Levansucrases enzyme

Hestrin, Feingold, and Avigad (195a) found that the levansucrase enzyme system of Aerohacter levanicum which utilizes the D-fructose moiety of sucrose, forming a polysaccharide (levan) and D-glucose, possesses a complementary property of catalyzing the reversible transfer of the D-fruc-tosyl unit of jS-D-fructofuranosyl aldosides of different configurations to the anomeric carbon position of an aldose. 

Hestrin and Avineri-Shapiro98 have recently suggested a mechanism for levan production from sucrose and raffinose by levansucrase. The enzyme was used in the form of the autolyzate from A. levanicum (rendered sterile with chloroform and thymol9). This autolyzate was incubated for twenty-four hours at 37° with four volumes of 3% sucrose solution and one volume of phosphate buffer (pH 5.0). 

The exact nature of levansucrase activity08 is not clear. It differs in certain respects from invertase, polymerase, fructosaccharase, and phosphorylase. Possibly the aldoside part of the substrate molecule is replaced by an enzyme-linked group, and partial decomposition of this levan precursor to aldose and ketose may furnish the energy necessary for levan synthesis. 

L. K. Ozimek, S. Kralj, M. J. van der Maarel, and L. Dijkhuizen, The levansucrase and inulosucrase enzymes of Lactobacillus reuteri 121 catalyse processive and non-processive transglycosylation reactions, Microbiology, 152 (2006) 1187-1196. 

L. Hernandez, J. Arrieta, C. Menendez, R. Vazquez, A. Coego, V. Suarez, G. Selman, M. F. Petit-Glatron, and R. Chambert, Isolation and enzymic properties of levansucrase secreted by Acetobacter diazotrophicus SRT4, a bacterium associated with sugar cane, Biochem. J., 309(Pt 1), (1995) 113-118. 

Thiosucrose (98) was also a good inhibitor for the two enzymes which act on sucrose, the levansucrase from Bacillus subtilis and the yeast invertase [19]. 

That the same enzyme might catalyze transfer and hydrolytic reactions was suspected early and ultimately shown (16) for levansucrase however, the first clear demonstrations of this capacity in 1950 involved two well known hydrolases, yeast invertase (17, 18) and / -glucosidase (19). 

Fig. 8 Influence of affinity tags fused to levansucrase LevA773 on secretion by B. megaterium and enzyme activity. B. megaterium MS941 carrying the plasmids encoding LevA773 (lane 1),...

Levan has a backbone of p-(2 6) linked o-fructose and occurs as high molecular weight polysaccharide in microorganisms. It is accessible from sucrose by use of the enzyme levansucrase (sucrose 6-fructosyltransferase (FTF, EC 2.4.1.10)). Either culture broth of bacterial strains like Bacillus or Zymomonas or the cell-free supernatant can be used for the enzymatic reaction with sucrose. The molecular weight and the viscosity of levan depend on the strain used and the reaction conditions [131, 134]. Even though levan has interesting properties, it has never gained extensive industrial use up to now [134]. 

Levansucrase is a transfructosylase which converts sucrose to levan , a largely p(2 6)-linked fructofuranose polymer, and has been investigated because of its synthesis by oral bacteria. The levans and dextrans produced from dietary sucrose are sparingly water soluble and contribute to the build-up of dental plaque on the teeth. The enzyme from Bacillus subtilis was shown to have ping-pong kinetics,and the intermediacy of a covalent, fructosylated... 

The sucrose glycosyltransferases discussed in this section—sucrose phosphory-lase, B. subtilis levansucrase. Streptococcus glucosyltransferases, and Leuco-nostoc dextransucrase—share sucrose as a source of free energy in glucosyl or fructosyl transfer to an acceptor [ - 6600 cal/mol (146)]. The high specificity of the enzymes for sucrose has limited the variety of available substrates and consequently the extent of kinetic and mechanism analysis, although Chambert et al. (122) performed a remarkable series of kinetic experiments to establish the levansucrase kinetic mechanism. 

Bacillus subtilis levansucrase (sucrose 2,6-/8-D-fructan 6-)8-D-fructosyltrans-ferase, EC 2.4.1.10) catalyzes fructosyl transfer from sucrose to levan (154). In the absence of a fructosyl acceptor, the primary reaction is sucrose hydrolysis, although a limited amount of self-initiated levan synthesis occurs as well (155). As with sucrose phosphorylase, acceptor specificity is broad a number of saccharides and other nucleophiles are suitable fructosyl acceptors (154-158). The complete amino acid sequence of the approximately SO-kDa enzyme has been determined by both protein (159) and gene (160) sequence analyses. The three-dimensional structure at 3.8 A reveals a rod- or ellipsoid-shaped protein with a length some four times the diameter (161). 

There is no direct evidence on the form of the glycosyl-enzyme intermediate. Nonetheless, stabilization of a noncovalent oxocarbonium for the period between product fructose release and acceptor binding may not be realistic for slow reactions, given the extremely short lifetime of a glycosyl oxocarbonium ion (9). Levansucrase and particularly GTase-S are quite slow enzymes, with sucrose hydrolysis Heat of 48 sec 122) and 9.1 see" 213), respectively. Thus, the carbonium ion may well collapse to a more stable covalent complex or develop an equilibrium between the two forms. Nucleophilic catalysis is consistent with... 

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