Fermentation Condition Optimization Of Beta-Mannanase, Its Purification And Enzymology Characteristics

As thedevelopment of new resources hemicellulose and thediscovery on medical nutrition of mannan oligosaccharide, furthermore potential application value in many areas of beta-mannanase, corresponding research on beta-mannanase carried out step by step and achieved some results. In this study, fermentation conditions of Pichia Pastoris engineering strain Man12-17saved in our laboratory was optimized firstly. Then purification of induced fermented liquid under the best conditions to produce enzyme was carried out. At last, its enzymology characteristics were studied. It provides references for beta-mannanase researches and lays a foundation on the industrial production of low cost enzymes.(1) Through the single factor experiment and orthogonal experiment, we hadgot the best conditions of enzyme production. Fermentation temperature was29℃. Substrate concentration was0.01%. Initial fermentation liquid pH was6.00. Fermentation time was4d. Methanol concentration was0.5%. Value of OD600was1.0. The enzyme activity was3797.29U/mL before optimization. The enzyme activity reached5009.4U/mL after optimization which increased by31.93%.(2) The culture of Man12-17under the best conditions to produce enzyme was original.We achieved electrophoresis pure enzyme protein which had only one band after ammonium sulfate precipitation classification,dialysis,dEAE-Sepharose Fast Flow anion-exchange chromatography, Sephadexg-75gel filtration chromatography and so on. Specific activity of pured enzyme was8341.14U/mg. Yield coefficient reached61.68%and purification fold was743.42. It was in a leading level compared with other study.(3) In our study, optimum temperature of pured mannanase was60℃. Optimum pH was6.3and sodiumdihydrogen phosphate-disodium hydrogen phosphate buffer was choosed. Either in the condition of weak acid that pH value was between6.3and7.1, or in the condition of65℃below, enzyme stability wasgood.different metal ions, EDTA, SDS, organic reagents anddifferent concentration of NaCl haddifferentdegrees of influence on enzyme activity. According to Lineweaver-Burkdouble reciprocal plot and formula1/V=1/Vmax+(Km/Vmax)·(1/[S]), the enzyme kinetics parameters of konjac powder was worked out. Km was0.009mmol/L. Vmax was10000U/mg. Beta-mannanase obviously had better specificity of konjacgum compared with locust beangum.