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.
Primers and probes were established from the sequences of the alpha-amylase genes (amyA) of L. amylovorus CIP 102989 and of L. plantarum A6 (Giraud and Cuny 1997). They were successfully used for the detection of the amyA gene in L. manihotivorans strain LMG 18010T and a 2842 bp region, containing the entire gene (2706 bp) with its putative promoter has been sequenced. More than 98% nucleotide sequence identities was found with L. amylovorus and L. plantarum amyA genes. The deduced amino acid sequence shares more than 96% amino acid sequence identities with L. amylovorus and L. plantarum alpha-amylases, and also 65% and 59% identities with the alpha-amylases of B. subtilis and S. bovis, respectively. The 3' terminal part of L. manihotivorans LMG 18010T amyA gene contained four repeated sequences (SRU). The amyA genes of the three lactobacilli species differed mainly in the number of SRU and in the size of the flanking regions of the SRU.
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