Acceptor specificity and transglucosylation reaction of amylomaltase from Corynebacterium glutamicum

Abstract

Amylomaltase, a 4-α-glucanotransferase, catalyzes intramolecular transglucosylation reaction producing large-ring cyclodextrins (LR-CDs) and intermolecular transglucosylation reaction in which glucosyl units of starch or related oligosaccharides are transferred to suitable acceptors resulting in linear oligosaccharides or glucosides. This work aims at determination of specificity of acceptors in intermolecular transglucosylation reaction for the synthesis of glucoside products. The recombinant amylomaltase from Corynebacterium glutamicum was purified by His Trap affinity column. The purified enzyme showed a major protein band of 84 kDa on SDS-PAGE. Transglucosylation reaction catalyzed by amylomaltase using soluble potato starch as glucosyl donor and three types of acceptor: short chain alcohols, consisting of methanol, ethanol, propanol and butanol could not act as glucosyl acceptor at all concentrations tested. In contrast, glucose, maltose, maltotriose and maltotetraose were good saccharide acceptors indicating that this enzyme preferred hexose structure containing α-OH at C2, C4 and C6 with up to 4 glucose units Then glucoside products were analyzed by TLC and HPLC techniques. The glucoside products could not be detected when all of short chain alcohols and flavonoids (hesperidin, naringin, pinostrobin, fisetin, epicatechin and epigallocatechin gallate) were used as acceptor, but the products were detected when acceptors were maltooligosaccharides (G1-G4), mannose, sucrose and palatinose. Palatinose was chosen as suitable acceptor on account of possible getting new product and considerable amounts obtained. The optimal condition for synthesis of palatinose glucosides (PGs) was to incubate 5 U/ml amylomaltase with 7.5 mM palationse and 1.0% (w/v) soluble potato starch in 50 mM phosphate buffer. pH 6.0 at 30 ℃ for 24 hours and 67.2% yield was obtained. Then the reaction was up scaled and PGs were separated by Biogel-P2 column and analyzed by HPAEC. PG1-PG15 were detected with a trisaccharide PG1 as a main product. By MS and NMR, the size of PG was 504 Da and the linkage between palatinose and glucose unit was of the α- 1, 4 type The short chain and long chain PGs are less sweet than palatinose and sucrose, but have higher hygroscopic property than palatinose. However, prebiotic activity is the same. From PGs properties, they can thus be used to replace sucrose or palatinose in food and cosmetic products.