Purification And Characterization Of A Novel Chitosanase From Metarhizium Guizhouense In Solid-state Fermentation

Chitin is the second most abundant polysaccharide, less than cellulose but similarly in structure. Chitooligosaccharides, which are solube products hydrolyzed from chitosan, are of special interests for a variety activities, such as foodstuff, agriculture, medicine, and so on.We compared relative activity of chitosanase from fungal chitosanasee-producting strains. A high yield chitosanase fungus strain Metarhizium guizhouense AS3.4608 was screened out. We studied the chitosanase production of Metarhizium guizhouense AS3.4608 under solid state fermentation conditions. The results showed that wheat bran and soybean power at the ratio of 1:3 as the optimal culture medium with an initial pH 5.6, supplemented with 10g chitosan.L^-1, the optimal nitrogen source 1g KNO₃ concentration at 1g/L,temperature area at 27℃ 28℃ and 3ml of inoculation were the most suitable for chitosanase production at 120h.Additional rice husk and amino glucosealso effected on chitosanase production. Under the optimal conditions, maximum chitosanase activity reached 83.09±2.9U/g dry medium per gram when culture for 120h.A novel chitosanase was detected in extracellular lixivium of solid culture medium of Metarhizium guizhouense AS3.4608 and was purified to apparent homogeneity by SDS-PAGE through PEG6000 and acetone fraction, DEAE-Sepharose FF chromatography, CM-Sepharose FF chromatography and final Superdex 75 chromatography. The specific activity of purified enzyme reached 102. 65U/mg. The chitosanase had a molecular mass of 50.3kD.The enzyme showed a maximum activity at 50'C in pH 4.0,and its activity still retained 50% ability at 48°C for 60 min.It was stable below 50"C and pH 5.0-7.0.Kinetic parameter Km was 2.718g/L .The chitosanase activity was strongly inhibited by Hg2+;also markedly inhibited by Mn2*> Mg2+> Zn2\ Fe3\ Ag+; but it was activiated by Cu2+.The chitosanase is a kind of glycoprotein, it contains about 26.33% of carbohydrate.The enzyme didn't degraded flaked chitin, but showed a little activity toward CMC and colloid chitin;the most susceptible substrate was 90% deacetylated colloid chitosan.