Construction Of Metagenomic Library Of Arctic Deep-sea Sediment And Identification Of A Novel Chitin Deacetylase Gene

The Arctic Regions are nearby the66°34’in the arctic circle area, all year long cold and most of the Arctic Ocean surface is covered with sea ice. The special environment increased the diversity and specificity of the Arctic deep-sea microorganisms. At present, the Arctic deep-sea organisms are studied by the traditional pure-culture techniques. However,99%of the microbes in the natural environment can not cultured in the laboratory, inevitably there are a large number of uncultured microbial resources waiting to be discovered. Bypassing the pure culture of metagenomics technology directly on the microbial genome research, provides a way for uncultured microorganism study, widely used in environmental microorganism resources research.We extract S24sites of deep-sea sediments DNA. The DNA of deep-sea sediment at S24(E205°82.4’,N72°40.1’), derived from July2008to September, China’s3rd Arctic expedition, was extracted and partially digested with Sau3A I, then cloned into plasmid vector pUC19generating a metagenomic library of2750clones per gram sediment sample. Ten clones were chosen randomly and sequenced. And we found that in addition to ccs24-1and ccs24-3, the rest of the clones have the complete ORF by ORF Finder tool. Software SMART predict that ccs24-1may encode plastoquinone, ccs24-2is likely to encode polysaccharide deacetylase, ccs24-3may encode metal peptidase, ccs24-4may encode tRNA synthetase and poly-A synthase, ccs24-5may encode bacterial regulatory proteins, aldehyde dehydrogenase, and an unknown protein, ccs24-6is likely to encode the heavy metals associated with regional, ccs24-7may encode ferrochelatase, ccs24-8may encod transcriptional regulator family PadA gene and sugar glycosyltransferase,and ccs24-9may encod threonine synthase. Although the library storage capacity is limited, it also underlined the microbial and its genetic diversity of Arctic deep-sea sediment.ccs24-2clone containing the gene has all the functional areas of chitin deacetylase, polysaccharide deacetylase domain. But its N segment area is longer than other chitin deacetylase, and C segment was slightly shorter, displaying the specificity. Amino acid sequence analysis revealed that the protein shared the greatest homology with Sinorhizobium medicae WSM419and Magnetospirillum magnetotacticum MS-1both ofAlphaproteobacteria. It means that the gene is most likely sources of a strain of Alphaproteobacteria. The putative chitin deacetylase (CDAap-arc) was subcloned into pET-28a vector and expressed in E. coli BL21. The recombinant enzyme CDAap-arc exhibits an apparent molecular weight of43kDa. Its highest activity was in50mM PBS buffer, pH7.6, at28℃. CDAap-arc is stable on alkaline pH but not on acid pH and is thermally stable under50℃.