Breeding Of Agarase-producing Marine Microorganisms And Study On Its Enzymatic Properties

Agarase is a kind of polysaccharide hydrolase and researchers have paid much more attention to it than before and studied its properties and applications for the past few years. It is major source for industry fermentation production with marine microorganisms,as well as regarding as the base of industrial production of the agaro-oligosaccharides.It is a significant way to isolate agarase-producing bacterium from the ocean.(1)A agarase-decomposing strain was isolated from red agar and abalone viscera by transparent circle method and submerged fermentation method, which was identified as Vibrio sp. based on its morphological observation and phylogenetic analysis of 16S rDNA sequence. Vibrio sp. strain Ag-1 was treated repeatedly by using UV compound mutation, the best mutagenic dose of UV irradiation is 1 min. The mutant Ag-1-Y7 was obtained after two cycle of mutation, which had good genetic stability. The agarase activity of the mutant was increased to 19 U/mL, more than 1.71 times higher than that of the original strain.(2) Through single-factor experiments and orthogonal design, the optimum fermentation conditions for Vibrio sp. strain Ag-1-Y7 to produce agarase were obtained. The optimum medium for shake flask fermentation contained Agar powder 2 g/L, peptone 5 g/L, Yeast 7 g/L, NaCl 35 g/L,MgSO4-7H2O 6 g/L, FeSO4 7H2O 0.018 g/L, initial pH 6.5.The agarase activity of the optimized fermentation medium was increased to 41 U/mL, more than 1.16 times higher than that the original one.(3)The conditions for fermentation were about the combination of the inoculums size 1%,. medium volume 50 mL/250mL, temperature 33℃, and shaking at 200 r/min for 24 h. As a result, the maximum agarase activity of Vibrio sp.Ag-1-Y7 was at 24 h, more than 4.85 times higher than the Ag-1.(4) Researched on the enzymatic properties of agarase which product from Vibrio sp. Ag-1-Y7, we learned that the optimal pH and optimal temperature of agarase were 8.0 and 50℃; The pH stability was sTab. in the range of pH 5.0 to 8.0 which could keep 70% enzyme activity after deal with 1 hours under these pH; the temperature stability was sTab. when temperature below 40℃, Agarase was significantly activated by K+, Ca2+, Mg2+, Li+ and Fe3+ and strongly inhibited by Mn2+ and Cu2+. The optimal concentration of substrate was 1%-1.2%, and the enzyme kinetic parameters Km and Vmax were 0.58 mg/mL and 3.29 ug/mL·min, respectively.