Gene Cloning And Characterization Of β-1, 3-glucanase And β-mannosidase From Streptomyces Sp. S27

Streptomyces is an important resource-rich antagonistic microorganism and is widely used in industry, agriculture and environmental sciences.In this study,Streptomyces sp.S27 was selected as the research objects based on the evaluation of species-novelty and enzyme production capacity.Streptomyces sp.S27 was isolated from the Flaming Mountain in the Turpan Basin of Xinjiang Uygur Autonomous Region, China.By using bioinformatical methods,the amino acid sequence of Streptomyces producedβ-1,3-glucanase from glycoside hydrolase(GH) family 16 were aligned and analyzed.A set of degenerate primers were designed based on the conserved motifs.Gene fragments ofβ-1,3-glucanase from Streptomyces was cloned by using degenerated PCR method.The full length gene bglS27 was obtained by combinating with genomic library screening and TAIL PCR,and successfully expressed in Escherichia coli BL21(DE3). The bglS27 gene contains 1,362 bp and encodes a protein of 453 amino acids with a calculated molecular mass of 42.7 kDa.The mature protein comprises a catalytic module of glycosyl hydrolase(GH) family 16, a short glycine linker region,and a family 13 carbohydrate-binding module(CBM).The purified recombinant enzyme showed optimal activity at 65℃and pH 5.5,and preferentially catalyzed the hydrolysis of glucans withβ-1,3-linkage with an endolytic mode of action.Specific activity and Km value of BglS27 for laminarin was 304.2 U mg^-1 and 1.89 mg ml^-1,respectively.In antifungal assay,BglS27 had ability to inhibit the growth of some mycotoxin-producing fungi,such as Fusarium Graminearum, Fusarium crookwellense,and Paecilomyces variotii,and phytopathogenic fungus Rhizoctonic solani.These favorable properties make BglS27 a good candidate for use in feed and food preservation,plant protection, and other biotechnological applications.A pair of primers were designed based on the gene fragment ofβ-mannosidase from Streptomyces sp. S27 to clone the full length gene,manS27,by screening the genomic library from Streptomyces sp.S27. The gene manS27 contains 2,496 bp and encodes a protein of 832 amino acids with a calculated molecular mass of 92.5 kDa.The gene manS27 was inserted into the expression vector of pET-22b(+) and transformed into Eschetichia coli BL21(DE3) to express the recombinant protein ManS27. Characterization of ManS27 indicated that the optimum temperature and pH were 50℃and pH7.0,and the specific activity and Km value of ManS27 were 304.2 U mg^-1 and 1.89 mg ml^-1,respectively.The result of transglycosylatic experiment showed that ManS27 had the activity of transglycosylation.In this study,aβ-1,3-glucanase gene and aβ-mannosidase gene were cloned from Streptomyces sp. S27.By constructing and transformating expression vector to Eschetichia coli,we have obtained recombinants which are promising in applications of constructing the commercialized high-yielding strains and molecular biological research on enzymy.