Abstract
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 α-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°C.
1
H and
13
C nuclear magnetic resonance spectroscopy revealed the structures of two catechin glucosides, (+)-catechin-4′-
O
-α-
d
-glucopyranoside and (+)-catechin-4′,7-
O
-α-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.