| Chitosan, derived from the deacetylation of chitin, is rich in nature. Low-molecular-weight chitosans or water-soluble chitosans have special and unique physiological activity, which makes them more valuable as Pharmaceuticals. This paper aimed at the production of low-molecular-weight chitosans by the degradation of macromolecular chitosan catalyzed by Rhizopus delemar lipase. The conditions for producing lipase and for hydrolyzing chitosan were studied, and the products were separated and identified also. The main points of research work are as follows:1. Production of lipase by solid state fermentation of microorganismsA wild strain fungus Rhizopus delemar was screened and employed to undergo solid state fermentation so as to produce lipase. Factors such as carbon and nitrogen sources, inoculum, water content and temperature of culture were studied. The result showed that the soybean cake, an agriculture product residue, could be used as the basic culture medium, and the added starch and peptone as carbon and nitrogen sources would benefit the enzyme production. The water content in medium and temperature also influenced the fermentation process. Under optimal conditions, i.e., 12g soybean cake powder, l.Og starch, 0.5g peptone, 55.6% water content of medium and at 28 "C, lipase activity reached 320Iu/g dry medium after 48h cultivation. Extracting medium with pure water to produce crude enzyme solution, and through iso-electrical precipitation, Sephadex gel filtration, and freeze drying, pure enzyme powder was obtained. Its highest lipase activity reached 2430IU/g.2. Optimization of degradation of chitosan catalyzed by lipaseFactors such as temperature, pH value, substrate concentration, enzyme amount, reaction time and some metal ions were studied. The result indicated that temperature3and pH value had major effects on reaction. The suitable ranges for temperature and pH were 40癈~45癈 and 4.5-5.5, with the optimal values 45癈 and 5.0 respectively. The reaction rate dropped dramatically when both temperature and pH were out of the range. The reaction rate at 45 "C was five times greater than that at 20癈. Within the tested range, the enzymic reaction rate increased with the increase of enzyme amount, and a linear relationship was showed between two of them. The increase of substrate concentration in some degree also brought in the acceleration of degradation rate. However it was indicated on the reciprocal curve of concentration and rate that Rhizopus delemar lipase catalyzed hydrolysis of chitosan did not conform to the kinetic model of first-order reaction, which means the lipase used does not have the trait of Michaelis enzyme. Among the common metal ions, adding 0.02mol/L Cu2+ and Mn2+ would make chitosan solution flocculate and Ni * and Ca * would reduce lipase activity, but Na+, K+, Fe3+, Mg2+ did not affect the reaction. Under optimum conditions, the viscosity of chitosan solution reduced rapidly in the first course of reaction, which went down to 36% that of original solution in Ih. With time progress, the degradation rate slowed down gradually and after reaction for 6 hours, the viscosity decreased to 8% that of original solution. In addition, studies on chitosans with different deacetylated degrees displayed that chitosans with 82%~87% degrees of deacetylation would benefit the degradation of chitosans. 3. The preparation of immobilized lipase and its test on chitosan degradationThe combined method of adsorption and cross-linking was employed to produce immobilized lipase with chitin particles as the carrier and glutaric dialdehyde as the cross-linking agent. The immobilization conditions were optimized as follows: ratio of enzyme powder to carrier: 0.04:1 (w/w), pH value 6.0 , glutaric dialdehyde concentration: 0.6% , reaction time: 6h , temperature: 40癈. The immobilized lipase with the activity of 40IU/g was obtained.The degradation of chitosan by immobilized lipase was tested compared with free lipase of same activity. The result indicated that immobilized enzyme...
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