| Due to the difficulty in product purification, production of cyclodextrin is time-consuming and money-consuming. In the study of cyclodextrin glycanotransferase [1,4-a-D-glucan 4-a-D-(1,4-a-D-glucano)-transferase (cyclizing); CGTase; EC2.4.1.19 ] , efforts were made to help to increase the efficiency but the achievements were insignificant.The enzyme reaction mechanism and the product specificity of CGTase were the hot point of CGTase study. More than 30 kinds of CGTase have been cloned and expressed heterogeneously. The relationship between the structure and the function of some CGTase have been studied on the genetic level to reconstruct the enzyme.In order to optimize the method of CD-producing, the CGTase from Bacillus circulans strain 251 was expressed on the surface of yeast which is descnbed in the first part of the paper. In the second part, a novel method of CGTase reconstruction introduced.In the first part of the experiment, the gene of cyclodextrin glucanotransferase (CGTase) from Bacillus circulans 251 was cloned into plasmid pYD1, which allowed regulated expression, secretion and detection. The expression of cgt with a-agglutinin at the N-terminal end on the extracellular surface of Saccharomyces cerevisiae was confirmed by immunofluorescence microscopy. This surface-anchored CGTase endowed the yeast with the ability to directly utilize starch as the sole carbon source and to produce the anticipated product—cyclodextrins as well as glucose and maltose. The resulting glucose and maltose which are efficient acceptors in the CGTase coupling reaction could be consumed by yeast fermentation and thus facilitated the cyclodextrins production. On the other hand, ethanol produced by yeast may form complex with CD and shift equilibrium in favor of cyclodextrin production. The yeast immobilized with CGTase produced 24.07 mg/ml cyclodextrins when incubated in yeast medium supplied with 4% starch. The proportion of β -CD in the product is increased from 64% to 75% which... |
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