Screening And Identification Of Chitinase-Producing Marine Microbe And The Characterization Of Its Chitinase

Chitin, αβ-1,4-linked polysaccharide of N-acetyl-D-glucosamine (GlcNAc), is the second largest natural resources, second only to cellulose, and the main structural component of fungal cell walls, invertebrates, crustaceans and peritrophic membrane of insects. Decomposition products of chitin have various application. Chitinase can catalyse the reaction of hydrolyzing chitin into N-acetylglucosamine(GlcNac)and(GlcNac)n. Chitinase commonly exists in animals, plants and fungi. Therefore, work on screening chitinas-producing strains and studying its characteristics of the enzyme has significance in a wide variety of biological and biotechnological processes, agricultural and industrial production, natural resources utilization, biological control, environmental protection, food and medical exploitation.With the purposes of screening high-yield strains of chitinase, research was carried out on the following aspects:isolation of marine microbes producing chitinase from marine soil samples collected from Dalian Sea, screening and identification of the strains, separation of the chitinase and studying its characters. The main contents and conclusions are as follows:(1)9strains producing chitinase, including8bacteria and1actinomycetes, were isolated from the sea samples, among which, one with high enzyme activity, which was numbered Z-1was selected for the use of further research.The characters of morphological, physiological biochemical and16S rDNA sequence suggested Z-1belongs to Bacillus; so the strain was named Bacillus sp.Z-1tentatively.(2) The liquid fermentation conditions and medium components were optimized. The effects of carbon source, nitrogen source, salinity, inorganic salt, pH value and temperature on the production of chitinase were studied and the ratio of nutrition component was optimized by orthogonal experiment. The optimum culture medium components included:colloid chitin20%, Peptone1.5%, NaCl2.5%, K2HPO40.03%, old sea water. The optimum inoculation conditions were:initial pH7.0, seed cultural time42h,2%inoculation amount,28℃,140rpm for72h.(3) Chitinase crude solution was obtained through the following treatments of the fermetntation broth in the order of freezing centrifuging,40%-90%ammonium sulfate precipation, dialysis tube separation. The chitinase collected was characterized with the optimal temperature at50℃and pH5.0, while the chitinasc activity was stable in pH range of4.5-6.5under50℃. This enzyme did not only degrade colloidal chitin, but also chitin powder.