Search

2-O-α-D-glucopyranosyl-l-ascorbic acid (AA-2G) is a stable derivative of l-ascorbic acid (l-AA), which has been widely used in food and cosmetics industries. Sugar molecules, such as glucose and maltose produced by cyclodextrin glycosyltransferase (CGTase) during AA-2G synthesis may compete with l-AA as the acceptors, resulting in low AA-2G yield. Multiple sequence alignment combined with structural simulation analysis indicated that residues at positions 191 and 255 of CGTase may be responsible for the difference in substrate specificity. To investigate the effect of these two residues on the acceptor preference and the AA-2G yield, five single mutants Bs F191Y, Bs F255Y, Bc Y195F, Pm Y195F and Pm Y260F of three CGTases from Bacillus stearothermophilus NO2 (Bs), Bacillus circulans 251 (Bc) and Paenibacillus macerans (Pm) were designed for AA-2G synthesis. Under optimal conditions, the AA-2G yields of the mutants Bs F191Y and Bs F255Y AA-2G were 34.3% and 7.9% lower than that of Bs CGTase, respectively. The AA-2G yields of mutant Bc Y195F, Pm Y195F and Pm Y260F were 45.8%, 36.9% and 12.6% higher than those of wild-type CGTases, respectively. Kinetic studies revealed that the residues at positions 191 and 255 of the three CGTases were F, which decreased glucose and maltose specificity and increased l-AA specificity. This study not only proposes for the first time that the AA-2G yield can be improved by weakening the acceptor specificity of CGTase toward sugar byproducts, but also provides new insight on the modification of CGTase that catalyze the double-substrate transglycosylation reaction. [ABSTRACT FROM AUTHOR]

This study characterizes the acceptor specificity of levansucrases (LSs) from Gluconobacter oxydans (LS1), Vibrio natriegens (LS2), Novosphingobium aromaticivorans (LS3), and Paraburkholderia graminis (LS4) using sucrose as fructosyl donor and selected phenolic compounds and carbohydrates as acceptors. Overall, V. natriegens LS2 proved to be the best biocatalyst for the transfructosylation of phenolic compounds. More than one fructosyl unit could be attached to fructosylated phenolic compounds. The transfructosylation of epicatechin by P. graminis LS4 resulted in the most diversified products, with up to five fructosyl units transferred. In addition to the LS source, the acceptor specificity of LS towards phenolic compounds and their transfructosylation products were found to greatly depend on their chemical structure: the number of phenolic rings, the reactivity of hydroxyl groups and the presence of aliphatic chains or methoxy groups. Similarly, for carbohydrates, the transfructosylation yield was dependent on both the LS source and the acceptor type. The highest yield of fructosylated-trisaccharides was Erlose from the transfructosylation of maltose catalyzed by LS2, with production reaching 200 g/L. LS2 was more selective towards the transfructosylation of phenolic compounds and carbohydrates, while reactions catalyzed by LS1, LS3 and LS4 also produced fructooligosaccharides. This study shows the high potential for the application of LSs in the glycosylation of phenolic compounds and carbohydrates., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

The synthesis of complex oligosaccharides is desired for their potential as prebiotics, and their role in the pharmaceutical and food industry. Levansucrase (LS, EC 2.4.1.10), a fructosyl-transferase, can catalyze the synthesis of these compounds. LS acquires a fructosyl residue from a donor molecule and performs a non-Lenoir transfer to an acceptor molecule, via β-(2→6)-glycosidic linkages. Genome mining was used to uncover new LS enzymes with increased transfructosylating activity and wider acceptor promiscuity, with an initial screening revealing five LS enzymes. The product profiles and activities of these enzymes were examined after their incubation with sucrose. Alternate acceptor molecules were also incubated with the enzymes to study their consumption. LSs from Gluconobacter oxydans and Novosphingobium aromaticivorans synthesized fructooligosaccharides (FOSs) with up to 13 units in length. Alignment of their amino acid sequences and substrate docking with homology models identified structural elements causing differences in their product spectra. Raffinose, over sucrose, was the preferred donor molecule for the LS from Vibrio natriegens, N. aromaticivorans, and Paraburkolderia graminis. The LSs examined were found to have wide acceptor promiscuity, utilizing monosaccharides, disaccharides, and two alcohols to a high degree.

