Transglycosylation by Streptococcus mutans GS-5 glucosyltransferase-D: Acceptor specificity and engineering of reaction conditions

The acceptor specificity of Streptococcus mutans GS-5 glucosyltransferase-D (GTF-D) was studied, particular the specificity toward non-saccharide comp ounds. Dihydroxy aromatic compounds like catechol, 4-methylcatechol, and 3- methoxycatechol were glycosylated by GTF-D with a high efficiency. Transgly cosylation yields were 65%, 50%, and 75%, respectively, using 40 mM accepto r and 200 mM sucrose as glucosyl donor. 3-Methoxylcatchol was also glycosyl ated, though at a significantly lower rate. A number of other aromatic comp ounds such as phenol, 2-hydroxybenzaldehyde, 1,3-dihydroxybenzene, and 1,2- phenylethanediol were not glycosylated by GTF-D. Consequently GTF-D aromati c accepters appear to require two adjacent aromatic hydroxyl groups. In ord er to facilitate the transglycosylation of less water-soluble accepters the use of various water miscible organic solvents (cosolvents) was studied. T he flavonoid catechin was used as a model acceptor. Bis-2-methoxyethyl ethe r (MEE) was selected as a useful cosolvent. In the presence of 15% (v/v) ME E the specific catechin transglucosylation activity was increased 4-fold du e to a 12-fold increase in catechin solubility. MEE (10-30% v/v) could also be used to allow the transglycosylation of catechol, 4-methylcatechol, and 3-methoxycatechol at concentrations (200 mM) otherwise inhibiting GTF-D tr ansglycosylation activity. (C) 2000 John Wiley & Sons, Inc.