Gene Cloning, Characterization Of Two Novel Esterases Deirved From Mangrove Soil And Construction Of Metatranscriptomic Library Of Mangrove Water

Esterases （EC3.1.1.1） belong to the class of carboxylic esterhydrolases, they areable to hydrolyze triglycerides into fatty acid and glycerol, and thus are considered as animportant industrial enzyme. Due to their broad substrate specificity, stereoselectivity,highly catalytic activity, and low side reaction, esterases are widely used many fields,such as food, cosmetics, and medicine. Furthermore, they also have broad applicationpotential in bioconversion.More than99%microorganisms in the environment can not be cultured underconventional laboratory condition, therefore, the utilization for these unculturablemicrobes is limited.Metagenomics and Metatranscriptomic provide an efficient path tosolve the problem through direct extraction of total DNA or RNA of samp les andfollowing sequential screening and functional screening. This technique omits theprocess of pure culture, and thus expands the source of functional genes.In this study, a metagenomic library with high quality was successfully constructedusing mangrove soil samples firstly.Two esterase genes （estl and estz） were finallyidentified from the library through functional screening. The result from pBLASTanalysis revealed that estl had a length of1332bp and encoded a protein of443amino acidswith a predicted molecular weight of48kDa.The enzyme showed46%and38%amino acid identity to β-lactamase from Caulobacter sp.K31（YP₀01682220） andSpirosoma linguale DSM74（YP₀03388323）, respectively. Moreover, the enzymescontained SMTK motif of conserved region, and belonged to family VIII ofesterase/lipase; The gene estz contained a1407bp open reading frame （ORF） encoding apolypeptide of468amino acids with a predicted molecular weight of51kDa. Estzshowed54%and42%amino acid identity tophospholipase from Legionellapneumophilastr.Paris （YP₁23889） and Shewanell apealeana ATCC700345（YP₀01500179） respectively, which belongs to family LiPG phospholipase.estl and estz genes were then subcloned to the express vector pET32a（+）, andoverexpressed in Escherichia coli BL21.The recombinant Estl exhibited the highestactivity at51.7℃and pH8.2and Estz displayed the highest activity at40.8℃andpH7.8.The activities of Estz were greatly inhibited by Tween-20, Tween-80andTritonX-100. Interestingly, Estl exhibited strong tolerance to organic solvent andsalt,and its residual activities reached up to80%and110%when incubated in30%DMSO and30%ethanol for50min, respectively.In addition, Estz retained80%initialactivity after incubating at4M NaCl for1h. Estl displayed high tolerance to metal ions.When incubated at25mM solution of Mg²⁺,Mn²⁺and Ca²⁺for50min,the presence of25mM Mg²⁺and Mn²⁺only reduced the enzymatic activity to80%and90%of initialactivity. Moreover, addition of Ca²⁺slightly stimulated the enzyme activity andincreased its activity to110%of initial activity.These particular properties make Estzand EstL have high research value and industrial application potential.In addition, to obtain eukaryotic function genes from unculturable microorganismsof mangrove, a metatranscriptomic library of mangrove wa ter was constructedsuccessfully. The library was consisted of3000clones. The result of electrophoresisanalysis indicated that the recombinant rate of the library was about95%, and cDNAinserted fragments was about0.5–5kb with an average size of0.9kb, which suggestedthe quality of the metatranscriptomic library was good. The functional screening ofesterases and-glucosidases was performed, but no target gene was obtained from thelibrary. The reason was perhaps relative to low clone numbers of the library.More metatranscriptomic libraries with larger clone numbers should be constructed in thefuture.In addition, changing host strains used for library construction and screeningmetatranscriptomic library based on sequence are also urgently in demand for the studyof metatranscriptomics.