[Display omitted] • A molecular mechanism underlying the acceptor specificity of CGTases on ginsenosides is raised. • A series of ginsenosides with new structure features are produced by Bc CGTase. • The first report of a CGTase (Bc CGTase) that can glycosylate the C3-OH position of ginsenoside aglycons. Understanding the mechanisms of enzyme specificity is increasingly important from a fundamental viewpoint and for practical applications. Transglycosylation has attracted many attentions due to its importance in improving the functional properties of acceptor substrates both in vivo and in vitro. Cyclodextrin glucanotransferase (CGTase) is one of the key enzymes in transglycosylation, it has a broad substrate spectrum and utilizes sugar as the donor. However, little is known about the acceptor selectivity of CGTase, which greatly hampers efforts toward the rational design of desirable transglycosylated derivatives. In this study, we found that the CGTase from Bacillus circulans , Bc CGTase, was able to form glycosylated products with diverse ginsenosides. In particular, it not only carries out diverse mono-, di-, and even higher-order glycosylations via the transfer of glucose moieties to the C O Glc positions, but also can glycosylate the C3-OH position of ginsenosides. In contrast, another CGTase from Bacillus licheniformis (Bl CGTase) showed relatively specific acceptor preference with only several ginsenosides. Structural comparison between Bc CGTase and Bl CGTase revealed that the Arg74/K81 position within the acceptor-binding sites of Bc CGTase/ Bl CGTase was responsible for the differences in catalytic specificity for ginsenoside F1. Further mutagenesis confirmed their roles in the acceptor selection. In conclusion, our study not only demonstrates the acceptor selectivity of CGTases, but also provides insight into the catalytic mechanism of CGTases, which will potentially increase the utility of CGTase for biosynthesis of new, rationally designed transglycosylated derivatives. [ABSTRACT FROM AUTHOR]

Maple syrups with selected degree Brix (°Bx) (15, 30, 60) were investigated as reaction systems for levansucrase from Bacillus amyloliquefaciens . The enzymatic conversion of sucrose present in the maple syrup and the production of the transfructosylation products were assessed over a time course of 48 h. At 30 °C, the use of maple syrup 30°Bx led to the highest levansucrase activity (427.53 μmol/mg protein/min), while maple syrup 66°Bx led to the highest converted sucrose concentration (1.53 M). In maple syrup 30°Bx, oligolevans (10 < DP ≤ 30) were synthesized as major products (>80%). In maple syrup 66°Bx, the most abundant products were oligolevans at 30 °C and levans (DP ≥ 30) at 8 °C. The acceptor specificity study revealed the ability of B. amyloliquefaciens levansucrase to synthesize a variety of hetero-fructooligosaccharides (FOSs) in maple syrups 15°Bx and 30°Bx enriched with various disaccharides, with lactose being the preferred fructosyl acceptor. The current study is the first to investigate maple-syrup-based reaction systems for the synthesis of FOSs/oligolevans/levans. [ABSTRACT FROM AUTHOR]

Acceptor specificity for intermolecular transglucosylation reaction of amylomaltase from Corynebacterium glutamicum was investigated using starch as glucosyl donor and various saccharide acceptors. Maltooligosaccharides (G1–G4), mannose, palatinose and sucrose were efficient acceptors; the best one was glucose. This amylomaltase preferred hexose sugar containing the same configuration of C2-, C4- and C6-hydroxyl groups as glucopyranose. Palatinose was chosen as suitable acceptor for the synthesis of palatinose glucosides (PGs). The optimal condition was to incubate 5 U/ml amylomaltase with 7.5 mM palatinose and 1.0% (w/v) soluble potato starch at 30 °C for 24 h. In addition to PGs, maltooligosaccharides were also produced as by product. The product yield was 67.9%, in which the ratio of PGs to maltooligosaccharides was 1:1. Then PGs were separated by Bio-Gel-P2 column chromatography and analyzed by HPAEC. PG1–PG13 were identified with PG1 and PG2 as major products. NMR analysis showed that the PGs produced are novel products, PG1 and PG2 were a tri- and tetra-saccharide with the structure [ O -α- d -glucopyranosyl-(1→4)] n - O -α- d -glucopyranosyl-(1→6)- d -fructofuranose, where n = 1–2. PG was less sweet than palatinose and sucrose, more hygroscopic with similar prebiotic activity as palatinose. PGs thus have potential to replace sucrose or palatinose in food products for health benefits. [ABSTRACT FROM AUTHOR]

