Levan enzymic synthesis

One of the recent fundamental advances in carbohydrate chemistry has been enzymic synthesis in vtiro this has now been realized with dextran8 and with levan.8 However, since phosphate sugars are not involved in the enzymic syntheses of these two bacterial polysaccharides, it is obvious that phosphorylation is just one process for the natural synthesis of polysaccharides. 

Indirect evidence of the reversal of enzymic synthesis of levan has recently been obtained.10Ba Enzyme preparations from B. subtilis were shown to contain an enzyme capable of hydrolyzing levan (in addition to the levan-synthesizing enzyme) in a system composed of D-glucose, levan, yeast invertase (to hydrolyze sucrose formed) and the B. subtilis enzyme preparation. 

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. 

It is of interest that similar enzymes, capable of exchanging glycosidic linkages, appear to be involved in the synthesis of some polysaccharides. Thus the polysaccharide dextran is formed from sucrose by enzyme preparations from Leuconostoc46 while the polysaccharide levan is produced from sucrose or raffinose by enzymes of other bacteria.49 The reactions may be written as follows ... 

Thus when o-glucose was added to a solution containing levan, yeast invertase and an enzyme preparation from Bacillus subtUis, it was found that the decomposition of the levan became accelerated, presumably through the synthesis and decomposition of sucrose47 as follows ... 

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... 

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). 

The type of biosynthesis, exemplified by the levans and some dextrans, is generally associated with some Gram-positive organisms, usually cocci, and is the simplest type of saccharide synthesis to occur in bacteria. The enzymes involved are extracellular and do not require sugar nucleotide or lipid-phosphate sugars. 

Levan Synthesis. Oligosaccharides of the levan type (fructosyl-2-6-fructose) are synthesized by fructosidases. The synthesis of larger polysaccharides is accomplished by special enzymes that appear to have higher aflSnity for long chains than for short ones. Levan-synthesizing enzymes have been studied in preparations from Aerahacter levanicum. ... 

A second Gram positive system that has been studied in some detail is levansucrase synthesis in B. subtilis. This extracellular enzyme catalyses transfructorylation from sucrose to various acceptors which results in levan synthesis and sucrose hydrolysis. 

Inorganic phosphate is not required for levan synthesis, and it has no effect on the rate of reaction no esterification of phosphate can be detected. It is therefore considered that levan synthesis is the result of the transfer of fructose units from sucrose to a suitable acceptor through the mediation of a fructose-enzyme complex. 

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