Screening And Optimization Of Fermentation Conditions Of A Chitosanase-producing Strain And Study On Chitosan-degrading Enzymes

The microbial enzymatic degradation of chitosan to oligosaccharides andglucosamine has many advantages such as mild condition for reaction, homogeneityproduct, and avoiding pollution in environment and so on. Therefore, it is widely usedin industrial production. In this study, chitosanase and exo-β-D-glucosaminidase wereinvestigated. The main results are as following:The beach with abundant shrimp and crab was selected as the separate source.Initial screening and rescreening were done by using chitosan as the sole carbon sourcein the plate and the shake flask, respectively. Enzyme activity was determined by theDNS method. The Chitosanase-producing strain A502was obtained and identified asMicrobacterium sp. by morphological observation and16S rDNA identification.Single factor and orthogonal experiments were conducted for optimizingfermentation condition. Optimal fermentation medium (%,w/v):1.0chitosan+0.1glucose,1.0(NH4)2SO4,0.14K2HPO4·3H2O,0.5NaCl,0.13MgSO47H2O,0.03KH2PO4and0.3yeast extract. The maximal chitosanase activity could be124U/mLwhen the strain was incubated for96h with4%(v/v) amount of inoculum and initial pH5.5at30℃.According to the characterization of enzymatic properties, it is shown that theoptimum temperature of the enzyme is50℃, and the optimum pH is6.0. The thermalstability of chitosanase is not high. Mn2+could enhance the enzyme activitysignificantly, however, Cu2+, Fe2+and Fe3+could inhibit the enzyme activity. When Ca2+concentration was5mM, it could inhibit enzyme activity significantly. SDS couldseverely inhibit the enzyme activity, whereas EDTA had a weak inhibition on theenzyme activity.Research on the enzymatic properties showed that the optimum condition of theenzymatic reaction:10%(w/v) chitosan acetate solution (10mL) as the substrate,0.4mL enzyme solution (45U/mL enzyme solution),1.6mL distilled water,50℃, and6hreaction time, respectively.The gene (PF0363) from the hyperthermophilic archaeon Pyrococcus furiosus wasamplified by polymerase chain reaction, cloned into expression vector pET15b, andexpressed in E. coli BL21-codonPlus(DE3)-RIL. The soluble protein (PF0363) wasobtained. The recombinant protein has a higher thermal stability. By the TLC analysis, the enzyme(PF0363) was able to deacetylate chitosan to final product glucosamine,which showed that the expressed product of gene (PF0363) in E. coliBL21-codonPlus(DE3)-RIL displayed exo-β-D-glucosaminidase activity.