Streptococcus mutans dextran glucosidase (SmDG) belongs to glycoside hydrolase family 13, and catalyzes both the hydrolysis of substrates such as isomaltooligosaccharides and subsequent transglucosylation to form α-(1 → 6)-glucosidic linkage at the substrate non-reducing ends. Here, we report the 2.4 Å resolution crystal structure of glucosyl-enzyme intermediate of SmDG. In the obtained structure, the Trp238 side-chain that constitutes the substrate-binding site turned away from the active pocket, concurrently with conformational changes of the nucleophile and the acid/base residues. Different conformations of Trp238 in each reaction stage indicated its flexibility. Considering the results of kinetic analyses, such flexibility may reflect a requirement for the reaction mechanism of SmDG. [ABSTRACT FROM AUTHOR]

6-sulfate modified N-acetylglucosamine (6-sulfo-GlcNAc) is often found as part of many biologically important carbohydrate epitopes such as 6-sulfo-Le. In these epitopes, the 6-sulfo-GlcNAc moiety is extended by a galactose sugar in a β1-4 linkage. The β4GalT1 enzyme transfers galactose (Gal) from UDP-Gal to N-acetylglucosamine (GlcNAc) in the presence of manganese. Here we report that the β4GalT1 enzyme transfers Gal to the 6-sulfo-GlcNAc and 4-methylumbelliferyl-6-sulfo- N-acetyl-β-D-glucosaminide (6-sulfo-βGlcNAc-MU) acceptor substrates, although with very low efficiency. To understand the effect that the 6-sulfate group on the GlcNAc acceptor has on the catalytic activity of the β4GalT1 molecule, we have determined the crystal structure of the catalytic domain of bovine β4GalT1 mutant enzyme M344H-β4GalT1 complex with the 6-sulfo-GlcNAc molecule. In the crystal structure, the 6-sulfo-GlcNAc is bound to the protein in a way that is similar to the GlcNAc molecule. However, the 6-sulfate group engages in additional interactions with the hydrophobic region, residues 276-285, of the protein molecule, and this group is found wedged between the aromatic side chains of Phe-280 and Trp314 residues. Since the side chain of the Trp314 residue undergoes conformational changes during the catalytic cycle of the enzyme, molecular interaction between Trp314 and the 6-sulfate group might hinder this conformational change. Therefore, the lack of a favorable binding environment, together with hindrance to the conformational changes, might be responsible for the poor catalytic activity. [ABSTRACT FROM AUTHOR]

The production of levansucrase (LS) by thermophilic Geobacillus stearothermophilus was investigated. LS production was more effective in the presence of sucrose (1%, w/v) than fructose, glucose, glycerol or raffinose. The results ( Top 57°C; stable for 6 h at 47°C) indicate the high stability of the transfructosylation activity of G. stearothermophilus LS as compared with LSs from other microbial sources. Contrary to temperature, the pH had a significant effect on the selectivity of G. stearothermophilus LS-catalyzed reaction, favoring the transfructosylation reaction in the pH range of 6.0-6.5. The kinetic parameter study revealed that the catalytic efficiency of transfructosylation activity was higher as compared with the hydrolytic one. In addition to levan, G. stearothermophilus LS synthesized fructooligosaccharides in the presence of sucrose as the sole substrate. The results also demonstrated the wide acceptor specificity of G. stearothermophilus LS with maltose being the best fructosyl acceptor. This study is the first on the catalytic properties and the acceptor specificity of LS from G. stearothermophilus. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1405-1415, 2013 [ABSTRACT FROM AUTHOR]

Highlights: [•] Cellobiose phosphorylase mutants, C485A, Y648F, and Y648V, were produced. [•] C485A showed higher preference for d-glucosamine than the wild type. [•] Y648F more rapidly produced 4-O-β-d-glucopyranosyl-d-mannose than the wild type. [•] Y648V showed synthetic activity of 4-O-β-d-glucopyranosyl-N-acetyl-d-glucosamine. [ABSTRACT FROM AUTHOR]

