Isolation And Biological Function Of Excretion Of Marine Microorganism Producing Enzyme

Objective The aim of this study was to isolate marine bacterium and actinomycetes producing enzyme, classify to the microorganism, study growing property and explore biological function of excretion.Method Bacterium and actinomycetes which could produce enzyme from the sea area of Qingdao were isolated, using the methods of evaluating protease, amylase and collagenase activity. Appearance and physiological-biochemical characteristic of the microorganism were identified by based morphological. Genus was classified by genetic characterization. The optimal condition of growing temperature, pH, concentration of NaCl were tested. As the ability of enzymatic production in different condition of temperature and pH did, so that the factors of enzymatic production were grading-up. Molecular weight of the excretion was preliminary defined by SDS-PAGE. The optimal temperature and pH were detected. The biological function of the excretion on enzymatic activity, anti-carcinoma activity and immunocompetence was explored.Result A strain named XF-1 with highly proteolytic activity was successfully isolated from alimentary canal of butterfish. Based on the 16S rDNA sequence and phylogenetic analysis, the strain was classified to Aranicola proteolyticus. The results showed that XF-1 was gram negative, rod shaped, aerobic, with many flagellum, neither endospore nor capsule. The colony on milk medium were 1-2 mm diameter, circular, white, smooth, translucent and with regular edges. Based on the 16S rDNA sequence and phylogenetic analysis, the strain was classified to Aranicola proteolyticus XF-1 which could grow at 15-37℃, medium psychrotrophs by the growth condition test. The optimum growth temperature was about 25℃. The optimal pH for growth was 7.5 with pH range between pH 5.5 and pH9.0. It can grow at a wide range of NaCl concentration of 0-6 g/dL with the optimum concentration at 1g/dl. The activity of extracellular protease wasl45.0U/mL, with an optimized fermental condition of pH 7.5 for 28 hours at 25℃. Molecular weight of the protease was approximately 43-55KD by SDS-PAGE. The protease produced by XF-1 exhibited the highest activity at pH7.5 and 40℃. The strain XF-1 could secret multiple mas, which could resolve casein, agglutinate milk powder, exist anti-carcinoma activity and immunocompetence.Conclusion A strain named XF-1 with highly proteolytic activity is successfully isolated. The excretion exhibits favourable biologic function and might be a novel source of protease of marine microorganisms.