Genome Analysis Of High-agarase Production Bacterium Flammeovirga Sp. SJP92 And Study On Agarase Gene

Under mild conditions, agarases can hydrolyze agar to generate oligosaccharides.Most of the agarases were isolated from marine microorganisms which were able to secreted a series of agarases and can utilize agar as a rich carbon source. Agarases were widely used in different fields, such as food industry, aquaculture industry and medicine. So it is important to search agarase-producing bacteria.In this study, we analyzed an agarase-producing bacteria, Flammeovirga sp.SJP92, which was isolated in the previous research of our lab. In order to understand the metabolism characteristics, functional genes and agar degradation mechanism of Flammeovirga sp. SJP92, whole genome sequences were determined and protein coding genes were predicted. Sequencing results showed that the genome of Flammeovirga sp. SJP92 has only one circular chromosome of a total size of about 8,534, 834 bp with a 34.80% GC content, 6519 genes were predicted, of which 6291 genes were protein-coding genes. 272 Tandem Repeat, 144 Minisatellite DNA,1Microsatellite DNA and 99 RNA were also identified. The analysis of KEGG, COG and functional genes indicated that the strain has complete metabolic pathways,including carbohydrate, fatty acid, amino acid and so on; most genes were related to metabolism of energy and amino acid transport. It was also encoded multiple transporters and proteins of agar-degradation, such as agarase and sulfatase.In nature, agaeases were secreted out of the cell in the form of multi-agarases,and it was difficult to isolate and purify a single agarase, but the use of heterologous expression can overcome these disadvantages. In this study, we cloned and expressed an agarase, after purification and renaturation, a protein with agarase activity was obtained and it was named Aga B. The results of enzymatic properties showed that the optimum temperature and pH of AgaB were 45℃ and 8.0 respectively, it was stable at the pH between neutral and mildly alkaline, and remained more than 85% of its activity after treatment at these conditions for 1 h. Also, activity of enzyme was analyzed under various reagents conditions, the results showed that the enzymeactivity was strongly stimulated by β-Mercaptoethanol and DTT markedly.Meanwhile, AgaB was hardly inhibited by urea and SDS. Finally, the hydrolysis products of AgaB were analyzed by thin layer chromatography(TLC)and 13C-NMR,these results confirmed that Aga B is an endo-type β-agarase and the end enzymatic products of Aga B were neoagarotetraose and neoagarohexaose.The genome sequence of Flammeovirga sp. SJP92 will supply the genomics study of the genus Flammeovirga with more useful information. And the research of agarase could lay the foundation of its industrial applications.