title: A thermostable alpha-galactosidase from Lactobacillus fermentum CRL722: genetic characterization and main properties.
abstract: Alpha-galactosidase (alpha-Gal) enzyme, which is encoded by the melA gene hydrolyzes alpha-1,6 galactoside linkages found in sugars, such as raffinose and stachyose. These alpha-galacto-oligosaccharides (alpha-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 alpha-Gal in the gut. The use of microbial alpha-Gal is a promising alternative to eliminate alpha-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 alpha-Gal gene of thermophilic microorganisms. The alpha-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 degrees C) and stable at wide ranges of temperatures and pH. The thermostable alpha-Gal from L. fermentum CRL722 could thus be used for technological applications, such as the removal of alpha-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 alpha-GOS in situ. Both strategies would improve the overall acceptability of soy-derived products by improving their nutritional value.
authors.affiliation: Centro de Referencia para Lactobacilos (CERELA-CONICET), Chacabuco 145, San Miguel de Tucumán, Tucumán, T4000ILC, Argentina.
authors.forename: E A
authors.initials: EA
authors.lastname: Carrera-Silva
authors.forename: A
authors.initials: A
authors.lastname: Silvestroni
authors.forename: J G
authors.initials: JG
authors.lastname: LeBlanc
authors.forename: J-C
authors.initials: JC
authors.lastname: Piard
authors.forename: G
authors.initials: G
authors.lastname: Savoy de Giori
authors.forename: F
authors.initials: F
authors.lastname: Sesma
mesh_terms: D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D052197:Limosilactobacillus fermentum / Q000201:enzymology*; D009844:Oligosaccharides / Q000378:metabolism; D013696:Temperature; D000519:alpha-Galactosidase / Q000235:genetics* / Q000378:metabolism
chemical_list: D009844:Oligosaccharides; D000519:alpha-Galactosidase