The mechanism of polysaccharide production from sucrose. 3. Donor–acceptor specificity of levansucrase from Aerobacter levanicum

Abstract

An improved procedure for extraction of A. levanicum levansucrase and a micro test for levansucrase activity are described. Levan produced from sucrose by cell-free levansucrase solution was methylated. Examination by paper chromatography of the products of hydrolysis of the methylated polysaccharide indicate that it has a branched structure similar to that of levan from cultures of A. levanicum and other bacterial species. This conclusion was further supported by infrared analysis. The effect of levansucrase on representatives of several classes of oligofructosides was examined. All compounds examined with a terminal [beta]-fructofuranosidic group linked to the anomeric carbon of an aldose formed levan, whereas compounds with the same group linked to a carbinol carbon were not attacked by levansucrase. It is suggested that the fructosidic linkage in the former group, typified by sucrose, has the higher free-energy, content. Members of this class are therefore described by a high-energy symbol fr[image]R, whereas compounds of the second class are designated fr<R. Formation of levan from compounds of the type fr[image]R has been observed with R as follows: glucose, galactose, xylose, 6-[alpha]-galactosylglucose (melibiose), 6-B-fructosylglucose and 6-[alpha]-galactosylmelibiose. Substitution of the fructosidic moiety of fr[image]R at position C-1, C-3 or C-6 by a glycosyl group led to loss of the capacity to form levan. Formation of levan from fr[image]R was accompanied in all cases by concomitant formation of oligosaccharides (largely frn<fr[image]R), free fructose and free aldose (R). Constituents of a sucrose-levansucrase reaction mixture differed markedly from each other in activity as fructosyl acceptors. The acceptor activities (molar basis) were: waters sucrose L-[beta]-fructosylsucrose caverage terminal group of the growing levan molecule. The findings conform to the suggestion that the transformations catalysed by levansucrase comprise a readily reversible, step: fr[image]R + enzymefr[image]enzyme + R. This is followed by a not readily reversible step: fr[image]enzyme + acceptor[forward arrow]fr<acceptor + enzyme, where the acceptor can be either water or carbinol.