Enhanced (+)-catechin transglucosylating activity of Streptococcus mutans GS-5 glucosyltransferase-D due to fructose removal.
The (+)-catechin transglucosylating activities of several glucosyltransferases (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 coli were studied. It was shown that after removal of alpha-glucosidase activity, GTF-D transglucosylated catechin with the highest efficiency. A maximal yield (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 sucrose (K(m), 13 mM) in 125 mM potassium phosphate, pH 6.0, at 37 degrees C. (1)H and (13)C 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 glucosyl transfer from sucrose to the acceptor competitively inhibited catechin transglucosylation (K(i), 9.3 mM), whereas glucose did not inhibit catechin transglucosylation. The addition of yeasts was studied in order to minimize fructose inhibition by means of fructose removal. For this purpose, the yeasts Pichia pastoris and the mutant Saccharomyces cerevisiae T2-3D were selected because of their inabilities to utilize sucrose. Addition of P. pastoris 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 GTF-D.
Meulenbeld
G H
GH
Division of Industrial Microbiology, Department of Food Technology and Nutritional Sciences, Wageningen University, 6700 EV Wageningen, The Netherlands. gerwin.meulenbeld@imb.ftns.wau.nl
Zuilhof
H
H
van Veldhuizen
A
A
van den Heuvel
R H
RH
Hartmans
S
S
D002392:Catechin / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005632:Fructose / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D005964:Glucosyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D006868:Hydrolysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D011994:Recombinant Proteins / Q000378:metabolism; D013295:Streptococcus mutans / Q000201:enzymology*
D011994:Recombinant Proteins; D005632:Fructose; D002392:Catechin; D005964:Glucosyltransferases; C113848:glucosyltransferase D; D005947:Glucose