Construction Of Recombinant Escherichia Coil With Expressing β-CGTase And Studying On Application

β-CD was extensively used in many fields. β-CGTase is an industrially importantenzyme in forming β-CD, so the study of β-CGTase is a popular issue.A β-CGTase gene from Bacillus cereus was amplified by PCR methods using thepbvcgt plasmid as template. The gene was cloned in pET-22b (+) plasmid to constructpETcgt. Then the recombinant plasmid was transformed into E.coli DH5α. Afterrestriction analysis and sequencing, the recombinant plasmid was transformed intoE.coli BL21(DE3) for expression. Finally, E.coli BL21(DE3)/pETcgt was obtainedsuccessfully.The enzyme was overexpressed in E.coli BL21(DE3) by optimizing fermentationconditions. The optimal medium component is0.03%CaCl2,1%NaCl,0.5%yeastextract,1%tryptone; the optimal conditions for the expression in shaking flask were25°C and0.2mmol/L IPTG induction at OD600of1.7for12h. The highest activity was5239U/mL and increased by24.6times. In addition, the induced temperature was oneof the key factors on activity of β-CGTase.The molecular weight of the purified enzyme was about68kDa. The optimumtemperature and pH of the enzyme were60°C and9.0, respectively. The enzyme wasstable between pH7.0and9.0and temperature up to70°C. Ca2+could slightly enhanceenzyme activity while Mg2+and Mn2+showed medium inhibitory effects. Moreover, wefound that the activity was inhibited greatly by Cu2+, Zn2+, Fe2+, Al3+and Fe3+. Km andVmax of β-CGTase was calculated to be2.8439mg/mL and0.1874mg/mL·min-1,respectively.The main products from soluble starch as the substrate were β-CD with negligiblequantity of α-CD (0.62%) after12h of reaction at60°C, pH9.0with enzyme dosage of300U/g, without adding any selective agent. The highest ratio of β:γ was6.87(86.75%:12.63%).The constructed engineering bacteria from our reseach has higher activity byoptimization fermentation conditions. The ratio of β-CD was high in catalysate, so theresearch made a foundation for expanded production and industrial application ofβ-CD in future.