Identification And Characterization Of Novel Esterases Isolated By Functional Screening Of A Chestnut-grove Soil Metagenomic Library

The lipid hydrolases,principally catalyze the hydrolysis and synthesis of ester compounds.Lipases and esterases,the two major families of lipolytic enzymes.Due to their inherent thermal stability and resistance to organic solvents,combined with their high regio-and chemo-,enantioselectivity,these enzymes could be functional in the industrial production process with harsh conditions,and make them very attractive biological catalyst in a variety of industrial applications.Similarly,lipolytic enzymes sourced from the extreme microbes can provide some additional features such as activity at extremely high or low temperatures,extremely acidic or alkaline conditions or high salinity levels,which may cause interest of certain industrial objection.Esterases can be found in plants,animals and microorganisms,and most industrial esterases are of microbial origin.At the same time,esterases are economic and effective biocatalysts whose extensive use in food industry including nutrition improving,flavor formation,and food fermentation.Traditionally,novel lipases and esterases were found by separation of the pure-cultivating microbial strains harbouring lipolytic activity.Entering into the Era of Genome Project,gene mining was available,and making use of homology with known lipases and esterases to search the new ester hydrolase was allowed.The Era of Metagenomic meant that the area of genome had took a step forward by researching metagenome,while metagenome meant a collection of all the genes of microbial communities in certain environment.At present,molecular biology techniques can be used to construct libraries of total environmental DNA,including the uncultured microbial genomes.For instance,the broad field of discovering unknown enzymes which has novel and unique properties had been opened a new window.In addition,the function-based screening by metagenomic libraries can be used to discover positive clones with lipolytic activity.This screening method may be succeed depending on the compatibility between the transcription and translation components of the cloned gene and the heterologous host,Escherichia coli is usually selected as such host.In this study,we constructed a metagenomic cosmid library from a chestnut-grove soil sample in Yunnan,China.Two esterases EstGX1 and EstGX2 were identified based on function-based screening of the library.Multiple sequence alignments and phylogenetic tree analysis revealed that both predicted enzymes were new members of bacterial lipolytic family ?.The SDS-PAGE analysis illustrated that the purified EstGX1 and EstGX2 showing almost a single belt possessed corresponding estimated molecular weight of 31 kDa and 33 kDa,followed by the biochemical characterization of the purified EstGX2.Enzyme properties including optimum pH,optimal temperature,tolerance to organic solvents and metal ions were measured.The optimal temperature of EstGX2 was 50?,while its optimum pH was alkaline pH 9.0.However,in extremely acidic or alkaline(>pH 10.0)condition,the esterase would lose the ability to hydrolyze p-nitrophenol butyrate.It is especially noteworthy that the activity of EstGX2 could maintain about 40%after incubated at 99? for 55 min,and could be increased in presence of 15%ethanol.The unique properties of EstGX2,high thermo stability and stability in the presence of organic solvents,may make it a promising enzyme candidate in food industry.