The hydrolysis of maltodextrins by a β-amylase isolated from leaves of Vicia faba

Abstract

The action pattern of a β-amylase (α-1,4-glucan maltohydrolase, EC 3.2.1.2) which was extensively purified from extracts of leaves of Vicia faba has been investigated. The enzyme, which was purified by (NH 4) 2SO 4 fraction, precipitation with acetone and chromatography on Sephadex G-200 and Bio-gel P-100 columns had a specific activity of 100 μmoles/min per mg and was obtained in an over-all yield of 13%. The final preparation was homogeneous on the basis of polyacrylamide gel electrophoresis at several pH values, sedimentation in the ultracentrifuge and its elution profile on Sephadex G-200 columns. A symmetrical peak with s 20, w = 6.5 was found on ultracentrifugal sedimentation analysis. The molecular weight of the leaf β-amylase, calculated from data obtained by sucrose density gradient centrifugation, chromatography on Sephadex G-200 and ultracentrifugation, was 107 000. The purified enzyme had a Stokes radius of 31.8 Å and a diffusion constant of 6.49·10 −7 cm 2/s. Information obtained from a complete analysis of the amino acid composition indicated the presence of comparatively large amounts of aspartic and glutamic acids, and the partial specific volume was calculated to be 0.733. The action pattern of the purified leaf β-amylase upon hydrolysis of a number of maltodextrins was the same and was independent of the molecular size. The only products detected during the course of hydrolysis of maltodextrins with chain lengths of from 9 to 198 glucose residues were maltose and very small amounts of glucose and maltotriose. No evidence for the release of other intermediates was obtained, even when the hydrolysis was carried out essentially to completion. The extent of binding and rate of hydrolysis increased with chain length and reached a maximal level at a chain length of about 40–50 glucose units. The K m of the enzyme for linear maltodextrins decreased with chain-length up to about 50 glucose units, and with longer chain lengths it remained constant at about 1.4·10 −4M. The maximum velocity of the reaction was not significantly influenced by the chain-length of the maltodextrin used as substrate. The findings reported are consistent with a mechanism in which the maltodextrin combined with β-amylase to form a loose complex, which then rearranges to form a tightly bound intermediate which is hydrolyzed completely to maltose and small amounts of glucose and maltotriose.