Isolation And Identification Of Beta-mannanase Producing Strain, Optimization Of Fermental Conditions And Its Enzymatic Characterization

?-mannanase is an endo glycoside hydrolase,which can catalyze random hydrolysis of ?-1,4-mannosidic linkages in the main chain of mannan,and it is a kind of hemicellulases.It existed widely in animals,plants and microorganisms,and when synergying with other hemicellulases,it could hydrolyse hemicellulose effectively.It had been broadly used in many trades,and was becoming a hot research in recent years.But in general, the low yields and the high cost seriously limited the scale and scope of its application,and the most direct way for change was to isolate and cultivate high-yield wild (3-mannanase producing strains.The purpose of this study was to isolate a ?-mannanase producing strain from soil samples, identify the strain by various means, optimize its medium compositions and fermental conditions, purify its ?-mannanase and investigate its enzymatic properties.This would lay a foundation for the construction of P-mannanase genetically engineered strain and the ultimate realization of large-scale industrial production.The results were as follows:1.20 ?-mannanase producing strains were isolated, after enriching culture and initial screening by Congo red selective medium, from the soil samples gathered from coniferous forests on laobanshan mountain and konjac base in the farm in Sichuan Agricultural University.And then 6 strains which got the higher H/C value were selected for liquid fermentation, and finally NO.6, named J6, with the highest enzymatic activity (11.36 U/mL) was chosen for the subsequent experiments. By morphological observation, physiological and biochemical identification and 16S rDNA sequence analysis, the isolated strain J6 was identified as Paenibacillus polymyxa.2. Through single factor tests and two orthogonal experimental designs,including the type and concentration of the carbon source, the nitrogen source, and metal ions,as well as the initial pH, incubation temperature, liquid amount and the shaking speed,the optimised medium compositions and fermental conditions was got:Konjac powder 1.0%, peptone 1.0%, yeast extract 0.5%, K+1 mmol/L, Mn2+4 mmol/L,initial pH 5.5, the amount of fluid 70/250 mL, incubation temperature 35? shaking speed 160rpm. Under this condition, the highest bacterial production activity (62.92 U/mL) is 5.54 times than that before optimization (11.36 U/mL).3. The ?-mannanase was purified gradually by ammonium sulfate salting, acetone precipitation and the glucan gel filtration, and finally resulted in an electrophoresis pure ?-mannanase,which was approximately 48.0 KD. The optimum reaction temperature and pH of the ?-mannanase seperately was 65? and pH 6.5, and it showed a high level of stability in the range of 60-80? and pH 5.0-8.0. Final concentration of 1 mmol/L K+, Ca2+, Mn2+, Ba2+activated the activity of the p-mannanase,while the same concentration of Fe3+, Mg2+, Cu2+, Zn2+, Na+ inhibited. Take konjac powder as substrate,its kinetic constants Km was 10.42 mg/mL, Vmax was 136.97 ?mol/(min-mL).