Purification,Characterization,Gene Cloning And Expression Of Cyclodextrin Glycosyltransferase From Geobacillus Caldoxylosilyticus CHB1

To investigate the potential for industrial application of CGTase from Geobacillus caldoxylosilyticus CHB1 which was screened by our laboratory, purification, characterization, gene cloning and expression of CGTase from Geobacillus caldoxylosilyticus CHB1 were investigated in this dissertation. The main results are as follows:l.The CGTase of Geobacillus caldoxylosilyticus CHB1 was purified to homogeneity by 90% (NH4)2SO4 precipitation, DEAE-SepharoseTM Fast Flow column chromatography, Sephadex G-100 column chromatography, and preparative electrophoresis. The specific activity of the CGTase was raised approximately 350.56 fold, from 1.29 U/mg proteins to 452.22 U/mg proteins, with a recovery of 9.34%.2.The basic enzymatic properties of CGTase from Geobacillus caldoxylosilyticus CHB1 were investigated using the purified CGTase. The results showed that the enzyme was a monomer with a molecular weight 70 kDa estimated by SDS-PAGE. The enzyme was stabled below 50℃ with an optimum activity at 65℃, and was stable at a pH range of 5.5-9.5 with an optimum pH at 5.5. The enzyme activity was completely inhibited by SDS, and EDTA had no influence on the enzyme activity, when the final concentration of the metal ion was 10 mM, the enzyme activity was strongly inhibited by most of metal ions, but the enzyme activity was stimulated by Ni2+, Mg2+ and Li+. On the condition of 60℃ and pH6.0, the Km and Vmax values for the purified enzyme were 13.4 mg/mL and 0.02416 mmol/L·min, respectively, with soluble starch as the substrate, so with a low affinity for soluble starch. The enzyme produced α,β and y-CD after 21 h incubation with 3% soluble starch at 60℃, the yield of α,β and γ-CD were respectively 2.25%,9.13% and 7.04%, and the total yield of CD was 18.42%.3.The CGTase gene of Geobacillus caldoxylosilyticus CHB1 was cloned by the PCR technology, the secretory expression plasmid (pEASY-E2-OmpA-cgt) was constructed and the recombinant CGTase was secreted into the culture medium successfully. Optimal conditions for extracellular expression in shaking flasks were as follows:induction temperature 25℃, IPTG concentration 0.6 mM, induction OD600nm=0.4, under this condition, extracellular activity reached 12.23 U/mL after 72 h fermentation. Adding 0.7% glycine into the culture medium could significantly promote extracellular expression, and the extracellular activity increased 1.36 times than the blank. The extracellular recombinant CGTase in fermentation broth can purify to homogeneity by Ni affinity chromatography. The pure recombinant CGTase were generated α,β and y-cyclodextrin after 21 h incubation at 60℃ with 3% soluble starch, potato starch and maltodextrins, respectively. In addition, other undetermined substances were equally produced. The products from the reaction of pure recombinant CGTase on both potato starch and maltodextrins were consistent. When the soluble starch as substrate, the yield of α,β and y-cyclodextrin were 20.83%, 39.39% and 7.65%, respectively. The total yield of cyclodextrin was 67.87%, which were significantly higher than the substrate of potato starch and maltodextrin.