The Preparation And Application Of Chitosan Enzyme

Chitooligosaccharide is a low-molecar-weight chitosan with average molecular weight between 500 to 10000. It has many good physical properties, such as good solubility and high absorption, and has promising uses in the field of medicine,food and agriculture. Up to date,the physical, chemical and enzyme degradations have been applied in chitosan oligosaccharide preparation. But acid degradation and oxidation synthesis were currently primary methods leading to the technical process complexity, serious environment pollution,high molecular weight of products. Comparably, the biological synthesis of chitosan oligosaccharide is more competitive because of its environmental friendliness,cost-effectiveness and high yield.Tn this study, The recombinant chitosanase gene from the wild Bacillus cereus shs-0903 T with high-yielding chitosanase was recombined and expressed with pET28 a by Escherichia coli BL21.The optimization of cultrure medium was carried out. After the fermentation on the optimal conditions, 1500U/ml of crude enzyme activity was obtained.And then the crude enzyme was purificated by 20%-70% ammonium sulfate. The characterizations of chitosanase were shown the optimum temperature and pH were 55? and4.8, respectively. The enzyme structure was stable under 60? and the enzyme activity was still above 90% at 4? for 30 days. The Mn2+,converse to the Cu2+and Fe3+, could improve the enzyme activity. The results showed that this enzyme was likely to be a promising enzyme for industrialization.The parameters of chitosanase catalysis were studied containing the temperature, pH,substrate concentration, enzyme titer and reaction time. By centrifugation and spray, some quality standards of chitooligosaccharide was obtained at 53-57?,pH 4.8-5.3, 6.5% of chitosan and 10u/g of Chitosanases for 100 min. The physical and chemical properties of chitooligosaccharide were as follows: appearance: light yellow powder; pH5.74, water insoluble: 0.015%; moisture: 10.1%; ash: 1.7%; color: 5800; average molecular weight: 1920;yield 97.2%.