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Various glycosyl hydrolase enzymes of the clan GH-H (http:// www.cazy.org/Glycoside-Hydrolases.html) catalyse transglycosylation reactions, mostly by using starch (e.g. , CGTase, family GH13) or sucrose (e.g. , glucansucrase, family GH70) as the donor substrate, thus transferring glucose. Some alternative biocatalysts for glycosyl transfer reactions have also been discovered; these include glycosynthases and glycosyltransferases. In recent years we have introduced the concept of sucrose analogues. Sucrose analogues such as a-d-glycopyranosyl bd-fructofuranosides have been reported as promising tools for the synthesis of functional fructo-oligosaccharides. In recent review articles we have suggested that these analogues can be used as glycopyranosyl donors for artificial transglycosylation reactions, which are not performed in nature. 9] Sucrose analogues are readily available from the cheap and abundant starting material sucrose. They are obtained through a transfructosylation reaction catalysed by levansucrase from Bacillus subtilis or Bacillus megaterium, by transferring fructose from sucrose to mannose, galactose, xylose or fucose. a-d-Allopyranosyl b-d-fructofuranoside (1, All-Fru, Scheme 1) is an allose that contains a sucrose analogue derived from sucrose by dehydrogenisation to 3-ketosucrose and subsequent hydrogenation to All-Fru. This synthesis has been established at an industrial scale. Allose is a rare sugar in nature; only a few plant species, for example, Passiflora edulis, Sideritis grandiflora and Protea rubropilosa, are known to contain glycoconjugates with blinked allose residues. Allose and allosylated glycoconjugates show promising properties for pharmaceutical use in cancer therapy, in immune suppression, for inhibition of neutrophil production and as antioxidants. To the best of our knowledge no a-d-allosyltransferase is known, however, research by Thorson and co-workers indicated a possible b-allosyl transfer performed by the glycosyltransferase OleD enzyme (family GT1). In recent work, we sought to identify enzymes able to transfer d-allose from the sucrose analogue All-Fru as the donor substrate. For this, All-Fru was incubated with various enzymes from the glycosyl hydrolase clan GH-H. As this sucrose analogue is a-linked and enzymes of the GH-H clan generally retain the configuration at the anomeric centre, the formed products should exhibit a-linkage. The sucrose isomerase (SI, GH13) from Protaminobacter rubrum showed no activity towards All-Fru. In contrast, incubation of All-Fru (292 mm) with amylosucrase (NpAs, GH13) from Neisseria polysaccharaea at pH 6.6 and 30 8C showed slow hydrolysis : even after 80 h, consumption of All-Fru was incomplete. Glucansucrases from Lactobacillus reuteri strains 121 (GTFA) and 180 (GTF180), and GTFR from Streptococcus oralis (all GH70) were able to hydrolyse 292 mm All-Fru. As hydrolysis activity on All-Fru was highest for GTFA (complete hydrolysis after 80 h), this enzyme was used in further studies. The glucansucrase GTFA from L. reuteri 121 is an extracellular enzyme that produces the polymer reuteran (mainly a(1!4)-linked glucan, with minor a(1!6) and branches) from sucrose. In contrast, with sucrose as donor, no formation of allose oligoor polysaccharides was observed by TLC. The transferase activity of GTFA-DN-CHis (a N-truncated version was used in all experiments) with All-Fru was observed with several acceptor substrates (note: concentration depended on solubility in the solvent system), thus obtaining a variety of functionalities (Table 1) such as sugars (methyl a-d-glucopyranoside (a-d-Me-Glcp, 2) and methyl 6-O-p-toluenesulfonyl-ad-glucopyranoside (a-d-Me-6-Ts-Glcp, 4)), amino acids (e.g. , N-(tert-butoxycarbonyl)-d-serine methyl ester (N-Boc-d-serineOMe, 6)) and ( )-epicatechin (8). Incubating GTFA with All-Fru (292 mm) and a-d-Me-Glcp (772 mm, 2) as the acceptor resulted in methyl a-d-allopyranosyl-(1!4)-a-d-glucopyranoside (3) as the main product (50% yield). The a-configuration of the glycosidic bond was determined by H NMR (H-1’: d= Scheme 1. The sucrose analogue All-Fru.

Two long-standing questions about glucansucrases (EC 2.4.1.5) are how they control oligosaccharide versus polysaccharide synthesis and how they direct their glycosidic linkage specificity. This information is required for the production of tailor-made saccharides. Mutagenesis promises to be an effective tool for enzyme engineering approaches for altering the regioselectivity and acceptor substrate specificity. Therefore, we chose the most conserved motif around the transition state stabilizer in glucansucrases for a random mutagenesis of the glucansucrase GTFR of Streptococcus oralis, yielding different variants with altered reaction specificity. Modifications at position S628 achieved by saturation mutagenesis guided the reaction toward the synthesis of short chain oligosaccharides with a drastically increased yield of isomaltose (47%) or leucrose (64%). Alternatively, GTFR variant R624G/V630I/D717A exhibited a drastic switch in regioselectivity from a dextran type with mainly alpha-1,6-glucosidic linkages to a mutan type polymer with predominantly alpha-1,3-glucosidic linkages. Targeted modifications demonstrated that both mutations near the transition state stabilizer, R624G and V630I, are contributing to this alteration. It is thus shown that mutagenesis can guide the transglycosylation reaction of glucansucrase enzymes toward the synthesis of (a) various short chain oligosaccharides or (b) novel polymers with completely altered linkages, without compromising their high transglycosylation activity and efficiency.