Cloning And Expression Of β-glucanase Gene From Bacillus Amyloliquefaciens

β-1,3-1,4-glucanase can hydrolyzeβ-glucan in the plant and microorganism cell wall efficiently. Lower wort's filtration speed, worse beer non-biological stability in beer brewing and lower nutrition absorptivity in feed industry could be resolved effectively by adding exogenousβ-glucanase. Nowadays, the key point of the research is how to obtain the strains with highβ-glucanase activity. This paper used a strain of Bacillus amyloliquefaciens which producesβ-glucanase high as original strain, constructed a recombinant Escherichia coli which could expressβ-1,3-1,4-glucanase efficiently, and studied the separation, purification and characteristic of the recombinantβ-glucanase.β-1,3-1,4-glucanase gene (has been registered in GenBank, accession number is EU623974) was amplified from a strain of Bacillus amyloliquefaciens which produceβ-glucanase very high by PCR and was cloned into three vectors pEGX-4T-1, pET20b(+) and pET28a(+) respectively to construct pEGX-4T-1-bgl, pET20b(+)-bgl and pET28a(+)-bgl recombinant plasmids. The pEGX-4T-1-bgl was transformed into three different E. coli host strains. The pET20b(+)-bgl and pET28a(+)-bgl were transformed into E. coli BL21(DE3) respectively. Recombinantβ-glucanase was expressed by IPTG inducement in these recombinants.All the recombinant strains could produce target protein by SDS-PAGE analyzing and determination of enzyme activity. The enzyme activity produced by E. coli BL21(DE3)-pET28a(+)-bgl was highest. The total enzyme activity was 322.0±8.8 U/mL, which was higher than other four recombinant strains obviously and 40.1% of original strain in optimal shaking flask condition. It may be fit to be used for directed reconstruction in future.To study the enzyme's applied value, the effect of several methods for breaking cell wall on recombinant enzyme activity was compared. It was shown that the freezing and thawing's method was the best. The crude recombinantβ-1,3-1,4-glucanase produced by E. coli BL21(DE3)-pET28a(+)-bgl was purified with ethanol classification precipitation and DEAE-650M. The purified enzyme was analyzed by SDS-PAGE and showed a single band, which showed pure recombinant protein maight be obtained. The final recovery rate of the total enzyme activity was 20.60%, the activity increase of 19.85 folds, the specific activity was 13437 U/mg, and the MW was about 30 kDa.The characteristic of recombinant enzyme produced by E. coli BL21(DE3)-pET28a(+)-bgl was researched after purfied. The optimal temperature and pH of the recombinant enzyme were 50℃and 6.0, respectively. It was relatively stable in 40-50℃and pH 4.5-6.0. The enzyme activity was inhibited by Cu2+ and Fe2+ with 10 mmol/L seriously. These characteristic were same as original enzyme mainly, which showed the recombinant enzyme could be used in beer brewing.