Breeding, Identification And Optimized Fermentation Of An Alpha Cyclomaltodextrin Glucanotransferase Producing Strain And Its Enzymatic Characterization

Cyclodextrin(cyclodextrin, CD) is circular, nonreducing oligosaccharide, formed by the connection of α-1,4- glycosidic bond. Cyclodextrin is usually composed of 6, 7 or 8 glucose residues, are known as the alpha, beta, and gamma cyclodextrin. In addition to these three kinds of cyclodextrin, there are some cyclodextrins, such as delta, epsilon, zeta, eta cyclodextrin(composed of 9~12 glucose units), which were rarely used in bussiness. Cyclodextrin can be embedded a variety of chemical substances in, leading to a change in the physical and chemical properties of these compounds. Due to the propertie, cyclodextrins were so widely used in the pharmaceutical, food, chemical, cosmetics, industry and agriculture as well as in analytical chemistry. The cyclodextrin was mainly prepared by enzyme rather than chemical synthesis. But the production by enzyme was a mixture of a variety of cyclodextrins, which was not beneficial to separation and purification of cyclodextrin. Therefore, producting a specific type of CGTase is very meaningful, using the microorganism fermentation.In this study, a bacterial strain capable of producing α-CGTase was obtained. 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:Isolaiton, breeding and indentfication of Y112. The strain 112 was found producing α-CGTase at the highest level. Thus, the strain 112 was selcted as an intial strain to be exposed to chemical pressure of nitrosoguanidine and rifampicin for further mutation for better production of the α-CGTase. An eventually selected mutant named Y112 was capable of produting the α-CGTase 37% times more than the initial strain. The mutant strain Y112 was finally identified as Bacillus based on its morphological, physiological and biochemical characteristics and sequences alignment of its 16 S r DNA.Optimized fermentation coditions for α-CGTase production. Based on the single factor experiment and the response surface experiment for fermention coditions, the optimited liquid medium for the production of α-CGTase by Y112 was composed of corn starch 7.5 g/L, peptone 17.8 g/L, yeast extract 9.1 g/L, Mn2+ 0.6 mmol/L, Na Cl 50 g/L, Na2CO3 15 g/L(p H 9.6). Incubated on a shaking bed(230 r/min) at 27 oC for 50 h, 250 m L flasks with each containing 25 m L of the liquid medium inoculated with 4% seed culture(v/v) result a high level of α-CGTase production.Purificaiton of α-CGTase. The specific acivity of the α-CGTase produced by Y112 in liquid cultures was enhanced by 12.4 folds after a series of purification steps including ethanol precipitation, Superdex chromatography and DEAE-Sepharose ion-exchange. Polyacrylamide gel electrophoresis(PAGE) analysis indicates that the purified enzyme was homogeneous with its subunit molecular mass being estimated as 90 k Da in SDS-PAGE.Characteristics of the purified α-CGTase. The optimal conditions for reaction of the purified α-CGTase produced by Y112 was p H 8.5 at 55 oC. The enzyme was stable in range of p H 7.0-9.0 at the temperature below 45 oC. The Km and Vmax values using soluble starch were estimated to be 5.5 g/L and 0.25 μmol/L/min, respectively. The α-CGTase was inactivated in under the action of Ag2+,whereas it was activated in under the action of Mn2+, Mg2+.