Comparative characterization of complete and truncated forms of Lactobacillus amylovorus alpha-amylase and role of the C-terminal direct repeats in raw-starch binding.
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.
Laboratoire de Biotechnologie Microbienne Tropicale, Institut de Recherche pour le Développement, Montpellier, France.
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Rodriguez Sanoja
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Morlon-Guyot
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Jore
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Pintado
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Juge
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JP
Guyot
D001426:Bacterial Proteins; D017353:Gene Deletion; D006863:Hydrogen-Ion Concentration; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D008855:Microscopy, Electron, Scanning; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013213:Starch / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature; D020079:Terminal Repeat Sequences / Q000235:genetics*; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D013213:Starch; D000516:alpha-Amylases