Purification And Characterization Of A Novel Chitosanase From Metarhizium Guizhouense And Breeding For The High-yield Strains By Mutagensis

Chitosan and its N-acetylated analogue, chitin, are among the most abundant glycans in nature. Conservative estimates of the amount of crustacean shells produced worldwide fall in the range of 100×10³ metric tons per annum. Chitosanases are hydrolytic enzymes acting on chitosan, a polymer composed ofβ-(1→4)-linked glucosamine residues. Chitosanases find application in the generation of size-specific chitosan oligomers. Such oligomers and their derivatives possess biological properties, such as induction of callose, immunopotentiation and inhibition of growth of plant pathogens. Chitosanases are found in bacteria, fungi and plants. For furthermore use, It is necessary to find new source of chitosanases, simplify its purification, and breeding for the high-yield strains.We found a new source of chitosanase from Metarhizium guizhouense. We studied the chitosanase production under solid state fermentation, and separated it from crude enzyme with affinity chromatography and molecular sieving. Cross-linked chitosan resins adsorbed chitosanase from crude enzyme, then eluted with 5‰Cu²⁺. The eluting was dialyzed,concentrated and then separated by Superdex 75 chromatography. Under the optimal conditions, maximum chitosanase activity reached 83.09±2.9U/g dry medium when cultured for 120h. The chitosanase was purified, and the enzyme activity was 43.52% of original activity. The specific activity of purified enzyme reached 106.39U/mg. The chitosanase had a molecular mass of 50.3kD. The enzyme showed a maximum activity at 50℃in pH 4.0. It was stable below 50℃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 most susceptible substrate of the enzyme was 90% deacetylated colloid chitosan.We carried out UV & LiCl as mutagensis treatment to study the effectiveness of increasing the productivity of chitosanase producing strain Metarhizium guizhouense. By this mean, mutant Y0320 was obtained, and its productivity of chitosanase was increased by 600% over the parent strain.Furthermore, the ingredients of culture media were optimized. Many influencing factors and fermentation conditions were studied, such as carbon/nitrogen sources, chitosan, and so on. And then by designed orthogonal experiments, an optimum culture medium was established.