Gene Cloning,Expression And Characterization Of Novel Lipid Hydrolase From Deep-Sea Hydrothermal Vents

The deep-sea hydrothermal vents possess unique high-temperature habitats,and they contain rich microbial and enzyme resources with extreme characteristics.Therefore,deep-sea hydrothermal vents have gradually become a new hot spot for mining enzyme resources.In this study,based on the metagenomic data of deep-sea hydrothermal vent sediment sample from the East Pacific Rise,eight suspected lipolytic genes(est-OK series)were selected for further study by using the COG coding(COG2267)as the entry point as well as the conserved regions of active site(GXSXG)and oxygen hole(HGX)as breakthroughs.Then,the selected est-OK series of lipolytic genes were cloned,expressed and enzymatic properties identified using modern enzyme gene mining techniques.Firstly,we cloned eight novel lipolytic genes from the deep-sea hydrothermal vent metagenomics though the method of sequence driven.The est-OK series of lipolytic enzymes were analyzed by bioinformatics,and the results indicated that est-OKH,est-OKJ,est-OKK,est-OKN and est-OKO were belong to family V;est-OKM was belong to family VIII;est-OKL was belong to family ?;est-OKI was belong to family ?.The transmembrane region predictions indicate that the lipolytic enzymes est-OKJ,est-OKM and est-OKN were transmembrane proteins.The prediction of amino acid secondary sequences showed that all the est-OK series of lipolytic enzymes as well as the thermostable lipohydrolases PLP and FCLipl had higher a-helix ratios.Secondly,the eight novel lipolytic genes was successfully expressed in Escherichia coli BL21(DE3).Among them,est-OKM and est-OKK were successfully obtained soluble expression.Then,affinity chromatography of the est-OKK with crude enzyme activity was performed on the nickel column.The results showed that the histidine-tagged est-OKK was efficiently purified Enzymatic kinetic study showed that est-OKK was belong to esterase,the optimal substrate was p-nitrophenyl butyrate,and the kinetic parameters Vmax and Km values were 161.3 U/mg and 483?M,respectively.Thirdly,enzymatic properties study showed the optimum reaction temperature of est-OKK was 50?,and the optimum pH was 9.0.The half-life of est-OKK at 55? was greater than 30 min.indicating that it had temperature stability to a certain extent.In addition,est-OKK could maintain high activity after 4 h treatmem in pH 10.0 buffer,which indicated that it has extremely strong alkali stability.Furthermore,est-OKK could be activated by low salt ions,indicating its higher salt tolerance.Finally,according to the intrafamily cluster analysis,the cluster analysis between the higher homologous proteins,and the homology modeling of the three-dimensional structure,we found that est-OKK might have a catalytic tetrad as its active center.The site-directed mutagenesis experiments confirmed that the amino acid residues 85S,109D,214S,and 242H were the key active site of est-OKK,which formed a catalytic tetrad structure.Further metal ion inhibition kinetics experiments showed that the IC50s of Fe3+ and Mg2+ to est-OKK were 0.72 mM and 3.09 mM,respectively.The inhibitory mechanisms of Fe3+ and Mg2+ on est-OKK were both reversible inhibition,and their types were non-competitive inhibition and mixed inhibition,respectively.