DOI: 10.14457/tu.the.2019.1304
title: Application of 4-alpha-glucanotransferases for the synthesis of functional glycosides and oligosaccharides

description: Cyclodextrin glycosyltransferase (CGTase) and amylomaltase (AMase) are the enzymes in 4-alpha-glucanotransferase family (4-alpha-GTase). Both enzymes play many important as a catalyst in transglycosylation reaction to produce functional glycosides and oligosaccharides. In this research, the main purpose of this study was to synthesize pinoresinol-alpha-D-glycosides (PGs) and xylose-alpha-D-glycosides (XGs) using recombinant CGTase and AMase, respectively. The influence of various parameters on the productions of PGs and XGs was investigated. For PGs, the optimal conditions for synthesizing were to incubate 0.5% (w/v) beta-cyclodextrin, 1.5% (w/v) pinoresinol and 80.0 U/mL of CGTase in 20 mM Tris-HCl buffer, pH 9.0 at 50 oC for 60 hours. Under these conditions, two PG transfer products were detected by TLC and mass spectrophotometer with molecular weights of 543.54 and 682.19 Daltons, corresponding to pinoresinol monoglycoside (PG1-I and PG1-II) and pinoresinol diglycoside (PG2), respectively. The PGs were also prepared in large scale and purified by preparative TLC and HPLC. The structures of PG1 and PG2 were confirmed as pinoresinol-alpha-D-glucopyranoside and pinoresinol-alpha-D-diglucopyranoside by 1H-NMR and 13C-NMR analysis. The free-radical scavenging and anti-inflammatory activities of PG1 and PG2 were comparatively studied with the original pinoresinol using DPPH radical scavenging reaction and beta-glucuronidase inhibition assay. The results found that the free-radical scavenging and anti-inflammatory activities of PG1 and PG2 reduced when compared to original pinoresinol. The loss of antioxidant and anti-inflammatory activities might result from the addition of sugar in the position 4-OH of pinoresinol that may have disturbed its electron rotation. However, in fact, the PGs need to be converted to the pinoresinols before they are absorbed and so loss of activity may be minimal. Interestingly, the alpha-glucosylated compounds which showed the better change of physicochemical properties such as solubility and sweetness could promote a positive effect on the bioavailability of original pinoresinol.In part of XGs, they were synthesized using tapioca starch (TS) and xylose through the transglycosylation reaction of recombinant AMase from p19bAMY cell. The optimal conditions for XGs synthesis were to incubate 1.5% (w/v) TS, 2% (w/v) xylose and 4 U/mL of AMase in 20 mM Tris-HCl buffer, pH 7.0 at 70 oC for 120 hours. Upon analysis of the products by TLC, at least 2-4 xylose glycosides were observed. These transferred products were formed with molecular weights of 335.10 and 497.20 Daltons as determined by mass spectrometry analysis; these values were in accordance with xylose monoglycoside (XG1) and xylose maltoside (XG2), respectively. The XG1 and XG2 were also prepared on a large scale and subsequently purified by preparative TLC and HPLC. The structure of XG1 and XG2 were confirmed as xylose-alpha-D-monoglucopyranoside and xylose-alpha-D-diglucopyranoside by 1H-NMR and 13C-NMR analysis. The long carbohydrate chains of xylose were less effective in sweetness property and acidic resistance than original xylose and sucrose. However, both XG1 and XG2 showed more solubility, maillard reaction, and prebiotic properties than original xylose. Thus, the glucosides of xylose have the potential of being incorporated into foods for health benefits.