Cloning,Expression And Characterization Of A β-glucanase Gene From Triconderma Reesei

The β-glucanase enzymes are a class of hydrolase, widely present in plants and microorganisms. They can hydrolyze P-glucan in the β-1,3 and P-1,4 glycosidic bond into oligosaccharides and restore monosaccharide. P-glucanases are widely used in brewing industry, feed industry, sugar industry, paper industry and other fields. Therefore scientists from various countries pay an grumous interest in it.In this study, a β-glucanase gene(EG) was cloned from Trichoderma reesei previously isolated in our laboratory. The primers were designed based on the gene, accession number EU149644, from GeneBank of NCBI. The 657bp-length mature peptide coding sequence EG was cloned from the Trichoderma reesei genome using the overlap extension PCR method. The EG gene was constructed into pET-32a and pPIC9K and expressed in Escherichia coli BL21(DE3) and Pichia pastoris GS115 respectively. The results showed that Pichia pastoris maybe more suitable for the expression of EG than Escherichia coli. The mutEG(A35V) gene was also expressed in Pichia pastoris GS115. The optimal enzyme production conditions of the Pichia pastoris recombinate strain was 2% methanol induction, initial pH of 6.0, initial induced concentration of OD600 2.0. The P-glucanase activity of 47.738 U/ml was obtained under the optimal fermentation conditions, after shaking for 192h at 30℃. The glycosylated P-glucanase was purified after a series treaments of salting-out, desalting, ion exchange chromatography, hydrophobic chromatography. The yield was 62.46%, and the specific activity of the glycosylated β-glucanase towards P-glucan was found to be 1194.22U/mg. The treatment of Endo Hf demonstrated the recombinant protein contained glycosylated part and non-glycosylated part. And the proportion of the glycosylated one was rather larger than the non-glycosylated one. The enzymatic properties of the recombinant β-glucanase(EG) and mutational β-glucanase(MEG) was measured. The optimal temperature and pH of the glycosylated β-glucanase was 60 ℃ and 6.0 respectively, and its pH stability was good, while its temperature stability was relatively poor:the activity of the enzyme lost quickly under the treatment at 60 ℃ and above. Comparing with the natural P-glucanase, the mutational one has a better temperature stability, and its optimal temperature, optimal pH and pH stabality had not changed.Summarily, we succeeded in cloning the natural and mutagenic β-glucanase gene, constructing, the recombinat strain to produce the β-glucanase. And the enzymatic properties of the natural and mutational P-glucanase was characterized. Our research is a good theoretical guidance for expression, purification and industrial applications of EG, and promote a cetrain extent on the industrialized production of EG.