Purification and Characterization of a Third Glucosyltransferase from Streptococcus Mutans Serotype g

Abstract

Streptococcus mutans strain AHT (serotype g ) secretes at least two glucosyltransferases with different PI values. A novel glucosyltransferase with a PI of 5.8 was purified 244-fold from the ammonium sulphate fraction by DEAE-cellulose chromatography, FPLC (Mono Q column, Pharmacia) and hydrophobic chromatography. The enzyme preparation gave a single protein band on analysis by both PAGE and SDS-PAGE, and did not form multiple protein bands detectable by IEF. The M , was estimated to be about 130000 by SDS-PAGE and about 135000 by ultracentrifugal analysis. The apparent K , value and pH optimum of the enzyme were 3-9 f 0.2 mM (mean ~S D ) and about 4.7, respectively. The enzyme synthesized water-soluble glucan from sucrose, and the glucan consisted of over 90 mol% 1,6-a-~-glucosidic linkages. The enzyme activity was not stimulated by primer dextran. Anti-enzyme serum produced a single precipitin band with the purified enzyme preparation, whereas it did not react with either of the other two known glucosyltransferases.

Purification of a fourth glucosyltransferase from Streptococcus sobrinus

“…GTF-SiN synthesized a product with molecular weights smaller than 5,000, as described previously (23), while the product of GTF-Si was eluted in the void volume. We have already found that the product of GTF-SiN has only a 1,6-ot-Dglucoside linkage (23) and that the product of GTF-Si has 8.6% 1,3-cx-D-glucoside linkage (8). Although GTF-Sd is known to be accelerated by the addition of dextran as a primer, it is able to synthesize glucan even in the absence of a primer.…”

“…GTF-Si and GTF-Id and antisera against the enzymes were prepared as described previously (7,8). GTF-Sd was purified by a minor modification of the procedure of Mooser et al (15).…”

“…The resulting purified enzyme had 47 U of activity per mg at pH 6.0 and 37°C in the presence of 1 mg of dextran T10 per ml and no detectable dextranase activities. Antiserum against the purified enzyme was prepared as described previously (7,8).…”

“…These types were separated by DEAE-cellulose column chromatography; and the four GTFs were then designated P1, P2, P3, and P4 by their order of elution from the DEAE-cellulose column. We have previously purified P1 and P3 and have observed that P1 is a primer-independent, water-soluble glucan synthase (GTFSi) (8) and that P3 is a primer-dependent, water-insoluble glucan synthase (GTF-Id) (7). P4 is thought to be a primerdependent enzyme (GTF-Sd) which synthesizes highbranched, water-soluble glucan, as described previously (18).…”

Nucleotide sequence analysis of the gtfT gene from Streptococcus sobrinus OMZ176

“…Nucleotide sequence analyses of the three types of gtf genes derived from S. mutans GS5 have been completed by Kuramitsu and his coworkers (12,22,27). On the basis of previous biological studies (10,13,23,29,31), four types of gtf genes were expected to be isolated from S. sobrinus (Table 1). Russell et al (19,20) isolated the gtf7 and gtfS genes from S. sobrinus (which is now classified as Streptococcus downei) MFe28.…”