Cyclodextrin glycosyltransferase: Its kinetics and overproduction in a T7 promoter system
Abstract
The major applications of cyclodextrins (CDs) are in food and pharmaceutical industries. Even though more than 850 metric tons of CDs were produced in 1989 and the production of CDs were expected to grow 7-fold by 1995, CDs are still fairly expensive especially $\alpha$- and $\gamma$-CDs. CDs are produced by cyclodextrin glycosyltransferase (CGTase) from starch by its cyclization reaction. CGTase from B. macerans was used as the model to examine the structure/function relationships of this class of biocatalyst. Because of the simultaneous production of more than one type of CD, the difficulties in isolating individual CD leads to their high production cost. This research shows the first detailed study of the inhibition kinetics of CGTase by substrate and its products. It was found that the CGTase reaction was inhibited at substrate concentration $>$0.6 mg/mL, and by all types of CDs. The product inhibition follows uncompetitive inhibition kinetics and the ordered bi bi mechanism. It was found that the present study supports the hypothesis that CGTase has at least two binding sites and the induced fit model. The cgt gene from B. macerans was isolated and amplified by polymerase chain reaction (PCR). Because of the low protein (CGTase) production from lac promoter system, the gene was isolated and subcloned into the expression vector. As a result, CGTase was overproduced in this system and most of the protein was produced intracellularly in the form of inclusion bodies. About 30% of the total cellular protein was produced as CGTase. By means of a simple process, the inactive CGTase was renatured to active CGTase. A higher CGTase production was obtained when the clones were cultivated in a tryptone-phosphate medium. Mutant CGTase were obtained by designing suitable pairs of oligonucleotides for PCR or by restriction enzyme digestion of the wild-type cgt gene. These were again cloned into the two systems and were confirmed with restriction map analysis. However, active mutant CGTases were not detected in any of these clones.
Degree
Ph.D.
Advisors
Tao, Purdue University.
Subject Area
Chemical engineering
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