AUTHOR=Lee Yong-Suk TITLE=Isolation and Characterization of a Novel Cold-Adapted Esterase, MtEst45, from Microbulbifer thermotolerans DAU221 JOURNAL=Frontiers in Microbiology VOLUME=7 YEAR=2016 URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2016.00218 DOI=10.3389/fmicb.2016.00218 ISSN=1664-302X 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 K_m and V_max 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.