Cloning And Characterization Of A New Organic Solvent-tolerant Esterase Derived From Metagenomics

In the past decade, advances in the field of metagenomics have dramatically revised our view of biodiversity. Considering the estimation that more than99%of microorganisms in most environments are not amenable to culturing, very little knowledge is known about their genomes. The isolation, archiving, and analysis of environmental DNA (or so-called metagenomes) have enabled us to mine microbial diversity, allowing us to access their genomes. In which researchers have found a large number of valuable new active substances.In this study, An environmental DNA library approximately50,000metagenomic clones was constructed using soil sample from Fanjingshan of Tongren, Guizhou. Function-based metagenomics screening, a new esterase designated EstC23was isolated, which evolutionary relationship analysis showed that this enzyme belongs to the lipase IV family (Hormone-sensitive lipase family, HSL). The protein was amenable to overexpression in Escherichia coli under control of the T7promoter, resulting in expression of the active, soluble protein that constituted30%of the total cell protein content. Characterization of enzymatic properties show that the specific activity of EstC23on p-nitrophenyl butyrate was254U mg-1at25℃and pH8.0, EstC23showed optimal activity at40℃and retained about50%maximal activity at5-10℃. EstC23showed remarkable stability in up to50%(v/v) benzene and alkanes (high logP solvents). When incubated for7days in the presence of50%benzene or alkanes, the enzyme maintained its92%-274%elevated activity. The purified enzyme also cleaved sterically hindered esters of tertiary alcohols. These results indicate that EstC23has potential for using in ester synthesis, chiral compounds separation, unstable intermediate compound synthesis and peptide synthesis.In subsequent research work, based on EstC23specific primers, we employed Metagenomic Gene Specific PCR (MGS-PCR) and Truncated Metagenomic Gene Specific PCR (TMGS-PCR) to screen homologous family genes of EstC23with65metagenomics DNA samples derived from domestic different geographies. We identified seven lipolytic activity full-length genes and fifteen homologous family core fragments, sequences analysis showed a broad sequence diversity and a wild rang of sequence identity (50-97%), which constitute a new esterase subfamily.This study provides a valuable experimental material for the following work such as function of research on esterase subfamily or evolution in vitro DNA family Shuffling.