A Levansucrase from Streptococcus mutans

Abstract

One of the predominant bacteria in early dental plaque, Streptococcus mutans, has been shown to synthesize levansucrase. This enzyme was obtained from the supernatant fluid of a glucose-broth culture. Purification of the enzyme was achieved in two steps by adsorption on hydroxylapatite followed by isoelectric focusing and fractionation. This resulted in a 10% yield and a 200-fold purification. Sucrose as well as raffinose could serve as substrate for the enzyme. In addition to levan (MW > 25 × 10⁶), free fructose and oligosaccharides were formed from sucrose. The enzyme had a pH optimum at 6.0, optimal activity around 40° C and isoelectric point at pH 4.2. Iodoacetamide, cysteine, Tris and sodium fluoride in a concentration of 0.05 m had no effect on the enzyme activity. The enzyme was totally inhibited by 0.1 mM Hg⁺⁺ and to about 40% by 1 mM Pb⁺⁺, Ag⁺, Ni⁺⁺ and Cu⁺⁺. EDTA in a concentration of 1 mM reduced the over-all activity of the enzyme by 75% and specifically interfered with the synthesis of levan. Most divalent cations could restore the activity of the enzyme in the presence of EDTA. The properties of this streptococcal levansucrase were similar to those previously described for levansucrases from Aerobacter levanicum and Bacillus subtilis. The finding, that EDTA interfered with the synthesis of levan, may give new possibilities in elucidating the mechanism of this enzyme reaction.

Purification of a novel fructosyltransferase from Lactobacillus reuteri strain 121 and characterization of the levan produced

“…reuteri levansucrase showed that there were two dominant size fractions present, one of approximately 150 kDa and one larger than 2 MDa. GFC analyses of the levan produced by S. mutans SacB [13] revealed only one size fraction with a molecular mass larger than 2 MDa. Two products with distinct sizes also have been observed for the levan of B. subtilis SacB [21].…”

“…The remaining fructose (as fructose-6-phosphate after HK action) was measured by adding phosphoglucose isomerase (PGI; Roche Biochemicals). Levansucrase activity was assayed at 37³C in a sodium acetate bu¡er (25 mM; pH 5.4) with 100 mM sucrose (unless stated otherwise) and 1 mM calcium chloride [10,11,13]. Samples were taken at 3 min time intervals and added to 1/10 volume 1 M sodium hydroxide to stop the reaction.…”

“…reuteri strain 121 were subjected to NMR and GFC analysis. The one-dimensional (1D) 13 C NMR spectrum of the FTF product (Fig. 1) displayed six carbon signals ( Table 2).…”

“…The C-2 resonance (N = 104.0) indicates the presence of L-fructofuranose. Comparison with the published 13 C chemical shifts of fructan produced by Lb. reuteri strain 121 grown on MRSr media [8] and Z. mobilis levan [14] indicated that the FTF fructan had a [C6)-L-D-Fruf-(2C] n structure, a levan.…”

Regulation of Synthesis of -Fructofuranosidase (Invertase) in Streptococcus mitis

“…glucosyl-and fructosyltransferases (EC 2.4.1 . 5 and EC 2.4.1 .lo) and p-fructofuranosidase (EC 3.2.1 .26) have been done with the oral streptococcus Streptococcus mutans (Carlsson, 1970;Tanzer, 1972;Fukui et al, 1974;Janda & Kuramitsu, 1978). The regulation of P-fructofuranosidase in strains of S. mutans has been studied by Tanzer et al (1973) who claimed that p-fructofuranosidase in S. mutans SL-1 was inducible by sucrose.…”