Characterization And Functional Analysis Of β-1,3-glucanases From Insect And Bacterium

β-1,3-glucanase(Laminarinase),which is widely distributed in bacteria,fungi,plants and invertebrates,hydrolyzes internal β-1,3-glycosidic linkages in β-glucan.β-1,3-glucanase is an important cell wall hydrolase,which can degrade the cell wall of fungi and play an important role in the prevention and treatment of fungal diseases.It is one of the hotspots in the research of antifungal genetic engineering.Different from the plant β-1,3-glucanase which belongs to the glycoside hydrolases GH17 family,insect and bacterium β-1,3-glucanase belongs to the GH16 family and the researches about its functional and antifungal activity are limited.Therefore,this thesis analyzes the enzymatic and biological activities of Asian corn borer(Ostrinia furnacalis)β-1,3-glucanase and Bacillus circulans β-1,3-glucanase,and provides new enzyme sources to the fungal biocontrol strategies which targets to fugual cell wall.The main work of this thesis includes:(1)The cDNA sequence of the Ostrinia furnacalis β-1,3-glucanase OfLam was obtained.The length of CDS was 1128 bp coding 375 amino acids,the theoretical molecular weight was 42.2 kD,and the isoelectric point p I = 5.16.Phylogenetic analysis demonstrated that the OfLam was closely related to other insect β-1,3-glucanases and its catalytic amino acid was highly conserved.The result of real-time quantitative PCR showed that the expression level of OfLam in midgut is much higher than that in other tissues,and the expression level in larval period is much higher than that in egg stage,pupal stage and adult stage.(2)Expression vector pET28a-Of Lam was constructed and Of Lam was successfully obtained in 250 mM imidazole solution by metal chelate chromatography,and the yield was 2.5 mg / L fermentation broth.SDS-PAGE showed that the accurate molecular weight of recombinant Of Lam was around 30 kD,which was lower than the theoretical value,and recombinant OfLam had β-1,3-glucanase activity.Characterization of enzymatic properties showed that the optimum pH of Of Lam against laminarin was 4.5,the optimum temperature was 50 ℃,the Km was 1.59 ± 0.28 mg/mL and the kcat was 15.8 ± 0.66 s-1s.OfLam had a broad substrate specificity and can hydrolyze fungal cell walls.(3)Recombinant Bacillus circulans β-1,3-glucanase BcLam was successfully expressed,the target protein was eluted in 250 mM imidazole solution and the yield was 50 mg / L fermentation broth.SDS-PAGE and mass spectrometry showed that the molecular weight of recombinant BcLam was 43.29 kD,The optimum pH was 6.0,the optimum reaction temperature was 50 ℃.It has broad substrate specificity and the highest hydrolytic activity when compared with β-1,3-glucanases from different sources.Bc Lam showed better activity than commercialized enzymes in the fungal cell wall degradation experiment with pretreatment,and when BcLam was synergistic with Aspergillus fumigatus chitinase B in hydrolyzing fungal cell walls,the hydrolysis efficiency increased.BcLam and Af ChiB also showd synergistic effects on inhibition of the growth of Fusarium oxysporum spores,in which BcLam played a major role.(4)The crystallization conditions of the recombinant BcLam were screened,and three suspected successful crystallization conditions were found in the total of 400 target protein concentrations and the next step was optimized.