Site-directed mutagenesis of Cyclodextrin Glucanotransferase from Bacillus macerans：expression and characterization of mutant CGTase, W101F/L304W, D196H, D196N and D196Y.

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Cyclodextrin glucanotransferases（CGTase）（EC 2.4.1.19）catalyze the cyclization of starch to cyclodextrins（CD）, coupling and disproportionation reactions. All known CGTases produce a mixture of α-, β- andγ-CDs. In our previous study, a clear change in product specificity was observed in W101F/L304W CGTase. In this study, we used a recombinant plasmid-pHG, a shuttle vector, to express cgt gene in both Bacillus subtilis and Escherichia coli. Analysis of the products by HPLC revealed no altered specificity in the mutant（W101F/L304W-pHG）CGTase. In order to understand if there would be any host effects on the cgt gene expression, we produced CGTases from B. subtilis（pHG）and E. coli（pHG）, and then analyzed the expression products. The biochemical properties of purified enzymes were also determined. It was found that B. macerans and B. subtilis（pHG）were both able to secrete CGTases directly into medium, while E. coli（pHG）produced CGTase mainly in the periplasmic space. Some of the CGTase produced from E. coli was insoluble, with the greater insolubility found in E344Y mutant. E. coli secreted less （E344D-pHG）CGTase into periplasmin space and retained most of （E344Y-pHG）CGTase in the cytoplasm. The specific activity of β-CD coupling reaction by E. coli CGTase was higher（18.2 U/mg）than that by B. macerans（1.1 U/mg）or B. subtilis （1.3 U/mg）CGTase. Moreover, E. coli CGTase was less stable than the two mutant CGTases at 50∼60℃. These results indicate that CGTase produced from B. subtilis can be used to study the mechanism of the enzyme. It is proposed that the conserved amino acid residue Asp196 was located nearby the sugar binding subsite -3 of B. circulans strain 251 CGTase. To study the importance of the distant substrate binding subsite for the product ratios of α-, β- and γ-CD in the various CGTases, we have constructed three single mutants, D196H, D196N, D196Y, by using site-directed mutagenesis. The mutants showed changes in the high pH slopes of the optimum curve which resulted in profound decrease in the thermal stability for coupling when compared with the wild-type enzyme. The mutations affected the cyclization, coupling; disproportionation and hydrolyzing reactions of the enzyme. The initial major product of the wild-type CGTase and mutant CGTase catalyzed reaction wasα-CD. The mutant D196H and D196Y underwent a drastic change in its cyclization characteristics: they produced considerably more α and γ-CD from starch. Changes in sugar binding subsite -3 thus resulted in mutant proteins with changed CD production specificity. It is suggested that Asp196 function as a significant part of the subsite -3 for the binding of the substrate in CGTase and therefore the mutants might possibly change the ratio of the produced CDs by altering the affinities for glucose residue at these sugar binding subsites.