The Study On The Fermentation Conditions And Crude Enzymatic Characteristics For A β-mannanase Producing Strain

β-mannanase (β-1,4-D-mannanohydrolase, EC.3.2.1.78) is an endo-type hydrolase capable of hydrolyzingβ-1,4-D-manno-pyanosyl linkages of linear mannan,glucomannan,galactomannan,galactoglucomannan, and has wide applications in food,pharmaceutical,pulp and paper,textile,oil exploitation and detergent industries. In this dissertation, FO-232 was screened out as the best producer ofβ-mannanase among 53 bacterial strains from our laboratory,β-mannanase activity was improved through fermentation medium and culture condition of FO-232, and the characterizations of enzyme solution and the purification of theβ-mannanase were studied. The main result is as follows:1)β-mannanase producing strain FO-232 was screened from 53 bacterial strains using method of shaking culture. It was identified as Bacillus subtilis using 16Sr DNA Squence.2) The fermentation medium and the culture conditions of Bacillus subtilis FO-232 were optimized by shaking culture. The optimal fermentation medium ingredients were as follows:konjac flour 3.5%, KNO 3100 mmol/L, peptone 1%, KH2PO4 0.1%, MgSO4 0.01%, MnCl2·4H2O 9 mmol/L. The optimal culture conditions were initial pH=7.0, medium volume 30 mL in the 250 mL flask. Bacillus subtilis FO-232 grew best when cultured at 37℃with the optimal fermentation medium for 32 h, the enzyme activity was improved to 23 U/mL from initial 2.9 U/mL. The results showed that metallic ion Mn2+ promotedβ-mannanase production.β-mannanase production was highest when the Mn2+ concentration was 9 mmol/L, when that was 50 mmol/L, it showed the intense inhibitory action to this enzyme production.3) The optimal temperature and pH forβ-mannanase activity was 55℃and pH 6, respectively. The enzyme was stable between a wide pH range from 4 to 10, below 50℃. The results indicated that konjac gum is the best substrate forβ-mannanase. In addition, the enzyme could reduce the viscosity of konjac gum,guar gum and locust bean gum, obviously. Theβ-mannanase was purified by acetone precipitation and sephadex G-100 gel filtration chromatography, The results of SDS-PAGE electrophoresis showed that there were three protein bands in obtained enzyme.