| Cyclodextrins (CDs; also called schardinger dextrins or cycloamylose) are cyclic, nonreducing oligosaccharides formed by α-l,4-glycosidic linkages of varying numbers of number of D(+)-glucopyranose units. Different CDs are usually distinguished with a Greek letter to indicate the number of glucose units, e.g., α- for 6 units, β- for 7,γ for 8, etc. CD production can be achieved by actions of cyclodextrin glucanotransferases (CGTases; EC 2.4.1.19) on starches. Central gaps in CD molecules can be filled with chemical compounds, forming complex inclusions and leading to improvement of physical and chemical properties of the compounds. Therefore, CD products have been widely used as emulsifiers, antioxidants or stabilizing agents in food, cosmetic, pharmaceutical, and plastic industries.In this study, a bacterial strain capable of producing α-CGTase was obtained and identified as Bacillus licheniformis. An emphasis was placed on optimization of fermentation conditions for production of the α-CGTase by the bacterial strain, its purification and enzymatic characteristics. The results are summarized as follows:Isolation, breeding and identification of a Bacillus strain producing a-CGTase. A total of 115 bacterial strains capable of producing CGTases were isolated from 20 soil samples collected in Jiangxi Province. Of those, the strain numbered as 403 was found producing α-CGTase at the highest level. Thus, the strain 403 was selected as an initial strain to be exposed to UV radiation and chemical pressure of diethyl sulfate for further mutation for better production of the α-CGTase. An eventually selected mutant named CLS403 was capable of producing the α-CGTase 1.43 times more than the initial strain. The mutant stain CLS403 was finally identified as Bacillus licheniformis based on its morphological, physiological and biochemical characteristics and sequences alignment of its 16S rRNA.Optimized fermentation conditions for α-CGTase production. Based on trials for optional sources of carbon, nitrogen and minerals as additives and an orthogonal experiment for medium composition, the optimized liquid medium for the production of α-CGTase by B. licheniformis CLS403 was composed of soluble starch 14.1 g/L, soybean cake 8 g/L, NH4NO3 8 g/L, KH2PO4 0.5 g/L, MgSO47H2O 0.1 g/L, and CaCl 2 0.01 g/L with a final pH6.5. Incubated on a shaking bed (180 r/min) at 35°C for 96 h, 250-ml flasks with each containing 30 ml of the liquid medium inoculated with 1% seed culture (v/v) resulted a high level of a-CGTase production.Purification of a-CGTase. The specific activity of the a-CGTase produced by B. licheniformis CLS403 in liquid cultures was enhanced by 63 folds after a series of purification steps including ammonium sulfate precipitation, DEAE-Cellulose ion-exchange, and Sephacryl S-200 chromatography. Polyacrylamide gel electrophoresis (PAGE) analysis indicates that the purified enzyme was homogeneous with its subunit molecular mass being estimated as 123 kDa in SDS-PAGE.Characteristics of the purified a-CGTase. The optimal conditions for reaction of the purified a-CGTase produced by B. licheniformis CLS403 was pH 6.5 at 50°C. The enzyme was stable in a range of pH 6.0-8.0 at the temperature below 45°C. The Km value for its reaction on soluble starch was 3.04 mmol/L. The enzyme was inactivated at the presence of Ba2+, Mn2+ or Ca2+ at the level of 1 mmol/L whereas it was activated at the same level of Ag+, Hg2+ or SDS. However, Zn2+, Fe3+, Li+, Cr3+, Cu2+ and NaN3and two surfactants Tween 20 and Triton X-100 had insignificant effect on the activity of the a-CGTase concerned.
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