A thermostable α-galactosidase from Lactobacillus fermentum CRL722:: Genetic characterization and main properties
A thermostable α-galactosidase from Lactobacillus fermentum CRL722:: Genetic characterization and main properties
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DOI：
10.1007/s00284-005-0442-y
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发表时间：
2006-11-01
影响因子：
2.6
通讯作者：
Sesma, F.
中科院分区：
文献类型：
--
作者：
Carrera-Silva, E. A.;Silvestroni, A.;Sesma, F.
α -半乳糖苷酶（α - gal）是由melA基因编码的酶，它能水解糖（如棉子糖和水苏糖）中的α -1,6半乳糖苷键。这些α -半乳糖寡糖（α - gos）大量存在于蔬菜（如大豆）中，可导致敏感个体的胃肠道疾病，因为单胃动物（包括人类）肠道中不含α -半乳糖。使用微生物α - gal是消除大豆衍生产品中α - gos的一种有前途的替代方法。利用简并引物对发酵乳杆菌CRL722的melA基因进行了鉴定。利用聚合酶链反应技术获得了melA的全基因组序列（2223 bp）和melA两侧的基因，结果显示melA与嗜热微生物的α - gal基因有很强的相似性。克隆了L. fermentum CRL722的α - gal基因，并利用离子交换色谱、盐沉淀、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和超滤等技术从原菌株和重组菌株的无细胞提取物中纯化了α - gal基因；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范围内具有稳定性。因此，从L. fermentum CRL722中获得的耐热性α - gal可用于技术应用，例如去除豆制品中的α - gos。完整的melA基因也可以插入到其他微生物中，这些微生物可以在肠道的恶劣条件下存活并原位降解α - gos。这两种策略都将通过提高大豆衍生产品的营养价值来提高其总体可接受性。
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.