Specificity of glycosyl acceptor in coupling and transglycosylation reactions of cyclodextrin glycosyltransferase from Bacillus circulans A11

Abstract

Cyclodextrin glycosyltransferase (CGTase) from Bacillus circulans A11 is able to catalyze coupling and transglycosylation reactions. Characteristics of the intra-, and inter-molecular transglycosylations are the ability to form interesting and useful linear opened-chain oligosaccharides (LOCO) and glycosides. The CGTase-catalyzed coupling reaction using beta-CD as glycosyl donor and various acceptors were analyzed. When kinetic parameters were determined, the result shows that efficienct acceptors, saccharides or their derivatives or natural glycosides composed of the same configuration of C2-, C3-, and C4-OH as D- glucopyranoside with six carbon units sugar monomer such as glucose, sorbose, ascorbic acid, lactose, cellobiose, and hesperidin. Among LOCO-forming acceptors, cellobiose was the best acceptor according to its highest Vmax/Km values. Besides kinetic parameters, the transglycosylation yield which measured the consumption of acceptor by HPLCtechnique can also be used to follow how well the reaction proceeds. The transglycosylation yield of the coupling and transglycosylation reactions using beta-CD or soluble starch donor with the good acceptor candidates judging from kinetic values were compared. When cellobiose, sorbose, and glucose were used as glycosyl acceptor with beta-CD donor, the transglycosylation yield of 78, 57, and 54% were obtained. When glycosyl transfer was from soluble starch, good acceptors were sorbose and ascorbic acid with the transglycosylation yield of 63 and 57%, respectively. It was thus concluded that cellobiose was the best acceptor when using beta-CD as glycosyl donor in the coupling reaction whereas sorbose was the best when soluble starch was used as glycosyl donor in the transglycosylation reactions. The optimal condition for coupling reaction using cellobiose as the glycosyl acceptor with beta-CD donor was also determined.The condition was performed with 2% beta-CD, 0.5% cellobiose, incubated with 16 U of CGTaseat pH 6.0, 30 ํC for 2 hrs. The transglycosylation yield obtained was 78%, with two main transfer products, PC1 and PC2, observed at Rt of 3.81 and 4.42 min, respectively. The product ratio was 1:1 as determined by peak area. In the identification of the type and the structure of the transfer products, each was collected from the separate peak of HPLC chromatogram and subjected to mass spectrometer and NMR analyses. The molecular mass of PC1 and PC2, the two transfer products were 504 and 666 daltons. The structures suggested by NMR were a trisaccharide of the structure glc(alpha1->4) glc(beta1->4)glc and a tetrasaccharide of the structure glc(alpha1->4)glc(alpha1->4)glc(beta1->4)glc.