Soo In Ryu

Biochemical characterisation of a glycogen branching enzyme from Streptococcus mutans: Enzymatic modification of starch.

A gene encoding a putative glycogen branching enzyme (SmGBE) in Streptococcus mutans was expressed in Escherichia coli and purified. The biochemical properties of the purified enzyme were examined relative to its branching specificity for amylose and starch. The activity of the approximately 75kDa enzyme was optimal at pH 5.0, and stable up to 40°C. The enzyme predominantly transferred short maltooligosyl chains with a degree of polymerization (dp) of 6 and 7 throughout the branching process for amylose. When incubated with rice starch, the enzyme modified its optimal branch chain-length from dp 12 to 6 with large reductions in the longer chains, and simultaneously increased its branching points. The results indicate that SmGBE can make a modified starch with much shorter branches and a more branched structure than to native starch. In addition, starch retrogradation due to low temperature storage was significantly retarded along with the enzyme reaction.

Characterization of UDP-glucose 4-epimerase from Pyrococcus horikoshii: regeneration of UDP to produce UDP-galactose using two-enzyme system with trehalose.

A gene encoding a putative UDP-glucose 4-epimerase (pGALE) in Pyrococcus horikoshii was cloned and expressed in Escherichia coli. The purified enzyme could reversibly catalyze both the synthesis of UDP-Gal and UDP-Glc but preferred the binding of UDP-Gal by approximately 10-fold. The optimum pH and temperature were 6.5 and 65°C. The enzyme acted effectively without the addition of nicotinamide adenine dinucleotide (NAD(+)), possibly due to the presence of tightly bound NAD(+). In particular, pGALE could be coupled with trehalose synthase (TreT) from P. horikoshii to regenerate UDP-Gal from UDP. The possible byproduct of glycosyltransferase, UDP, was capable of being converted to UDP-Glc with trehalose by TreT, and UDP-Glc was simultaneously converted to UDP-Gal by pGALE. Conclusively, the results suggest that pGALE and TreT with trehalose is an effective one-pot two-enzyme system for the regeneration of UDP-Gal, a high-cost substrate of galactosyltransferase, to complete a sugar nucleotide cycle.

Synthesis of nucleotide sugars and α-galacto-oligosaccharides by recombinant Escherichia coli cells with trehalose substrate

Useful nucleoside diphosphate (NDP)-sugars and α-galacto-oligosaccharides were synthesized by recombinant Escherichia coli whole cells and compared to those produced by enzyme-coupling. Production yields of NDP-glucoses (Glcs) by whole cells harboring trehalose synthase (TS) were 60% for ADP-Glc, 82% for GDP-Glc, and 27% for UDP-Glc, based on NDP used. Yield of UDP-galactose (Gal) by the whole-cell harboring a UDP-Gal 4-epimerase (pGALE) was 26% of the quantity of UDP-Glc. α-Galacto-oligosaccharides, α-Gal epitope (Galα-3Galβ-4Glu) and globotriose (Galα-4Galβ-4Glu), were produced by the combination of three recombinant whole cells harboring TS, pGALE, and α-galactosyltransferase, with production yields of 48% and 54%, based on UDP, respectively. Production yields of NDP-sugars and α-galacto-oligosaccharides by recombinant whole-cell reactions were approximately 1.5 times greater than those of enzyme-coupled reactions. These results suggest that a recombinant whole-cell system using cells harboring TS with trehalose as a substrate may be used as an alternative and practical method for the production of NDP-sugars and α-galacto-oligosaccharides.

Glucosylation of flavonol and flavanones by Bacillus cyclodextrin glucosyltransferase to enhance their solubility and stability

Enzymatically modified isoquercitrin (EMIQ), oligoglucosyl naringenin-7-(glucose [G]), and oligoglucosyl hesperetin (H)-7-G were produced via oligoglucosylation of quercetin-3-glucose, naringenin-7-G (prunin), and H-7-G, respectively, by cyclodextrin glucosyltransferase from Bacillus macerans. The aim was to explore the oligoglucosylation and the resulting changes in physicochemical properties. Water solubility of EMIQ, oligoglucosyl prunin, and oligoglucosyl H-7-G enormously increased in comparison with that of their aglycones. Glycosylation of an aglycone generally enhances its solubility. Resistance of the aglycones to oxidative degradation by the Cu2+ ion was strongly increased by the oligoglucosylation. This is probably because oligoglucosylation may protect sensitive parts of an aglycones molecule from the Cu2+ oxidation. Only EMIQ maintained its structure during thermal treatment much longer than quercetin did. Degradation of flavonoid aglycones by ultraviolet light C irradiation at 254nm was not affected, and their antioxidant activities gradually decreased with the greater extent of oligoglucosylation.

Coupling reactions of trehalose synthase from Pyrococcus horikoshii: cost-effective synthesis and anti-adhesive activity of α-galactosyl oligosaccharides using a one-pot three-enzyme system with trehalose.

A new sugar nucleotide cycling (SNC) process was established in a one-pot three enzyme-coupled reaction using disaccharide trehalose. Trehalose synthase from Pyrococcus horikoshii could be applied to the SNC process for the synthesis of functional α-galactosyl oligosaccharides, α-galactose (Gal) epitopes and globotriose, using the effective regeneration of UDP-Gal. The α-Gal epitopes and globotriose were found to attach to the cell-surface of enteropathogenic Escherichia coli O127 (EPEC) which were bound to human Caco-2 cells. These α-galactosyl oligosaccharides were able to prohibit the attachment of EPEC, which could have resulted in colonization and disease. The α-Gal epitope III with a lactulose acceptor showed the most inhibitory activity of anti-adhesion. The results suggest that the α-galactosyl oligosaccharides may be alternative anti-adhesion molecules that overcome antibiotic resistance.

Enzymatic Synthesis and Properties of Trehalose Analogues as Disaccharide and Trisaccharide

Abstract Trehalose analogue, non-reducing dissacharide of 1-α-D-glucopyranosyl α-D-galactopyranoside, was synthesized by Pyrococcus horikoshii glycosyltransferase transglycosylation reaction with sugar nucleotides and galactose. This disaccharide analogue was effective inhibitor for several disaccharidases including rat intestinal trehalase and sucrase. Trehalose was also modified by Escherichia coli β-galactosidase transglycosylation reaction with lactose to give trehalose trisaccharide analogues. These trisaccharide analogues have been supposed to be indigestible oligosaccharides exhibiting enhanced hygroscopic, cryoprotective, anti-cariogenic, and prebiotic effects. The enzymatic techniques using glycosyltransferase and glycosidase might lead to create more trehalose-based analogues with a wide variety of acceptor and donor sugars.