Poly-N-acetyllactosamine extensions on N- and O-linked glycans are increasingly recognized as biologically important structural features, but access to these structures has not been widely available. Here, we report a detailed substrate specificity and catalytic efficiency of the bacterial β3-N-acetylglucosaminyltransferase (β3GlcNAcT) from Helicobacter pylori that can be adapted to the synthesis of a rich diversity of glycans with poly-LacNAc extensions. This glycosyltransferase has surprisingly broad acceptor specificity toward type-1, -2, -3 and -4 galactoside motifs on both linear and branched glycans, found commonly on N-linked, O-linked and I-antigen glycans. This finding enables the production of complex ligands for glycan-binding studies. Although the enzyme shows preferential activity for type 2 (Galβ1-4GlcNAc) acceptors, it is capable of transferring N-acetylglucosamine (GlcNAc) in β1-3 linkage to type-1 (Galβ1-3GlcNAc) or type-3/4 (Galβ1-3GalNAcα/β) sequences. Thus, by alternating the use of the H. pylori β3GlcNAcT with galactosyltransferases that make the β1-4 or β1-3 linkages, various N-linked, O-linked and I-antigen acceptors could be elongated with type-2 and type-1 LacNAc repeats. Finally, one-pot incubation of di-LacNAc biantennary N-glycopeptide with the β3GlcNAcT and GalT-1 in the presence of uridine diphosphate (UDP)-GlcNAc and UDP-Gal, yielded products with 15 additional LacNAc units on the precursor, which was seen as a series of sequential ion peaks representing alternative additions of GlcNAc and Gal residues, on matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. Overall, our data demonstrate a broader substrate specificity for the H. pylori β3GlcNAcT than previously recognized and demonstrate its ability as a potent resource for preparative chemo-enzymatic synthesis of complex glycans. [ABSTRACT FROM AUTHOR]

Abstract: The kinetic parameters and the product spectra of purified Bacillus amyloliquefaciens levansucrase (LS) were investigated in the present study. LS was highly transfructosylic than hydrolytic with high k cat value of 1136.5s−1 for its transfructosylation activity. Contrary to the hydrolytic activity, the kinetic of the transfructosylation activity of LS was best fitted to the Hill model with low Hill coefficients (n H , 0.74), indicating the negative binding cooperativity of substrates. These results reveal the competition of sucrose with the growing (oligo)polymer chain as a fructosyl acceptor. A shift of the transfructosylation reaction further towards the polymerization side was observed in the presence of raffinose as a fructosyl donor as compared to sucrose. The acceptor specificity studies reveal the ability of LS to synthesize a variety of hetero-fructooligosaccharides from various saccharides as fructosyl acceptors. Disaccharides were more favourable fructosyl acceptors as compared to monsaccharides. Contrary to disaccharides acceptor reactions, LS-catalyzed transfructosylation of monosaccharides did not yield a quasi equilibrium state. Contrary to lactose, galactose, xylose and glucose acceptor reactions, a lower accumulation of levan was detected in the presence of maltose. When sucrose and raffinose coexisted, they were both used as fructosyl donor, revealing the low acceptor specificity of LS towards raffinose. The structures of products were analysed NMR and MS. [Copyright &y& Elsevier]

Glycosylation can significantly improve the physicochemical and biological properties of small molecules like vitamins, antibiotics, flavors, and fragrances. The chemical synthesis of glycosides is, however, far from trivial and involves multistep routes that generate lots of waste. In this review, biocatalytic alternatives are presented that offer both stricter specificities and higher yields. The advantages and disadvantages of different enzyme classes are discussed and illustrated with a number of recent examples. Progress in the field of enzyme engineering and screening are expected to result in new applications of biocatalytic glycosylation reactions in various industrial sectors. [ABSTRACT FROM AUTHOR]

