Characterization And High-efficiency Expresssion Of Recombinant Cyclodextrin Glycosyltransferase From Geobacillus Caldoxylosilyticus CHB1

To investigate the potential for industrial application of CGTase from Ge obacillus caldoxylosilyticus CHB1,CGTase gene from Geobacillus caldoxylosilyti cus CHB1 was cloned and expressed,the characterization was investigated in t his dissertation.Several strategies including signal peptide and fermentation cond ition optimization,additon of chemical additives were employed in this study to enhance the extracellular secretion efficiency of recombinant CGTase.The mai n results were as follows:1.The CGTase gene of Geobacillus caldoxylosilyticus CHB1 was cloned into the downstream of Pe1B signal sequence in the secretory expression plasmid pET22b?+?.Thus the recombinant expression plasmid pET22b?+?-cgt was constructed and transformed into the host E.coil BL21?DE3?,the recombinant CGTase was secreted into the culture medium successfully.2.The recombinant CGTase from Geobacillus caldoxylosilyticus CHB1 with six His-tags in C-terminal could be purified a nickel affinity chromatography and the relative high yield was obtained successfully.The characterizations of recombinant CGTase from Geobacillus caldoxylosilyticus CHB1 were investigated using the purified CGTase.The results showed that the optimum cyclization reaction temperature and pH of recombinant CGTase was 60? and 5.0.It was stable at 40?and the cyclization activity could not be effected within one hour.Moreover it retained 50%of its initial cyclization activity after incubation for 35min at 50?,25min at 60?,20min at 70?,15min at 80?.The cyclization activity was obviously enhanced by Ca²⁺,Mg²⁺,Li+,Ni+,the most metal cofactor including Na+,Zn²⁺,Cu²⁺,Fe³⁺ inhabited the cyclization activity.The cyclization activity was completely inhibited by SDS.On the condition of 60? and pH6.0,the Km and Vmax values for the purified enzyme were 3.73 mg/mL and 0.0202 mmol/L min.3.The recombinant expression plasmid pEASY-E2-CHB1-cgt which contained the signal peptide of Geobacillus sp.CHB1 and pEASY-E2-PelB-cgt which contained the signal peptide PelB were constructed by routine PCR,so did the recombinant expression plasmid pEASY-E2-cgt without signal peptide.At the same time the recombinant expression plasmid pEASY-E2-OmpA-cgt which contained the signal peptide OmpA was an control.They were successfully expressed in E.coil BL21?DE3?.The results showed that OmpA was the optimal signal peptide under the same conditions,the extracellular CGTase activity could reach 7.44U/mL,which was 2.04-fold and 11.27-fold that of the PelB and Geobacillus sp.CHB1 signal peptide.The extracellular activity of recombinant CGTase without signal peptide could not be detected.However,a large amount of recombinant CGTase existed in the form of inclusion bodies.4.The secretory plasmid pEASY-E2-OmpA-cgt which contained the signal peptide OmpA was taken as the research object,the result showed that recombinant CGTase could be induced by IPTG and lactose respectively.But IPTG apparently inhabited the growth of bacteria,lactose promoted the growth as the carbon source.After 72h of cultivation,the extracellular CGTase activity reached 8.05 U/mL under the lactose inductin,of which IPTG inducting was 4.55 U/mL.Lactose could be used as an inducer instead of IPTG for the expression of recombinant CGTase.Optimal conditions for extracellular expression in shaking flasks were as follows:induction temperature 25?,lactose concentration 0.5%,induction OD600nm=1.4.There was no significant difference in the expression amount between addition of lactose in batch and one time.The best fermentation condition is that the initial pH of 7.0,the inoculum size of 5%,a medium content of 50mL medium in 250mL flask.The extracellular CGTase.activity could be stimulated by two steps temperature induction,which was shifted 30 ? after 24h of induction at 25 ?.After the above optimization,the extracellular CGTase activity was 19.87 U/mL.5.SDS and Tween-80 inhabited extracellular secretion of recombinant CGTase.The optimal condition to get the extracellular activity of recombinant CGTase was added 0.6%glycine and 0.3%Triton X-100 togethere into the culture.The extracellular activity was 14.27U/mL,which was about 2-fold that of the control group without any chemical additives.