Purification And Characterization Of Chitosanase Which Was Used To Prepare Chitooligosaccharides And Analysis Of It

Chitosanases which catalyze the hydrolytic degradation of chitosan can be used for preparation of functional chitooligosaccharides. They also can be used as biological control agent in agriculture to improve plant anti-disease abilities.A bacterium with high chitosan degrading activity is preserved in our laboratory. Producing chitosanase is incubated at 30℃, 100 rpm, pH 6.0, aeration 8L/min and the highest chitosanase activity is 24U/mL after 72 hours later. The chiotsanase recovered from culture supernatant is purified to electrophoretic homogeneity by a procedure of centrifugal effect, Q-sepharose Fast Flow ion exchange and Sephacryl S-100 gel filtration chromatography. The molecular mass of the enzyme determined by SDS-PAGE is 66.2kD .The optimum pH and temperature for enzyme activity are pH6.0 and 55 ℃, respectively. The enzyme is stable at temperature below 45℃, and remains 95% activity if it is put at 4℃ for half a month. Mn2+enhances the enzyme activity, whereas Cu2+, Fe3+and Hg2+inhibite the activity. The chitosanase activity is strongly inhibited by EDTA and SDS, but IAA and 2-mercaptoethanol enhance its activity. The chitosanase shows substrate specificity. Kinetic parameter .Km is 5.15mg/mL. The activity of the chitosanase purfied in this experiment is obviously higher than that of others reported so far. It can be applied in industry to produce chitooligosaccharids.This paper also describes the enzymatic hydrolysis process for preparing chitooligosaccharides. The effects of temperature, pH value, concentration, deacetylation degree of substrate, quantity of enzyme and reaction time on chitosan hydrolysis are studied. It also analyzes the degraded products by HPLC and separatesthem by Bio-Gel P4. Those separated enzymatic hydrolysis products are analyzed by TLC. Experiment results demonstrate that this chitosanase is an endo-type cleave enzyme. It decreases viscosity of reaction solution, hydrolyzs (3-( 1,4) glucosidic bond of chitosan. The optimal temperature, pH value, concentration of substrate and reaction time are 45 °C, 6.0, 6% and 12 hours, respectively. The chitooligosaccharids molecular weight become smaller with the increase of substrate DD by HPLC analysis. 9.08U enzyme can degrade 1 g chitosan at least and the reaction ended after 24 hours. We obtain chitooligosaccharides with degree of polomerization over 20,between 10~~20, below 10, respectively by Bio-Gel P4. Chitooligosaccharides are prepared by enzymatic hydrolysis with the partly purified chitosanase. Some quality standard specifications of chitooligosaccharides are made. Detailed specifications indexes are made as follow: appearance, light yellow powder; pH,7.35; water,12.1%; ash, 1.65%; water solubility,>99.9%; heavy metal plumbum, 2.06ppm; average relative molecular weight, 2400.The physiological functions of fermented broth are also tested. It can inhabit growth of pathogenicbacteria in the plant. Chitooligosaccharides have't any effects in Hela cell culture.