Abstract: Glycoside phosphorylases are a special group of carbohydrate-active enzymes, with characteristics in between those of glycoside hydrolases and glycosyl transferases. The phosphorylases from family GH-112 are exceptional because they employ galactose-1-phosphate instead of glucose-1-phosphate as glycosyl donor. Different acceptor specificities have been observed in this family, ranging from l-rhamnose to GlcNAc, GalNAc and a combination of the latter. Three new phosphorylases from previously unexplored branches of the phylogenetic tree of family GH-112 have now been characterized to shed more light on this divergence in acceptor specificity. The enzymes from Erysipelothrix rhusiopathiae and Streptobacillus moniliformis were found to prefer GalNAc as acceptor, while that from Anaerococcus prevotii displays similar activities on GalNAc and GlcNAc. These results confirm the correlation between the amino acid residue at position 162 and the enzyme''s specificity, i.e. a threonine in the former group and a valine in the latter. However, mutagenesis of residue 162 did not allow the rational transformation of the substrate preference, as the substitution of valine by threonine in the enzyme from Bifidobacterium longum did not tighten its specificity towards GalNAc. Unexpectedly, introducing an isoleucine at position 162 increased the preference for GlcNAc as acceptor, which illustrates that the structure–function relationships in β-galactoside phosphorylases are not yet completely understood. Several other positions have also been examined by mutational analysis but true determinants of the acceptor specificity in family GH-112 could not be identified. [Copyright &y& Elsevier]

The α-galactosidase from Aspergillus nidulans (AglC) belongs to a phylogenetic cluster containing eukaryotic α-galactosidases and α-galacto-oligosaccharide synthases of glycoside hydrolase family 36 (GH36). The recombinant AglC, produced in high yield (0.65 g·L−1 culture) as His-tag fusion in Escherichia coli, catalysed efficient transglycosylation with α-(1→6) regioselectivity from 40 mm 4-nitrophenol α-d-galactopyranoside, melibiose or raffinose, resulting in a 37–74% yield of 4-nitrophenol α-d-Gal p-(1→6)-d-Gal p, α-d-Gal p-(1→6)-α-d-Gal p-(1→6)-d-Glc p and α-d-Gal p-(1→6)-α-d-Gal p-(1→6)-d-Glc p-(α1→β2)-d-Fru f (stachyose), respectively. Furthermore, among 10 monosaccharide acceptor candidates (400 mm) and the donor 4-nitrophenol α-d-galactopyranoside (40 mm), α-(1→6) linked galactodisaccharides were also obtained with galactose, glucose and mannose in high yields of 39–58%. AglC did not transglycosylate monosaccharides without the 6-hydroxymethyl group, i.e. xylose,l-arabinose,l-fucose andl-rhamnose, or with axial 3-OH, i.e. gulose, allose, altrose andl-rhamnose. Structural modelling using Thermotoga maritima GH36 α-galactosidase as the template and superimposition of melibiose from the complex with human GH27 α-galactosidase supported that recognition at subsite +1 in AglC presumably requires a hydrogen bond between 3-OH and Trp358 and a hydrophobic environment around the C-6 hydroxymethyl group. In addition, successful transglycosylation of eight of 10 disaccharides (400 mm), except xylobiose and arabinobiose, indicated broad specificity for interaction with the +2 subsite. AglC thus transferred α-galactosyl to 6-OH of the terminal residue in the α-linked melibiose, maltose, trehalose, sucrose and turanose in 6–46% yield and the β-linked lactose, lactulose and cellobiose in 28–38% yield. The product structures were identified using NMR and ESI-MS and five of the 13 identified products were novel, i.e. α-d-Gal p-(1→6)-d-Man p; α-d-Gal p-(1→6)-β-d-Glc p-(1→4)-d-Glc p; α-d-Gal p-(1→6)-β-d-Gal p-(1→4)-d-Fru f; α-d-Gal p-(1→6)-d-Glc p-(α1→α1)-d-Glc p; and α-d-Gal p-(1→6)-α-d-Glc p-(1→3)-d-Fru f. [ABSTRACT FROM AUTHOR]

Sucrose phosphorylase is a bacterial transglucosidase that catalyzes conversion of sucrose and phosphate into α-D-glucose-1-phosphate and D-fructose. The enzyme utilizes a glycoside hydrolase-like double displacement mechanism that involves a catalytically competent β-glucosyl enzyme intermediate. In addition to reaction with phosphate, glucosylated sucrose phosphorylase can undergo hydrolysis to yield α-D-glucose or it can decompose via glucosyl transfer to a hydroxy group in suitable acceptor molecules, giving new α-D-glucosidic products. The glucosyl acceptor specificity of sucrose phosphorylase is reviewed, focusing on applications of the enzyme in glucoside synthesis. Polyhydroxylated compounds such as sugars and sugar alcohols are often glucosylated efficiently. Aryl alcohols and different carboxylic acids also serve as acceptors for enzymatic transglucosylation. The natural osmolyte 2- O-(α-D-glucopyranosyl)- sn-glycerol (GG) was prepared by regioselective glucosylation of glycerol from sucrose using the phosphorylase from Leuconostoc mesenteroides. An industrial process for production of GG as active ingredient of cosmetic formulations has been recently developed. General advantages of sucrose phosphorylase as a transglucosylation catalyst lie in the use of sucrose as a high-energy glucosyl donor and the usually weak hydrolase activity of the enzyme towards substrate and product. [ABSTRACT FROM AUTHOR]

