Screening Of Chitosanase-producing Strain And Study On Its Enzymatic Properties

Purposes：To obtain high-yield Chitosanase strains, produce bioactive oligochitosanand provide the laboratory foundation for future Chitooligosaccharides marketapplications.Background and Significance：Chitosan is a derivative of chitin deacetylation, existsin nature shrimp shells, crab and fungal cell wall. It has the biological activity ofantibacterial, anti-tumor and improve immunity. Because of its high molecular weightand poor water soluble, chitosan is not readily absorbed in the human body and itsapplications are limited. Chitooligosaccharides is a degradation products of chitosan,it has the same functions with Chitosan, as well as low molecular weight, easilyabsorbed in the human body, which makes it widely applied. Current methods forpreparing Chitooligosaccharides include chemical, physical and biological enzymedegradation. The first two methords are too acidic, degradation is slow, difficult tocontrol the degree of polymerization in product, the production is complex,butbiological method have mild conditions, high yield, the reaction is easy to control, andthe product have moderate degree of polymerization. Chitosanase is a key enzyme ofbiological method, but currently beacause the lacking a kind of enzyme with highactivity and substrate specificity, how to get an efficient chitosanase becomes the keyto chitooligosaccharides commercial production success. Methods：Three strains with chitosanase activity were isolated from soil and shellssamples by inducing and enrichment with chitosan, one of the strains QS7with thehighest chitosanase activity was collected and identified as Penicillium oxalicum.DNS method was used for the determination of enzyme activity, which is32.8U/mL.The enzyme activity was up to47.6U/Ml after mutated by ultraviolet ray, and thestrain named QS7-6. Through single-factor experiment and orthogonal design, theoptimal compositions of the medium producing chitosanase were as follows (g/L):1%colloid chitosan,0.06%yeast extract,0.1%MgSO4,0.14%K2HPO4·3H2O,0.06%KH2PO4.The optimum culture condition were as follows: in500mLerlenmeyer flasks containing100mL culture medium, and the medium wasinoculated with5%inoculum (V/V).The flasks were kept at30°C with120rpm for96h. Under the optimal culture conditions, the chitosanase activity of fermentationliquid could reach70.5U/mL. The chitosanase from QS7-6strain was an inducibleand secreting extracellar enzyme. The chitosanase of the strain was extracted byammonium sulfate precipitation（40-90%saturation）, and purified by ion-exchangechromatography, SDS-PAGE showed single band for the purified chitosanases and themolecular weight was estimated to be40kDa（ChiQ）. Using LC-MS/MS to identifythe purified ChiQ.After enzymolysis by trypsin, the peptide segmentsSALNKNYITNFSTLR and partial amino acid sequence of Penicillium oxalicumchitosanase match exactly.Studies on characterizationof chiQ showed the optimaltemperature and pH for hydrolysis of the chitosanasse were50℃and5.0. Mn2+, Zn2+,Mg2+, Ca2+could enhance the enzyme activity of ChiQ, whereas Ag+and Hg2+could inhibite the enzyme activity. ChiQ showed the highest activity for hydrolysis ofcolloid chitosan, and ChiQ also can hydrolyze chitosan powder, but could nothydrolyze chitin and glucan. The enzymolysis process for preparingchitooligosaccharides was studied.Experiment results demonstrated the optimalculture conditions were as follows: at50℃, pH5.0, concentration of substrate is6%,enzyme quantity were66.7U/g chitosanase,8hours later the reaction reached balance,Oxford Cup antifungal experiments show that the enzymolysis product have theactivity of resistant Candida albicansConclusion：From the natural environment screened a High Chitosanase fungi whichcan be used as a dominant strain in the future industrial of producing chitosanase, andprovide the laboratory basis for future chitooligosaccharides production.