Purification And Characterization Of Agarse Produced By Bacillus Sp. BI3from Indonesia Hot Spring

There exists unique geographic and ecological environment in hot springs, inwhich the enzymes secreted by microorganisms have special catalytic functions whichhave not been founded the advantages of enzymes at common temperature. Thereforethe microorganisms in hot springs are not only the excellent materials of studying thebiological adaptability, but also the best sources of thermostable enzymes. This thesiswould set basis for the promotion and application of microorganisms and their activemetabolites in hot springs.A kind of strain, named BI3, with high ability of agarase-producing, was screenedby a range of qualitative and quantitative methods including Lugo’s stain anddetermination of enzyme activities in Indonesia hot spring water saved in the laboratory.According to the identification of the16S rDNA and phylogenetic analysis of thephylogenetic tree, BI3was identified as Bacillus.sp. Bacillus.sp.BI3was used in thisresearch for its better agarase-producing ability.In order to optimize the fermentation conditions of Bacillus sp.BI3, the range ofsingle factors（temperature, pH, carbon sources, nitrogen sources, metal ions, agarcontent, NaCl addition and inoculum） were selected by single test firstly. Then threeimportant factors influencing agarase activity, which identified by initial experimentaldesign of Plackett-Burman by Design-Expert software were peptone, agar and calciumchloride. The path of steepest ascent was undertaken to approach the optimal region ofthe three significant factors. Box-Behnken design and response surface analysis wereadopted to further investigate the mutual interaction between the variables and obtainoptimal values that bring maximum agarase activity. The optimal conditions for themaximal activity discriminated from the regression equation were: Yeast extract0.4%,peptone0.665%, agar1.160‰, calcium chloride7.080mmol/L, NaCl added4‰,incubation temperature was55°C,1%of the inoculum size, initial pH7.5. Themaximum value of agarase was5.8U/mL. Compared with the agarase activity culturedin fermentation medium it increased2.5times under the optimal conditions.The enzyme was purified47.78-fold from the culture supernatant by80％sulfateprecipitation, Q-Sepharose Fast Flow anion exchange and Sephacryl S-200HR gelfiltration column chromatographic methods. The purified agarase appeared as a singleband on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Estimation of themolecular mass by SDS-PAGE gave values of about58KDa. The optimum temperature for the agarase was70℃, and more than60％of theagarase activity was retained after incubation at70℃for1h. The agarase prefer toactive in a bit of acid environment, the optimum pH was6.4. The activity of agarasewas lost by the addition of Cu²⁺, Zn²⁺, Fe3+, Co²⁺, Fe²⁺, Mn²⁺, and Ba²⁺stronglyinhibited agarase activities. The activity of agarase was increased by the addition of K+,Na+, Sr²⁺, Mg²⁺, Ca²⁺, Cd²⁺, and agarase activities were increased by about2.75-foldand3-fold by the addition of Na+, Sr²⁺, respectively.This agarase can only hydrolyzeagar.The main products were neoagarotetraose and neoagarohexaose after hydrolyzingreaction for15min, and the addition of neoagarobiose after hydrolyzing reaction for30min. What’s more, only neoagarobiose was found after hydrolyzing reaction for1h.