Isolation And Identification Of High Chitinase-producing Bacterium And The Properties Of Its Chitinase

Chitin is also called chitosan, it is one kind of nitrogen-containing polysaccharide biological polymer which is widely exists in the nature. The reserves of chitin is the second one of natural polymer on the earth, it also has a lot of advantages, such as renewable,biocompatibility, biodegradability, function etc. Chitinase is the enzyme specificity degrading chitin, catalytic hydrolysis of chitin, chitosan and N- acetyl glucosamine and other substances. Among them, chitosan oligosaccharide has many kinds of functions. It also called soft gold in foreign countries, and can be used in medicine, health care, food,daily chemical, agriculture and other fields. This study selected one strain WY-1 from the sediment of Tianjin Hangu shrimp farming which can produce chitinase, identification and optimization the fermentation conditions of WY-1, at the same time the chitinase was purified and its properties were studied. The main methods and results are as follows:(1) The chitinase producing bacteria were separated and purificated using the method of transparent circle plate, 20 strains had been screened, they could grow in the solid plate with chitin colloidal chitin as the sole carbon source of strain. Secondary screening was conducted by measuring the chitinase activity, and the strain WY-1 with a high chitinase activity and high ratio of the transparent circle and the strain diameter had been choose.(2) The strain WY-1 was determined as Paenibacillus alvei by the colony characteristics, microscopic observation, physiological and biochemical characteristics and 16S rDNA sequence homology analysis of the strain.(3) The enzyme production condition of the strain WY-1 was optimized using single factor method and response surface method, and the optimum conditions for enzyme production: colloidal chitin content in culture medium was 1.62 %, the temperature was 40 ?, the culturing time was 72 h, on these conditions, chitinase activity of the strain WY-1 was 13.07 U/mL.(4) The chitinase was separated and purificated by salting out, gel filtration chromatography, dialysis, ultrafiltration and freeze drying methods, SDS-PAGE analysis showed that the chitinase was a monomeric enzyme. At the same time,conditions of enzyme activity were confirmed using single factor method, the optimum conditions:temperature was 50 ?, pH was 7.0, and the colloidal chitin content was 1.5 %.These results not only enrich the biological data of Paenibacillus alve, but also laid the foundation for further development and utilization of the strain.