The specificity of the aglycone-binding site of Escherichia coliα-xylosidase (YicI), which belongs to glycoside hydrolase family 31, was characterized by examining the enzyme's transxylosylation-catalyzing property. Acceptor specificity and regioselectivity were investigated using various sugars as acceptor substrates and α-xylosyl fluoride as the donor substrate. Comparison of the rate of formation of the glycosyl–enzyme intermediate and the transfer product yield using various acceptor substrates showed that glucose is the best complementary acceptor at the aglycone-binding site. YicI preferred aldopyranosyl sugars with an equatorial 4-OH as the acceptor substrate, such as glucose, mannose, and allose, resulting in transfer products. This observation suggests that 4-OH in the acceptor sugar ring made an essential contribution to transxylosylation catalysis. Fructose was also acceptable in the aglycone-binding site, producing two regioisomer transfer products. The percentage yields of transxylosylation products from glucose, mannose, fructose, and allose were 57, 44, 27, and 21%, respectively. The disaccharide transfer products formed by YicI, α-d-Xyl p-(1→6)-d-Man p, α-d-Xyl p-(1→6)-d-Fru f, and α-d-Xyl p-(1→3)-d-Fru p, are novel oligosaccharides that have not been reported previously. In the transxylosylation to cello-oligosaccharides, YicI transferred a xylosyl moiety exclusively to a nonreducing terminal glucose residue by α-1,6-xylosidic linkages. Of the transxylosylation products, α-d-Xyl p-(1→6)-d-Man p and α-d-Xyl p-(1→6)-d-Fru f inhibited intestinal α-glucosidases. [ABSTRACT FROM AUTHOR]

Glycogen debranching enzyme (GDE) has two distinct active sites for its 4-α-glucanotransferase and amylo-α-1,6-glucosidase activities. The GDE 4-α-glucanotransferases of mammals show stringent donor specificity; only α-glucans with an α-1,6–linked maltotetraosyl or maltotriosyl branch function as donors of a maltotriosyl or maltosyl residue. In this study, we investigated the acceptor specificity of the 4-α-glucanotransferases using methyl α-maltooligosides, p-nitrophenyl α-maltooligosides, and pyridylaminated maltooligosaccharides of various sizes as the acceptor substrates, and phosphorylase limit dextrin as the donor substrate. High-performance liquid chromatography analysis of the transfer products indicated that maltotriosyl and maltosyl residues were specifically transferred from phosphorylase limit dextrin to acceptors with a maltopentaosyl residue comprising a nonreducing-end. These results suggest that the acceptor binding sites in the active sites of mammalian GDE 4-α-glucanotransferases are composed of tandem subsites that are geometrically complementary to five glucose residues. [ABSTRACT FROM PUBLISHER]

Two glucuronyltransferases (GlcAT-P and GlcAT-S) are involved in the biosynthesis of HNK-1 carbohydrate, which is spatially and temporally regulated in the nervous system. To clarify the enzymatic properties of the respective glucuronyltransferases, we established an expression system for producing large amounts of soluble forms of flag-tagged human GlcAT-P and GlcAT-S in Escherichia coli. Approximately 15 and 6 mg of enzymatically active flag-GlcAT-P and flag-GlcAT-S were purified from E. coli cells in 5 liters of culture medium, respectively. These recombinant enzymes transferred GlcA to a glycoprotein acceptor, asialo-orosomucoid (ASOR), as well as a glycolipid acceptor, paragloboside. The specific activity of the recombinant GlcAT-P (1100 nmol/min/mg) toward a glycoprotein acceptor, ASOR, was comparable to that of the enzyme (4300 nmol/min/mg) purified from rat brain. Phosphatidylinositol (PI) is specifically required for expression of the activity of the recombinant enzymes toward a glycolipid acceptor, paragloboside. The recombinant GlcAT-P was highly specific for the terminal type II structure, Galβ1-4GlcNAc, while the recombinant GlcAT-S recognized not only the type II structure, Galβ1-4GlcNAc, but also the type I structure, Galβ1-3GlcNAc. These acceptor specificities were similar to those of the native enzymes. [Copyright &y& Elsevier]

