Screening And Identification Of β-mannosidase-production Bacterial,Studying On Its Formation Conditions And Research Of β-mannosidase Properties

β-mannosidase is an exo-hydrolase, which can remove a mannose molecules from non-reducing end of mannose oligosaccharides and its hybrid oligosaccharides which are connectted by1,4-β-D-glycosidic bond,and then can degradate oligosaccharides thoroughly. β-mannosidases are being widely applied in plant metabolism, glycoprotein processing, oil and gas wells development, food, pharmacy and other industries, and the demand is still gradually expanding. But the production of domestic β-mannosidase can’t meet the needs. Therefore, the study of β-mannosidase is also increasingly important. At present, there have been many foreign scholars studying on β-mannosidases, but it’s almost a blank in this respect in the domestic. so there is a vital significance to screen high-yield and superior-quality strains of producing β-mannosidases for the large-scale industrial production and the engineering bacterium construction of β-mannosidase. For this, the thesis mainly had been studying on screening and identification, fermentation conditions optimization, enzymatic properties and other aspects of bacteria producing β-mannosidase, and got the following main research results, which provides experiment basis and new strains resources for breeding and molecular biology research of strains producing β-mannosidase.1. In the acquisition of the soil sample collectted under the pine tree in the Boss mountain of Sichuan Agricultural University, got12β-mannanase producing strains through initial screening. Gain a highest active bacterium of β-mannosidase producing strains, by further screening in strains having the larger ratio of hydrolysis circle diameter and colonies diameter (Dp/Dc), and the β-mannosidase activity is1.03U/mL. According to the morphological shape, the physiological and biochemical characteristics and16S rDNA sequence analysis of the strains, the bacteria was determined to be Paenibacillus polymyxa of Paenibacillus and named Paenibacillus polymyxa-A8.2. The culture condition of Paenibacillus polymyxa-A8was optimized by using single factor experiment and orthogona design together. The best medium composition was determined to be:konjac flour2%, peptone2%. K2HPO4·3H2O0.1%. MgSO4·7H2O 0.02%, NaCl0.5%, pH7.0, and the best conditions of shaking culture were:inoculation volume2%, temperature30℃, cultured for24h, rotation speed160r/min. Under such conditions, the β-mannosidase activity add to be1.83U/mL, the enzyme production capacity was improved80%compared with the initial strains’3. The study on enzymatic properties of β-mannosidase produced in Paenibacillus polymyxa-A8showed that:its maximal activity was observed at45℃and pH6.0, acidic. It had good stability in30～45℃and pH5.0-pH7.5. With concentration being O.Olmol/L, the metalions such as Ca2+ and Mg2+have a more significant role in promoting on β-mannosidase; but Mn2+, Zn2+, Cu2+and Hg2+have different degrees of inhibition on β-mannosidase.