| Cyclodextrin Glycosyltransferase (CGTase, EC2.4.1.19), can produce cyclodextrinsusing starch and starch derivatives as substrate in intramolecular transglycosylation reaction.Cyclodextrins (CDs) are cyclic oligosaccharides with hollow truncated cone and they arecomposed of α-1,4-glucans glucose units. Among CDs, α-, β-and γ-CD, composed of6,7or8α-1,4linked glucose units, are the most extensively studied and exploited. Through forminginclusion complexes with various guest molecules, CDs can change their physical andchemical properties, so they have a wide range of applications and research values related tofood, medicine andbiologicals, etc.At present, CDs are mainly produced via the enzymatic reaction in industry. In thepresent study, the γ-CGTase from Alkalophilic B. clarkii7364was expressed by Escherichiacoli. In addition, the characters of the purified recombinant γ-CGTase and factors onproducing CD were also systemically studied. The main contents are as follows:After constructing engineering bacteria E. coli BL21(DE3)/pET24a-γ-CGTase, it wasinduced to produce the recombinant γ-CGTase. Then the characters of recombinant γ-CGTasewere studied in detail including optimum pH and temperature, pH and temperature stability,kinetics, etc. As shown in the results, the recombinant γ-CGTase is an alkalophilic enzymeand not dependent on Ca2+or other metal ions. It remains stable under50°C, at which itshalf-life was about4h. The optimum pH and temperature was10.5and60°C, respectively.Meanwhile, the Km, Vmaxand kcatof the recombinant enzyme were4.83g L-1,5.83U mg-1and12.55s-1, respectively.Based on the results obtained above, various parameters, including organic silvents sortsand concentrations, starch sorts and concentrations, temperature, pH, enzyme dosage, andreaction time, etc, were optimized. The optimum conditions were as follows：15%(w v-1)potato starch and7.5U of γ-CGTase per gram starch were mixed at70oC about30minutesfor liquefaction; the reaction mixtures cooled down to55oC and were adjusted to pH10.0,another7.5U of γ-CGTase per gram starch with3%(w v-1) cyclododecanone were added forbioconversion reaction. After150r min-1water bath reaction for7h,51%(w w-1) yield of γ-CD was obtained with virtually no α-CD detected.In the next, to get high purity product, the separation and extraction process of γ-CD wasstudied. When the reaction was terminated, the mixture was carried through a series of means,including azeotropic steam distillation, evaporation, crystallization, drying, etc, and theproduct crystal was obtained. Then, identification of the γ-CD was perofmred. In contrastwith γ-CD standard from Sigma, various means were adopted to veritify the exsistence of γ-CD obtained by myself, including observing apperance, high perofrmance liquid chromatogra(HPLC), infrared spectra and mass spectra (MS). According to the results gained by themeans mentioned above, and the residual concentration of cyclododecanone was only3.0ppm, which matches the standard of safety production. In addition, the product crystal wasagain proved to be γ-CD.To further enhance the γ-CD yield, the synchronous bioconversion of CD by both γ-CGTase and isoamylase from Thermobifida fusca was exploited. Allowing for the enzyme dosage and pH of isoamylase, the mutant A223K of γ-CGTase, which has a wider tolerancerange of pH, was used in the present study. Various paramerters affecting γ-CD yield in thisapproach were optimized. The optimized conditions were as follows:15%(w v-1) potatostarch and7.5U of γ-CGTase (A223K) per gram starch were mixed at70oC about30minutesfor liquefaction; the reaction mixtures cooled down to50oC and were adjusted to pH7.0,another γ-CGTase (A223K) and isoamylase with4%(w v-1) cyclododecanone were added forbioconversion reaction. The optimum concentration of γ-CGTase (A223K) and isoamylasewas12.5U g-1and40U g-1, respectively. After150r min-1water bath reaction for12h,72.5%(w w-1) yield of γ-CD was obtained with virtually no α-CD detected. |
PDF Full Text Request