Beta-glycosidase Enzyme Gene And Endoglucanase Gene Were Coexpressed In E.Coli And Fermentation Conditions For Research

Cellulase is a sort of multienzyme compound which can degenerate cellulose into cellobiose, glucose as well as other monosaccharide. It mainly comprises three parts including exoglucanase, endo-glucanase and β-1,4-glucosiase. β-1,4-glucosiase, one of the significant part of cellulose, could intensively affect the ability of cellulose hydrolysis of cellulose. With the widely application of cellulose enzyme, cellulose preparation has drawn much attention and has been utilized in lots of fields such as textile, pharmacy, food and feed industry, indicating its enormous economic value. Hence, by employing genetic engineering technology, we successfully achieved high expression of these two genes, namely Bacillus subtilis derived endo-glucanase gene and Aspergillus oryzae derived gene, respectively in E.coli, and their co-expression in E.coli as well. What’s more, further experiments were conducted on the expression products characteristics and fermentation conditions.Using bioimformatics, we analyzed the protein structures of Bacillus subtilis derived endo-glucanase gene and Aspergillus oryzae derived gene, predicted their signal peptides* and synthesized both of their primers which excluded their signal peptide sequences but added corresponding restriction enzyme cutting sites at 5’ends.We obtained endo-glucanase gene which contains 1500bp and β-1,4-glucosiase gene which contains 2800bp by applying PCR amplification, and successfully constructed recombinant plasmids pET32a-EG and pET-30b-BGL. Then Escherichia coli BL21 was transformed using these two plasmids. Among all the transformants, the positive ones were screened out with Amp or Kan resistance together with enzymatic determination. They displayed high enzyme activities 821.5U/mL and 219.6U/mL respectively. SDS-PAGE analysis was carried for the expressed products, showing their approximate 55 kD and 90kD molecular sizes. Besides, their optimal reaction temperature is 60℃, and the optimal pH were 6.0 and 5.0.We transferred recombinant plasmid constructed based on endo-glucanase gene into Escherichia coli BL21 whose genome was integrated with β-1,4-glucosiase gene. Positive transformants are these who could resist Amp or Kan and display the enzyme activity. When induced, Select the highest enzyme activity of the strain named pET32a-EG-pET30b-BGL Ⅱ, enzyme activity as high as 1196.8U/mL. Through the analysis of the enzymatic properties of the enzyme Ⅱ produced by E.coli pET32a-EG-pET30b-BGL Other characteristics such as the optimal temperature and pH of the enzyme are 60℃ and 6.0. Besides, enzyme could sustain almost 80% of its highest enzyme activity even when treated at a wide range of temperature (range from 30℃ to 60℃) and pH (range from 5 to 7).As for fermentation condition analysis, six factors as inoculation density, temperature, loading volume, induction time, initial pH, concentration of inductor IPTG, were picked out for assessment following the design of software Plackett-Burman. The variance analysis revealed that temperature, initial pH and loading volume were three key factors of the sixes. Consequently, we optimized these three elements by applying Box-Behnken. Results disclosed an optimum condition, that is when they were 35℃,7.5 and 25mL (total volume 100mL)respectively. Under this condition, the engineering bacterium pET32a-EG-pET30b-BGL Ⅱ enzyme presented its highest activity 2080.5U/mL which was nearly twice high as before optimization.