Enzymatic synthesis of low molecular weight amyloses with modified terminal groups

Abstract

Low molecular weight amyloses with modified terminal groups were synthesized by cyclomaltohexaose (α-cyclodextrin) transfer using (1→4)-α-d-glucan: 4-α-d-(1→4)-α-d-glucopyranosyltransferase (cyclising) (EC 2.4.1.9) from Bacillus macerans. 4-Nitrophenyl α-malto-oligosaccharides d.p. 2–7 served as acceptors, and cyclomaltohexaose served as the donor. The reaction was optimized to obtain a majority of species of definite chain lengths in a range of d.p. 10–20, depending upon the chain length of the acceptor.The course of the coupling reactions, as well as the action of the enzyme in disproportionation, cyclisation, and hydrolysis of the products, were observed by h.p.l.c. analysis of the oligomer distributions. Using a 15-fold molar excess of cyclomaltohexaose and 0.5 units enzyme per μmol of acceptor at pH 5.2, the chromatograms revealed that the products of the coupling reaction were predominant during the first reaction period. By incubating the acceptors with the enzyme, but without the donor, the mechanism of disproportionation was elucidated as a transfer of malto-oligosaccharyl residues dependent upon the substrate chain length. The minimum chain length required for a direct cyclisation reaction was d.p. 7. The results were confirmed by separation and investigation of the products of hydrolysis and cyclisation, which were nonmodified α-malto-oligosaccharides and cyclomalto-oligosaccharides.