The new fructosyltransferase (FTase) from Bacillus macerans EG-6 showed a broad acceptor specificity, and resulted in the formation of fructosylxyloside (FX) with d-xylose being the most effective acceptor. The optimal FTase concentration for FX production was 0.6 unit per g sucrose, which gave the highest transfer ratio, 83%, of fructosyl moiety from sucrose to d-xylose. Maximum yield of FX was 114 g l-1 with 200 g sucrose l-1 and 300 g d-xylose l-1. [ABSTRACT FROM AUTHOR]

To clarify the acceptor specificity of Fuc-TVII, its activity toward various analogs of a 2-(trimethylsilyl)ethyl α2,3-sialyl lacto-N-neotetraose, an acceptor for both Fuc-TVII and Fuc -TVI, was examined in comparison with that of Fuc-TVI. Fuc-TVII required three portions of α2,3-sialylated type-2 oligosaccharide structures (i.e., the hydroxyl group at C-4 of Gal, the hydroxyl group at C-3 of GlcNAc, and the carbonylamino group at C-2 of GlcNAc) for its acceptor recognition. Fuc-TVI required the carbonylamino group at C-2 of GlcNAc for its acceptor recognition. Fuc-TVI I showed higher affinity toward two analogs, in which the hydroxyl group at C-6 of GlcNAc has been deoxygenated and the acetamide group of N-acetylneuraminic acid has been replaced with a glycolylamino group, respectively, than that toward the original compound. On the other hand, Fuc-TVI showed higher affinity toward an analog, in which the acetamide group of GlcNAc has been modified with a lauroylamino group, than that toward the original compound. Analysis involving mass spectrometry confirmed that both Fuc-TVII and Fuc-TVI could fucosylate these three analogs to yield sialyl Lewis x derivatives. [ABSTRACT FROM AUTHOR]

The synthesis of complex oligosaccharides is desired for their potential as prebiotics, and their role in the pharmaceutical and food industry. Levansucrase (LS, EC 2.4.1.10), a fructosyl-transferase, can catalyze the synthesis of these compounds. LS acquires a fructosyl residue from a donor molecule and performs a non-Lenoir transfer to an acceptor molecule, via β-(2→6)-glycosidic linkages. Genome mining was used to uncover new LS enzymes with increased transfructosylating activity and wider acceptor promiscuity, with an initial screening revealing five LS enzymes. The product profiles and activities of these enzymes were examined after their incubation with sucrose. Alternate acceptor molecules were also incubated with the enzymes to study their consumption. LSs from Gluconobacter oxydans and Novosphingobium aromaticivorans synthesized fructooligosaccharides (FOSs) with up to 13 units in length. Alignment of their amino acid sequences and substrate docking with homology models identified structural elements causing differences in their product spectra. Raffinose, over sucrose, was the preferred donor molecule for the LS from Vibrio natriegens, N. aromaticivorans, and Paraburkolderia graminis. The LSs examined were found to have wide acceptor promiscuity, utilizing monosaccharides, disaccharides, and two alcohols to a high degree. [ABSTRACT FROM AUTHOR]

The transglycosylation activity of amylosucrase (ASase) has received significant attention owing to its use of an inexpensive donor, sucrose, and broad acceptor specificity, including glycone and aglycone compounds. The transglycosylation reaction of recombinant ASase from Deinococcus radiopugnans (DRpAS) was investigated using various phenolic compounds, and quercetin-3- O -rutinoside (rutin) was found to be the most suitable acceptor molecule used by DRpAS. Two amino acid residues in DRpAS variants (DRpAS Q299K and DRpAS Q299R), assumed to be involved in acceptor binding, were constructed by site-directed mutagenesis. Intriguingly, DRpAS Q299K and DRpAS Q299R produced 10-fold and 4-fold higher levels of rutin transglycosylation product than did the wild-type (WT) DRpAS, respectively. According to in silico molecular docking analysis, the lysine residue at position 299 in the mutants enables rutin to more easily position inside the active pocket of the mutant enzyme than in that of the WT, due to conformational changes in loop 4.