Comparative characterization of complete and truncated forms of Lactobacillus amylovorus alpha-amylase and role of the C-terminal direct repeats in raw-starch binding

Abstract

Two constructs derived from the alpha-amylase gene (amyA) of Lactobacillus amylovorus were expressed in Lactobacillus plantarum, and their expression products were purified, characterized, and compared. These products correspond to the complete (AmyA) and truncated (AmyADelta) forms of alpha-amylase; AmyADelta lacks the 66-kDa carboxyl-terminal direct-repeating-unit region. AmyA and AmyADelta exhibit similar amylase activities towards a range of soluble substrates (amylose, amylopectin and alpha-cyclodextrin, and soluble starch). The specific activities of the enzymes towards soluble starch are similar, but the K(M) and V(max) values of AmyADelta were slightly higher than those of AmyA, whereas the thermal stability of AmyADelta was lower than that of AmyA. In contrast to AmyA, AmyADelta is unable to bind to beta-cyclodextrin and is only weakly active towards glycogen. More striking is the fact that AmyADelta cannot bind or hydrolyze raw starch, demonstrating that the carboxyl-terminal repeating-unit domain of AmyA is required for raw-starch binding activity.

FIG. 1

Restriction map of pLPCR2-3 and pLPCR2-3ΔB/X plasmids. (A) pLPCR2-3 plasmid, carrying the 2.8-kb amyA gene of L. amylovorus, ligated at the BglII site of the pLPCR2 plasmid (dashed line). (B) pLPCR2-3ΔB/X plasmid derived from pLPCR2-3 after deletion of the 2-kb BamHI-XhoI fragment.

FIG. 2

Effect of pH and temperature on the amylase activities of AmyA (A) and AmyAΔ (B).

FIG. 3

Thermostabilities of AmyA (A) and AmyAΔ (B) at 75 (○), 65 (⧫), 55 (▴), and 45°C (■).

FIG. 4

Adsorption of AmyA (⧫) and AmyAΔ (▴) on raw starch. The linear adsorption isotherms indicate the apparent equilibrium distribution of enzymes between the solid phase (bound protein) and the liquid phase (unbound protein) at various protein concentrations.

FIG. 5

Scanning electron micrographs showing the effects of α-amylases produced by recombinant L. plantarum strains on raw corn starch granules after fermentation. Shown are native raw starch (A and B) and raw-starch granules after 24 (C and E) or 48 (G) h of fermentation with L. plantarum(pLPCR2-3ΔB/X) and after 24 (D and F) or 48 (H) h of fermentation with L. plantarum(pLPCR2-3).