Screening And Identification Of Chitosanolytic Marine Microbes And Analysis Of The Fermentation Products

The marine microorganism has became a new resource of enzyme preparation, because it has the diversity of species, gene composition and ecological function. With the abundance of chitins in the marine environment, so it has tremendous possibilities to find the chitosanolytic microbes and new chitosanase.Chitosanase-producing strain XW-1002,which was isolated from Xuwang culture factory in Xiaosha town of zhoushan city by using the enrichment culture and the transparent circle methods,was classified into Arthrobacter based on morphology,analysis of physiology and biochemistry and molecular biology methods. It has grown well in the chitosan powder medium,the optimum fermentation conditions were:fermentation temperature30℃, chitosan concentration3%,250ml erlenmeyer flask containing50-100ml medium and inoculation amount3%. The chitosanase from Arthrobacter XW-1002strain was an inducible and secreted extracellar enzyme. The degree of polymerization was measured by terminal analysis, the antibacterical activity of chitooligosaccharides was tested by spectrophotometric method,and the effects to plant growthing were studied with water culture method. When enzymatic hydrolysis time was10、20and30minutes, the average degree of polymerization was5.4、2.66and1.7,the average molecular weight of the chitooligosacchairdes was870.16、446.25and292.04respectively. The results showed the chitooligosacchairdes inhibited certain bacteria and yeast to different extent,the antibiotic action of chitooligosacchairdes were to bacteria better than to fungi,and antimicrobial activities were increased with chitooligosacchairdes concentration. The physiological functions of Arthrobacter sp.XW-1002fermented chitosan were also tested. The chitosanoligosaccharides in fermented chitosan could stimulate growing of Oryza sativa seedlings with low concentraion.In order to increase the activity of enzyme producing, the strain Arthrobacter sp.XW-1002was treated with chemical mutagen diethyl sulfate (DES) and ultraviolet irradiation respectively, and two mutant strain YY-1and UV-1were obtained. Growth rate of strain YY-1was not distinctly raised, and the chitinolytic enzyme activity was stable on2.50U/ml,1.7time larger than the strain Arthrobacter sp. XW-1002(1.47U/ml). And optimal nutagenic concentration and treating time were2%and50min respectively.(?)ne enzyme activity of mutant strain UV-1was also2.50U/ml,which1.39time larger than the strain4rthrobacter sp. XW-1002, and the growth cycle of this new strain was shorten to7days from8days. The two mutant strains also showed the quite good genetic stability.