Abstract
A novel esterase, MtEst45, was isolated from a fosmid genomic library of Microbulbifer thermotolerans DAU221. The encoding gene is predicted to have a mass of 45,564 Da and encodes 495 amino acids, excluding a 21 amino acid signal peptide. MtEst45 showed a low amino acid identity (approximately 23–24%) compared with other lipolytic enzymes belonging to Family III, a closely related bacterial lipolytic enzyme family. MtEst45 also showed a conserved GXSXG motif, G131IS133YG135, which was reported as active site of known lipolytic enzymes, and the putative catalytic triad composed of D237 and H265. Because these mutants of MtEst45, which was S133A, D237N, and H265L, had no activity, these catalytic triad is deemed essential for the enzyme catalysis. MtEst45 was overexpressed in Escherichia coli BL21 (DE3) and purified via His-tag affinity chromatography. The optimal pH and temperature of MtEst45 were estimated to be 8.17 and 46.27°C by response surface methodology, respectively. Additionally, MtEst45 was also active between 1 and 15°C. The optimal hydrolysis substrate for MtEst45 among p-nitrophenyl esters (C2–C18) was p-nitrophenyl butyrate, and the Km and Vmax values were 0.0998 mM and 550 μmol/min/mg of protein, respectively. MtEst45 was strongly inhibited by Hg2+, Zn2+, and Cu2+ ions; by phenylmethanesulfonyl fluoride; and by β-mercaptoethanol. Ca2+ did not affect the enzyme's activity. These biochemical properties, sequence identity, and phylogenetic analysis suggest that MtEst45 represents a novel and valuable bacterial lipolytic enzyme family and is useful for biotechnological applications.
Highlights
Lipolytic enzymes, primarily esterase and lipases, belong to the α/β hydrolase superfamily of enzymes that catalyze the hydrolysis and synthesis of ester bonds (Holmquist, 2000)
The protein encoded by ORF1 shared high sequence identity with an acyl esterase from Microbulbifer agarilyticus (WP_010133327)
The protein encoded by ORF2 had 85% sequence identity (354/416) with TldD protein from Microbulbufer sp
Summary
Primarily esterase and lipases, belong to the α/β hydrolase superfamily of enzymes that catalyze the hydrolysis and synthesis of ester bonds (Holmquist, 2000). Lipolytic enzymes are widely used in the production of biofuels, foods, detergents, papers, textiles, pharmaceuticals, and chemicals (Fu et al, 2013; Li et al, 2013; Tran et al, 2013; Leis et al, 2015; Vici et al, 2015), Novel Esterase from Microbulbifer thermotolerans DAU221 in part due to their chemo- and region- selectivity, and their ability to catalyze reactions without cofactors These properties make them an important class of enzymes for biotechnological applications and an important group of biocatalysts in organic chemistry (Jaeger and Reetz, 2002; Kim et al, 2007). The most recently established bacterial lipolytic family is Family XV, which includes the thermostable esterase EstGtA2 from Geobacillus thermodenitroficans (Charbonneau and Beauregard, 2013)
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