Studies On The Production, Purification And Characterization Of β-mannanase From Bacillus Sp. M-21

β-mannanase is an enzyme capable of hydrolyzingβ-1,4-D-manno-pyanosyl linkages of mannans with main product manno-oligosaccharide. Mannanase has wide commercial applications in industry processes such as foodstuff, pharmaceutical, feed, paper and pulp, and oil industry.β-mannanase producing bacterial strain M-21 was isolated from the soil samples. After identification of the strain, the fermentation conditions were optimized and the purification and characterizations of theβ-mannanase were studied. Theβ-mannanase was also applied to hydrolyze several mannan gums yielding manno-oligosaccharides.The results showed that:1.β-mannanase producing strain M-21 was isolated from the soil samples using the methods of enriching culture, isolation and selective plate culture, first screening by clear circles on selective paltes, and second screening by shaking culture. It was identified primarily as Bacillus sp.2. The fermentation conditions were optimized in order to improve the production of the enzyme. The optimal liquid medium consisted of 4 g/L guar gum as carbon source, 20 g/L soybean powder and 5 g/L (NH4)2HPO4 as nitrogen source and other inorganic salts(g/L): K2HPO4·3H2O 1, MgSO4·7H2O 0.5, NaCl 0.5, CaCl2 0.1, FeSO4·7H2O 0.001. The optimal culture conditions were initial pH 8.0, inoculation volume 4%, medium volume 50 mL in 250 mL flask, agitation speed 180 r/min. After incubation at 32℃for 36h, the enzyme showed the highest activity of 1487 U/mL.3. Theβ-mannanase was purified to homogeneity by acetone precipitation and Q-sepharose ion exchange chromatogramphy. The enzyme was shown to have a relative molecular weight of 33.4 kDa by SDS polyacrylamide gel electrophoresis.4. The maximum enzyme activity existed at 55℃and the optimized pH was 7.0. It was stable between a pH range of 5.0 and 11.0,below 50℃. The metal ions Zn2+, Pb2+, Fe2+, Cu2+, Hg2+ and Ag+ inhibitedβ-mannanase activity strongly, but Ba2+, Ca2+, K+, Mg2+, Na+ and Mn2+ activated the enzyme. Kinetics ofβ-mannanase was examined. The Km values for konjak gum, locust bean gum and guar gum were 3.97, 8.79 and 17.56 mg/mL, and Vmax were 9.02, 12.44 and 20.92μmol/(min·mL). Its optimal substrate was konjak gum.5. Konjak gum, locust bean gum and guar gum were hydrolyzed by the crudeβ-mannanase from Bacillus sp. M-21 to yield serial manno-oligosaccharides as the main products in different velocity and degree. The hydrolysate was separated by ethanol grading precipitation and gel filtration chromatography (Bio-Gel P-4 Extra Fine). By means of TLC and MS methods, the konjak oligosaccharides were identified as a series of di-, tri- and tetra-saccharides with little monosaccharide .This work might be helpful for both the industrial production ofβ-mannanase and manno-oligosaccharide hydrolysis catalysed byβ-mannanase.