoul 04312, Republic of Korea.
Bum Han
BH
Ryu
Department of Chemistry, Sookmyung Women's University, Seoul 04312, Republic of Korea.
T Doohun
TD
Kim
Department of Agricultural Chemistry, Sunchon National University, Suncheon 57922, Republic of Korea.
Jungho
J
Kim
Department of Pharmacy, and Research Institute of Life Pharmaceutical Sciences, Sunchon National University, Suncheon 57922, Republic of Korea.|Department of Agricultural Chemistry, Sunchon National University, Suncheon 57922, Republic of Korea.
Hoon
H
Kim
D000595:Amino Acid Sequence; D002087:Butyrates / Q000378:metabolism; D003001:Cloning, Molecular; D004950:Esterases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D005395:Fish Oils / Q000378:metabolism; D015723:Gene Library; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000378:metabolism; D054892:Metagenome; D000432:Methanol / Q000378:metabolism; D000069463:Olive Oil / Q000378:metabolism; D010802:Phylogeny; D016415:Sequence Alignment; D012987:Soil; D012988:Soil Microbiology; D013237:Stereoisomerism; D013379:Substrate Specificity; D014280:Triglycerides / Q000378:metabolism; D001618:beta-Lactamases / Q000378:metabolism
D002087:Butyrates; D005395:Fish Oils; D000069463:Olive Oil; D012987:Soil; D014280:Triglycerides; C033592:4-nitrophenyl butyrate; D004950:Esterases; D008049:Lipase; D001618:beta-Lactamases; C005830:tributyrin; D000432:Methanol
Compost metagenomic library; Enantioselectivity; Family VIII esterase; Methanol-activation; Short length p-nitrophenyl esters