Carrera-Silva E A, Silvestroni A, LeBlanc J G, Piard J-C, Savoy de Giori G, Sesma F
Centro de Referencia para Lactobacilos (CERELA-CONICET), Chacabuco 145, San Miguel de Tucumán, Tucumán, T4000ILC, Argentina.
Curr Microbiol. 2006 Nov;53(5):374-8. doi: 10.1007/s00284-005-0442-y. Epub 2006 Oct 16.
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.
α-半乳糖苷酶（α-Gal）由melA基因编码，可水解存在于棉子糖和水苏糖等糖类中的α-1,6半乳糖苷键。这些α-半乳糖寡糖（α-GOS）大量存在于大豆等蔬菜中，会使敏感个体出现胃肠道紊乱，因为单胃动物（包括人类）肠道中不存在α-Gal。使用微生物α-Gal是消除大豆制品中α-GOS的一种有前景的替代方法。通过简并引物，鉴定出来自发酵乳杆菌CRL722的melA基因。使用基于聚合酶链反应的多种技术组合，获得了melA（2223 bp）及其侧翼基因的完整基因组序列，其与嗜热微生物的α-Gal基因具有高度相似性。克隆了来自发酵乳杆菌CRL722的α-Gal基因，并使用多种技术（离子交换色谱、盐析、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和超滤）从天然菌株和重组菌株的无细胞提取物中纯化出该蛋白；测定了其主要生化特性。该酶在中等高温（55℃）下具有活性，在较宽的温度和pH范围内稳定。因此，来自发酵乳杆菌CRL722的热稳定α-Gal可用于技术应用，如去除大豆制品中发现的α-GOS。完整的melA基因也可插入其他能在肠道恶劣条件下存活的微生物中，以便在原位降解α-GOS。这两种策略都将通过提高大豆制品的营养价值来提高其整体可接受性。
