해양미생물 Vibrio sp. GMD509로 부터 신규 esterase의 클로닝과 특성분석

Abstract

Vibrio sp. GMD509, a marine bacterium isolated from an egg of the sea hare show a lipolytic activity on tributyrin plate. To characterize the lipolytic activity, a genome library of Vibrio sp. GMD509 was screened by detecting the activity on TBN plate and the sequence of a positive clone was determined. Sequence analysis revealed that the esterase gene (vlip509) consisted of 1017-bp (338 amino acids) with an estimated molecular mass of 37 kDa. The residues critical to the lipolytic activities such as G-X1-S-X2-G motif and catalytic triad were found. The sequence comparison of the deduced amino acids showed similarities to several lipases/esterases as well as dienelactone hydorolase family. The esterase was overexpressed in E. coli. and purified to homogeneity. The recombinant esterase (Vlip509) was further characterized. Vlip509 showed maximum hydrolyzing activity toward p-nitrophenyl butyrate (C4) among the p-nitrophenyl esters (C2 to C18), while no lipolytic activity was observed toward esters containing longer than 10 carbon atoms, indicating that enzyme is an esterase and not a lipase. Optimal temperature and pH were 30℃, pH 8.5, respectly. Most of metal ions and inhibitors did not affect the enzyme activity and organic solvents positively affected the GMD509 esterase. This work represents that marine bacteria could be a valuable resource to develope a biocatalyst