A thermostable α-galactosidase from Lactobacillus fermentum CRL722: Genetic characterization and main properties

Abstract

α-Galactosidase (α-Gal) enzyme, which is encoded by the melA gene hydrolyzes α-1,6 galactoside linkages found in sugars, such as raffinose and stachyose. These α-galacto-oligosaccharides (α-GOS), which are found in large quantities in vegetables, such as soy, can cause gastrointestinal disorders in sensitive individuals because monogastric animals (including humans) do not posses α-Gal in the gut. The use of microbial α-Gal is a promising alternative to eliminate α-GOS in soy-derived products. Using degenerate primers, the melA gene from Lactobacillus (L.) fermentum CRL722 was identified. The complete genomic sequence of melA (2223 bp), and of the genes flanking melA, were obtained using a combination of polymerase chain reaction-based techniques, and showed strong similarities with the α-Gal gene of thermophilic microorganisms. The α-Gal gene from L. fermentum CRL722 was cloned and the protein purified from cell-free extracts of the native and recombinant strains using various techniques (ion exchange chromatography, salt precipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and ultra-filtration); Its main biochemical properties were determined. The enzyme was active at moderately high temperatures (55°C) and stable at wide ranges of temperatures and pH. The thermostable α-Gal from L. fermentum CRL722 could thus be used for technological applications, such as the removal of α-GOS found in soy products. The complete melA gene could also be inserted in other micro-organisms, that can survive in the harsh conditions of the gut to degrade α-GOS in situ. Both strategies would improve the overall acceptability of soy-derived products by improving their nutritional value.