Isolation Of Chitinase-Producing Strains And Enzymatic Preparation Of Chitinoligosaccharides

A strain B22 producing chitinase was isolated from soil samples collected from Hainan Island, and was identified as Streptomyces celluloflavus. The optimized medium for strain B22 to produce chitinase was as follows(g/L): peptone 2, yeast extract 2, K2HP04 0.7, KH2P04 0.3, MgS04.7H20 0.5, FeSO4.7H2O 0.01g,chitin powder 12g, pH 6.8. After inoculated with 3% of starter in a 250mL flask containing 70mL medium, and incubated 96 h at 27℃and 180r/min,the chitinase activity reached 2585 U/mL.The chitinase of the strain was extracted by ammonium sulfate precipitation (50～70%saturation), and its properties were studied. The optimal temperature and pH for chitin hydrolysis were 45℃and pH 6.8, respectively; the chitinase activity was stable in pH range of 5.0～8.0 and temperature under 50℃; Ca2+,Mg2+ and Cu2+ enhanced the enzyme activity significantly, whereas Fe3+ and Zn2+ inhibited the enzyme activity.The enzyme can hydrolyze colloidal chitin, chitin powder and shrimp shell powder. The time course of chitin hydrolysis and effects of various pretreatments to shrimp shell powder were investigated. Some results were obtained as followed:The reducing sugar reached 3.716 mg/mL when 5mL 1% colloidal chitin was hydrolyzed 5h with 5mL crude enzymes, while the reducing sugar was only 2.547 mg/mL when 4 g shrimp shell powder was hydrolyzed 12h with 30 mL crude enzymes. The reducing sugar could reach 2.808 mg/mL when shrimp shell powder were steamed 5min at 0.15 MPa and then treated 1h by ultrasonic wave. The hydrolysis product of chitin was identified as trisaccharide by TLC analysis.