Induced Expression And Fermentation Of Recombinant E.coli BL21(DE3)-pET28a(+)-bgl Producing β-glucanase

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.Now,β-glucanase used in the beer industry and feed industry is mainly produced by fungi and Bacillus with aerobic fermentation.Cloning and expression of exogenousβ-glucanase gene in Escherichia coli with different plasmids have been studied widely recent years.The strain used in this paper is a recombinant Escherichia coli pET28a(+)-bgl,which cloned aβ-glucanase gene from a strain of Bacillus amyloliquefaciens prodeucingβ-1,3-1,4-glucanase efficiently.The recombinant starin could expressβ-glucanase by lactose and IPTG inducement.This paper studied the recombinant's performance according to the factors affecting expression,exp,growth characteristic of recombinant strain, inducing condition and composition of culture medium.The amplificatory fermentation in 7 litres fermenter was also junior studied.The conclusions are as follows:1.Seed liquid(OD₆₀₀=0.35) was inoculated into 25 mL LB medium with initial pH 7.0 in 10%volume.It was added the inducement when it was incubated in 37℃and 200 r/min to OD₆₀₀=1.The activity of recombinantβ-glucanase would be highest.2.The optimal inducing condition in LB medium were ensured by orthogonal experiment. 0.0336 mmol/L IPTG and 10 mmol/mL lactose was added as inductor.Inducing for 6 h in 24℃,the highestβ-glucanase activity was 336.33 U/mL in supernatant liquor,which was higher than non-optimization 44.3%.The activity was 4.85 fold higher than original strain in same conditions.3.Main effect analysis for initial medium using Plackett-Burman design was performed.The main effect factors were glycerol,yeast extract and NaCl.Using Box-Benhken design and Response Surface Regression(RSREG) of MATLAB 7.0 and NESS for datas of experiment, the optimal composition of culture medium was as follows:18.9 g/L glycerol,45.6 g/L yeast extract,5.3 g/L NaCl,5.0 g/L peptone,2.8 g/L KH₂PO₄ and 4.8 g/L K₂HPO₄.3H₂O.The recombinant strain fermented in the optimal medium for 12 h,the total activity could reach 2311.81 U/mL,which was higher than the forecast 2053.1 U/ml.4.Amplificatory fermentation in 7 litres fermenter for 13 h,β-glucanase activity was 4189.58 U/mL in supernatant,which was higher than shaking flask 81.23%and 4.91 fold higher than original strain in optimal condition.This paper showed the recombinant strain had great applied value in industry and layed the foundation for amplificatory fermentation.