| Chitosanases which catalyze the hydrolytic degradation of chitosan have been used for preparation of functional chitooligosaccharides. They have also been used as biological control agent in agriculture to improve plant anti-disease abilities, hi terms of bioengineering, chitosanases have been used for generation of fungal protoplasts along with other means, providing enzyme tool in cell molecular biology. Based on the chitosanases producer of Aspergillus sp.CJ22-3 screened from soil, this dissertation includes strain mutation and improvement, optimization of shake-flask culture medium and fermentation conditions, control of 10 L scale-up fermentation, isolation and purification of chitosanases, properties and hydrolysis action mode of purified enzyme, and enzymatic hydrolysis of chitosan. The main parts of this research work and results are presented below.1. By a screening method with chitosan as the only carbon resource, 45strains producing chitosanases were isolated from 65 samples of soil According to ratio between clear zone and colony diameter combined with shake-flask screening, enzyme activity was judged by means of DNS method and viscosity method. Hydrolysis products of strains secreting highly active chitosanases were analyzed by TLC, HPLC and MS assay. Results showed that hydrolysis products of strain 21-3 and 22-3 were similar, both of which were from glucosamine to chitohexaose. Strain 22-3 named CJ22-3 was chosen for test. According to the characterization of colony, vesicle, conidia head, and conidiospores of the strain CJ22-3, it was classified as Aspergillus sp.. Acute toxic tests of fermentation solution and enzymatic hydrolysis products were evaluated. The results showed that the product was nontoxic.2. Common utilized carbon sources such as glucose inhibited the secretion of chitosanases by Aspergilus sp. CJ22-3. Chitosanases generated by Aspergillus sp. CJ22-3 were also controlled by transcription factor. Mutant strain was screened by adding 0.1% Triton-100 inhibitor and 2% glucose to screening medium, Aspergillus sp. CJ22-3 as start strain. Strain CJ22-326 was obtained by UV and 60Co mutagenization, and its enzyme activity was 1.54 times of that of CJ22-3. After optimization of medium and fermentation conditions, enzyme activity in shake-flask was 3.06u/ml. Scale-up experiment in 10 L fermentation tank with mechanically agitated showed enzyme activity 3.65u/ml. It was higher than that in shake-flask.3. Chitosanases were extracted from culture filtrate of Aspergillus sp. CJ22-3 by using vacuum concentration, 75% alcohol precipitation, and the recovery of enzyme was above 75%. Two components of ChiA and ChiB were isolated by CM-Sepharose FF ion exchange chromatography, Sephacryl S-200 gel filtration chromatography and phenyl Sepharose CL-4B hydrophobic chromatography. Single band was obtained by SDS-PAGE and their relative molecular weights were 106.2 kDa and 29.0 kDa, respectively. Relative molecular weights determined by Sephacryl S-200 gel filtration chromatography were 96 kDa and 28.9 kDa, respectively. This showed that these enzymes were monomeric. Analysis of final purified ChiA and ChiB was done by inverse phase chromatography, and purities were 92% and 94%, respectively.4. Studies on characterization of ChiA and ChiB showed optimum pH 4.2 and temperature of 50癈for ChiA, and optimum pH 5.8 and temperature of 65 for ChiB. Stable pH range varied from 4.0 to 6.0 for ChiA and from 5.5 to 7.5 for ChiB. The two components showed stability below 50 . But stability of ChiB was better than that of ChiA above 60. Chitosanase activity of ChiA and ChiB were strongly inhibited, by 2.5mmol/L Ag+, Fe3+ and Immol/L Hg2+and strongly activated by Immol/L Mn2+. ChiB was also inhibited by 2.5 mmol/L Cu2+ and Pb2+. Other metal ions had little effects on ChiA and ChiB. In low substrate concentration, enzymatic reaction of ChiB conformed with Michaelis-Menten equation. Apparent Michaelis contents Km and Vmax were 0.7749 mg/ml and 0.272u/ml, respectively. The reaction velocity decreased with...
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