Optimization Of The Fermentation Process Of Extracellular Antibacterial Protein Of Lysobacter Sp. SNNU513

Pathogenic bacteria, fungi and nematodes are the main cause of plant disease. The prevention and control of plant disease has always been a top priority work in the process of agricultural production. Currently, although chemical pesticides can effectiv-ely prevent plant diseases, it can damage human health, pollute environment, and enhance the pathogen resistance. Benefiting from low residual, low toxicity and high efficient, biological control has achieved remarkable social and ecological benefits. Biopesticide is a kind of competitive or antagonistic metabolite, which secreted by biocontrol bacterias can suppress or kill different pathogens. Screening out active metabolites from biocontrol microorganism is realistic significance for biopesticide.L. sp.SNNU513, a new stain of L. genus, is a gliding organism with a high G+C content. It has been deposited in American Type Culture Collection (ATCC(?)BAA-26 21TM) and China General Micobioligy Culture Collection Center (CGMCC No.8375). L. sp.SNNU513 can secrete highly active proteins in LB medium, which can inhibit many plant pathogens actively. The results showed that it has tremendous potential application value. However, these active extracellular proteins of L. sp.SNNU513 were not clear. In addition, the low yield of total active proteins and the high cost of LB culture can not meet large-scale production of L. sp.SNNU513. Therefore, obtaining a number of low-cost and high-efficiency extracellular proteins is the key issue for L. sp.SNNU513 extensive application. In this study, we optimized both the fermentation medium composition and fermentation condition of L. sp.SNNU513 at the basis of LB medium. The superiority of the fermentation optimization process was verified from different aspects, such as total extracellular protein contents, the type of active protease, mRNA expression levels, et al. The main contents and results are as followings:1. The antimicrobial evidences of L. sp.SNNU513The inhibition results showed that L. sp.SNNU513 can inhibit both Botrytis cinerea and other plant pathogens. The total extracellular proteins from L. sp.SNNU513 have strong antagonistic effect to plant pathogens, and the mechanism of the antagonism attribute inhibiting the mycelium growth.2. Fermentation technology optimization of L. sp.SNNU513Three methods, single factor experiment, orthogonal experiment and response surface experiment, were used to optimize fermentation medium composition and fermentation conditions at the base of LB medium. The fermentation conditions include initial pH, culture temperature and rotate speed. The ultimate optimum fermentation conditions were:pH 7.0, temperature 30℃, speed 180 r/min. In this fermentation medium and fermentation conditions, the number of live bacteria reached at 5.69×108 CFU-ml’1 at 72h, which is the 1.35 times of the original LB medium, and the extracellu-lar proteins concentration is 2.48 times to original LB medium.3. Verification of the L. sp.SNNU513 optimization technology(1) The LC-MS/MS technology was used for construction and comparation of L. sp.SNNU513 extracellular proteome, which established at the base of genome database of L. sp.SNNU513. We screened out 20 antibacterial proteins. There are 10 kinds antib-acterial proteins in before and after optimization:chitinase, alpha-lytic protease 4, polyketide synthase and so on. Moreover, there are 8 and 2 antibacterial proteins only in optimization and LB medium respectively, such as alpha-lytic protease, lipase, glucose-dase, serine protease, and so on.(2) Real-time qPCR technology was used to detect chitinase gene, alpha-lytic protease 3 gene, polyketide synthase gene, glucosidase gene and phosphatase gene mRNA expressions. The results showed that the former 3 genes mRNA expressions were significantly up-regulated (p<0.01), glucosidase gene was notable up-regulated (p<0.05), while phosphatase gene was notable decreased (p<0.05) compared to the control group.(3) The influence of chitinase activity of L. sp.SNNU513 by fermentation medium was detected. The result showed that the strain’s chitinase activity in optimization medium was 1.45 times to LB medium.In general, after optimization, the number of bacteria, total extracellular proteins, and expressions of main active protein were increased significantly. The results can help furture develop the stain in the field of biological control.