Research On The Production, Purification And Characterization Of Mannanase

Mannans are the major constituents of the hemicelluloses fraction insoftwoods and show widespread distribution in plant tissues. The majormannan-degrading enzymes are β-mannanases, β-mannosidases andβ-glucosidases. The β-mannanases are endoacting hydrolases, attacking theinternal glycosidic bonds of the mannan backbone chain, releasing shortβ-1,4-manno-oligosaccharides.The experiment screened three strains (Bacillus subtilis4.37, Bacillussubtilis Coland w-0313.83, Bacillus licheniformis3.83) with highest enzymeactivity from seven kinds of Bacillus gained from Shandong Center of IndustrialMicroorganism Collection with the method of medium transparent circle.Rescreen the three strains by measuring the enzyme activity of the liquidfermentation. As a result the Bacillus has the highest enzyme activity. And theenzyme activity is907.486mg/mL·min. The bacillus subtilis s-88is used in thefollowing experiments.The fermentation conditions were optimized by single factor experimentwith the strain of bacillus subtiliss-88and the optimum fermentation conditionsare as follows: konjac flour is the best carbon source and the best content is4%.The best organic nitrogen source and inorganic nitrogen source are yeast extractand sodium nitrate. After the economic evaluation of nitrogen, the best organicnitrogen source and inorganic nitrogen source are corn gluten and ammoniumsulfare.The best enzyme production time is36h.An extracellular β-mannanase was isolated from crude extract samples ofthe Bacillus subtilis S-88when grown on konjac flour as the carbon source. Theenzyme was purified to single component by gradient of ammonium sulfatesalting, ultrafiltration,HiprepTM26/10Desalting, HiprepTM16/10DEAE FFand G-75gel filtration chromatography procedures. After purification, Bacillussubtilis produced β-mannanase about61697.52IU mL-1in culture medium.The purified β-mannanase had molecular mass of30kda determined bySDS-PAGE，which is much smaller than the other β-mannanase produced bybacteria.At last, we detected the nature of the mananase produced by bacillussubtiliss-88. The enzyme showed maximum activity at pH6.0and55℃and exhibited good stability over a broad pH range from4.4to6.8when bacteria is5.5-7.0. It retains>90%of its maximum activity in water bath for2h at pH6.0below55℃。 Thus the enzyme activity is sttable under55℃. The enzymeshowed high thermostability with more than60%enzyme activity when boiledfor10min and its enzyme activity is not vanished until boiled for70min. Mg2+can promote the activity of the enzyme, while EDTA can inhibit it. The othermental ions(Ca2+、Co2+、Cu2+、Al3+、Fe2+、Fe3+、Zn2+、Ba2+、Mn2+、Ag+、Hg2+) with certain concentration can inhibit it too. Mannanase produced bybacillus subtiliss-88is endo-hydrolase. The hydrolysis of the substrate withmannanase generate a large amount of oligosaccharides and little mannose aswell as mannobiose. It does not completely decomposed substrate. All of theseproperties provide valid data for the industrial production of β-mannanase.