Transglycosylation reaction catalyzed by cyclodextrin glycosyltransferase and amylomaltase for the synthesis of oligosaccharided with anticariogenetic property

Abstract

This work aims to synthesize maltosylsucrose using cyclodextrin glycosyltransferase (CGTase) from Paenibacillus sp. RB01 and recombinant amylomaltase from Corynebacterium glutamicum through the transglycosylation reaction with various glucosyl donors and sucrose acceptor. This is the first report on using amylomaltase for the synthesis of maltosylsucrose. In the analysis of product by TLC and HPLC on a Rezex RSO-Oligosaccharide column, the suitable glucosyl donor for CGTase and amylomaltase were soluble and raw tapioca starch, respectively. The optimal condition for the synthesis of maltosylsucrose from CGTase was 2.5% (w/v) sucrose, 20% (w/v) tapioca starch (soluble), 400 U/mL (dextrinizing unit) of CGTase at 40°C for 18 hours and that for amylomaltase was 2.0% (w/v) sucrose, 2.5% (w/v) tapioca starch (raw), 9 U/ml (disproportionation unit) of amylomaltase at 30°C for 48 hours. CGTase was more efficient than amylomaltase for this synthetic reaction in which the total percent yields of the transfer products from CGTase and amylomaltase were about 98% and 81%, respectively. The products were isolated by Biogel P-2 gel column, each peak was analyzed by HPLC and mass spectrometry. The major transfer products of both enzymes were G₂F, G₃F, G₄F and G₅F with different product ratio. The ratios were 1.0:1.2:1.2:1.3 and 1.0:0.9:0.8:0.7 for CGTase and amylomaltase, respectively. Higher ratio of larger products (G₇F, G₈F and > G₈F) could be obtained from both enzymes at low amounts of sucrose and enzyme. Low cariogenic property of maltosylsucrose products was confirmed by comparing the synthesis of water insoluble glucan, acid fermentation, plaque formation and cell aggregation of Streptococcus mutans to those exerted by sucrose. By adding sucrose to maltosylsucrose products in the ratios of 1:1, 1:2 and 1:4, inhibitory effects on glucosyltransferase activity of S. mutans by 7, 33 and 50% were observed. These results suggested that maltosylsucrose products were low cariogenic oligosaccharides.