Gene Cloning, Expression, And Characterization Of Two Novel Esterase: Screening Based On Functional Method

Esterase(EC3.1.1.1),also called carboxylesterase, is a hydrolase enzyme thatcatalyzes both the hydrolysis and the synthesis of ester bonds. Esterases are currentlyused in a broad array of industrial applications, they are used in food, medicine andcosmetic. BioH is one of the key enzymes to produce the precursor pimeloyl-ACPto initiate biotin biosynthesis de novo in bacteria. To date, very few bioH genes havebeen characterized. In this study, we used metagenomics and functional screeningmethod to find two esterase genes from aqueous environment. The two genes weobtained called bioHx and estx. The two genes, encoding enzymes that are capable ofhydrolysis of p-nitrophenyl esters of fatty acids, was expressed in Escherichia coliBL21using the pET expression system. The biochemical property of the purifiedBioHx and ESTx protein was also investigated.Screening of an unamplified metagenomic library with a tributyrin-containingmedium led to the isolation of two clones exhibiting lipolytic activity.One clone carried a4,570-bp DNA fragment encoding for six genes, designatedbioF, bioHx, fabG, bioC, orf5and sdh, four of which were implicated in the de novobiotin biosynthesis. The bioHx gene encodes a protein of259aa with a calculatedmolecular mass of28.60kDa, displaying24-39%amino acid sequence identity to afew characterized bacterial BioH enzymes. It contains a pentapeptide motif(Gly76-Trp77-Ser78-Met79-Gly80) and a catalytictriad (Ser78-His230-Asp202), bothof which are characteristic for lipolytic enzymes. BioHx was expressed as arecombinant protein and characterized. The purified BioHx protein displayedcarboxylesterase activity, and it was most active on p-nitrophenyl esters of fattyacids substrate with a short acyl chain (C4). Comparing BioHx with other knownBioH proteins revealed interesting diversity in their sensitivity to ionic and nonionicdetergents and organic solvents, and BioHx exhibited exceptional resistance toorganic solvents, being the most tolerant one amongst all known BioH enzymes. This ascribed BioHx as a novel carboxylesterase with a strong potential in industrialapplications.The other one clone carried a2,864-bp DNA fragment encoding for two genes,designated esterase and beta-lactamase. The estx gene encodes a protein of419aawith a calculated molecular mass of49.80kDa, displaying23-71%amino acidsequence identity to a few characterized bacterial Esterase enzymes. It contains apentapeptide motif and a catalytictriad, both of which are characteristic for lipolyticenzymes.After cloning and expression, and characterization, we finaly know that, the twoenzymes had the same suitable effect temperature and pH,30°C and pH8.BioHx andESTx are two estrases, they are most active on p-nitrophenyl esters of fatty acidssubstrate with a short acyl chain (C4). BioHx revealed interesting diversity in theirsensitivity to ionic and nonionic detergents and interesting diversity.ESTx revealedinteresting diversity to organic detergents. These interesting characters made themhave strong potential in industrial applications.