Screening Of High Chitinase Producing Strains And Exploring The Antibacterial Activity Of Fermentation Products

Tibet is known as the third pole of the Earth,with a relatively cold climate and the highest average elevation in the world,earning it the title of"Roof of the World".Linzhi City is located in the eastern part of Tibet,with abundant rainfall and lush vegetation,known as the"snowy Jiangnan".This study used soil from Linzhi City,Tibet as experimental samples to screen strains with high activity in degrading chitin,in order to enrich microbial resources in the Tibetan region and provide ideas for the development of biocontrol bacterial resources in special environments.After enrichment and cultivation,soil samples were separated and screened for chitinase producing bacteria using a tenfold gradient dilution method and a colloidal chitin plate transparent circle method.A total of 68 strains of chitinase producing bacteria were isolated after removing duplicates based on differences in colony color and shape.During the initial screening of the same dish,it was found that 6 strains of bacteria were able to stably produce chitinase.After fermentation,the ability to produce chitinase was re screened using DNS method.Finally,a strain named C81 was selected for further research.The strain had the largest transparent circle in the chitin dish,resulting in the strongest chitinase activity.Based on the spore color,colony morphology,and 16S r RNA gene sequencing results,the strain was preliminarily identified as Paemibacilli.sp.Optimization of the enzyme production time,initial pH,concentration of peptone,and concentration of colloidal chitin in the initial fermentation condition of strain C81 by controlling the single factor method.5 g/L peptone,1g/L Na Cl and 15%colloidal chitin,pH was adjusted to 8 and incubated at 37°C for 3 d,and the chitinase production was at the optimal level,approximately 3times of that before optimization.The crude enzyme liquor was prepared from the fermentation broth by the salting out method with ammonium sulfate,and the chitinase in the crude enzyme liquor was purified by the affinity purification method.The purified product was run against marker after SDS-PAGE electrophoresis,and its molecular weight size was determined to be approximately 68 k Da.The enzymatic characteristics of chitinase were investigated through temperature,pH,metal ions,and colloidal chitin concentration.It was found that the optimal reaction temperature of the enzyme was 40°C,heat resistance was good,its optimal reaction pH was 8,it was most stable at pH 7.Metal ions such as Mg²⁺,Cu²⁺,Fe²⁺all showed different degrees of inhibition on the Chitinase Produced by strain C81,Zn²⁺could promote this chitinase reaction.The antibacterial activities of the fermentation products against fungi and bacteria were detected by a flat dish to stand versus tube dish method.Results showed that the fermentation products of strain C81 were 72.72%,83.63%inhibitory against Rhizoctonia solani,Helmnthosporium trers Sacc.The fermentation products of C81 also showed obvious inhibitory effects on,in which Staphylococcus aureus>Escherichia coli.The above research results not only enrich the resources of biocontrol strains,but also provide data support for further research on chitinase producing strains,which is of great significance for the sustainable development of agriculture.