Breeding And Fermentation Of Chitosanase/Chitinase Producing Strains, Purification And Characterization Of Chitosanase

Chitin, the second largest renewable resource next to cellulose, is composed of β-1,4-linked N-acetylglucasamine residues which is major structure of fungi cell wall. Chitosan is partial and total deacetylation of chitin and is the largest alkaline polysaccharide in nature. The poor solubility of chitin and chitosan largely limited the application of chitin and chitosan. The oligosaccharides from chitin or chitosan can overcome this limit, and they have good solubility and bioactivities, such as antimicrobial, antioxidant, and antitumor activity. Using specific enzyme chitosanase and chitinase to hydrolyze chitin and chitosan to obtain their oligosaccharides has many advantages, such as the mild reaction, uniform degree of polymerization, and low environmental pollution. The microorganisms are the main source of chitinase and chitosanase. Hence, breeding of chitosanase/ chitinase producing strain from nature microorganism and improving the enzyme yield of the strains are very important.A chitosanase-producing strain was screened by clear zone from sewage of a waste water treatment work,which is identified as Bacillus cereus by 16S r RNA,physiology and biochemistry and named HMX-21.The fermentation conditions and components of medium were optimized by one factor experiment.The levels of medium components were optimized by Plackett-Burman design and Response Surface method.The supernatant was obtained by centrifugation and 70%saturation of(NH4)2SO4 was used to precipitate.The precipitation was desalted by Sephadex G-25 and purified by DEAE-纤维素anion exchange chromatography.A chitinase-producing strain was screened by clear zone from the sewage of a yielding chitin company which was identified as Aeromonas sp D5 through 16S r RNA.The strain D5 was mutated by UV and Li Cl to increase chitinase activity.The results were as follows:1 The culture conditions of Bacillus cereus HMX-21 were investigated and the maximum cultivating conditions were as follows: inoculum size 6%, fermentation temperature 35℃, initial p H 6.5, shaking speed 180r/min, loading liquid 100 m L/250 m L, optimal fermentation time 96 h, colloidal chitosan 3.45g/L, yeast extract 18.26g/L, K2HPO4 O 0.6g/L, KH2PO4 0.45g/L, Na Cl 2.5g/L, Mg Cl2 0.5g/L with which the chitosanase activity was 14.88U/m L.2 Chitosanase was purified throhgh salting out, desalting and anion exchange chromatography with which the yield was 2.4% and purification fold was 8.4. Molecular weight of chitosanase was determined by SDS-PAGE, which was approximately 43 k D. The chitosanase exbihited the optimum activity was at 50℃ and p H 5.5, respectively. The chitosanase was stable at 25℃~55℃, p H 4.5~7.0, respectively. The chitosanase was activated by Mn2+, while inbihited by Ba2+, Co2+, Cu2+ and EDTA.The most susceptible substrate was colloidal chitosan.3 The strain D5, which the chitinase activtity was 0.9U/m L, was treated by the first ultraviolet mutagenesis for 10 s and strain UV1-7 was obtained with which the chitinase activity was 1.33U/m L. The UV1-7 was conducted with second ultraviolet mutagenesis for 15 s and mutation UV2-7 was gained with which the chitinase activity was 2.84U/m L.The UV1-7 was treated by complicated mutation of second ultraviolet mutagenesis and Li Cl and mutation UVL-7 was obtained.The UVL-7 chitinase activity was 3.48 U/m L which was 3.87 time compard to D5, which showed higher genetic stability through stability experiment and was named HMX-16.