Enhanced (+)-catechin transglucosylating activity of Streptococcus mutans GS-5 glucosyltransferase-D due to fructose removal

The (+)-catechin transglucosylating activities of several glucosyltransfera ses (GTFs) from the genus Streptococcus were compared. For this purpose, a mixture of four GTFs from Streptococcus sobrinus SL-1 and recombinant GTF-B and GTF-D from Streptococcus mutans GS-5 expressed in Escherichia coil wer e studied. It was shown that after removal of alpha-glucosidase activity, G TF-D transglucosylated catechin with the highest efficiency. A maximal yiel d (expressed as the ratio of moles of glucoside formed to moles of catechin initially added) of 90% was observed with 10 mM catechin and 100 mM sucros e (K-m, 13 mM) in 125 mM potassium phosphate, pH 6.0, at 37 degrees C. H-1 and C-13 nuclear magnetic resonance spectroscopy revealed the structures of two catechin glucosides, (+)-catechin-4'-O-alpha-D-glucopyranoside and (+) -catechin-4',7-O-alpha-di-D-glucopyranoside. Fructose accumulation during g lucosyl transfer from sucrose to the acceptor competitively inhibited catec hin transglucosylation (K-i, 9.3 mM), whereas glucose did not inhibit catec hin transglucosylation. The addition of yeasts was studied in order to mini mize fructose inhibition by means of fructose removal. For this purpose, th e yeasts Pichia pastoris and the mutant Saccharomyces cerevisiae T2-3D were selected because of their inabilities to utilize sucrose. Addition of P. p astoris or S. cerevisiae T2-3D to the standard reaction mixture resulted in a twofold increase in the duration of the maximum GTF-D transglucosylation rate. The addition of the yeasts also stimulated sucrose utilization by GT F-D.