来自两歧双歧杆菌的具有转半乳糖基化特性的新型α-半乳糖苷酶：基因分子克隆与异源表达
A novel alpha-galactosidase from Bifidobacterium bifidum with transgalactosylating properties: gene molecular cloning and heterologous expression.
作者信息
Goulas Theodoros, Goulas Athanasios, Tzortzis George, Gibson Glenn R
机构信息
Department of Food Biosciences, School of Chemistry, Food Biosciences and Pharmacy, The University of Reading, P.O. Box 226, Whiteknights, Reading, RG6 6AP, UK.
出版信息
Appl Microbiol Biotechnol. 2009 Mar;82(3):471-7. doi: 10.1007/s00253-008-1750-5. Epub 2008 Nov 13.
A genomic library of Bifidobacterium bifidum (NCIMB 41171) DNA was constructed in Escherichia coli RA11r (melA(-)B(+)) and one alpha-galactosidase encoding gene was isolated. Conceptual translation combined with insertional mutagenesis analysis indicated an open reading frame (ORF) of 759 amino acid (aa) residues encoding an alpha-galactosidase (named as MelA) of 82.8 kDa. Partial purification and characterisation showed that the enzyme had an apparent native molecular mass of approximately 243 kDa and a subunit size of approximately 85 kDa. The enzyme belongs to glycosyl hydrolases 36 family with high aa sequence similarities (approximately 73%) to other known alpha-galactosidases of bifidobacterial origin. Under optimum pH conditions for activity (pH 6.0) and high melibiose concentration (40% w/v), the enzyme was able to form oligosaccharides with degree of polymerisation (DP) > or = 3 at higher concentration than DP = 2, with a total yield of 20.5% (w/w).
在大肠杆菌RA11r（melA(-)B(+)）中构建了两歧双歧杆菌（NCIMB 41171）DNA的基因组文库，并分离出一个α-半乳糖苷酶编码基因。通过概念性翻译结合插入诱变分析表明，一个由759个氨基酸残基组成的开放阅读框（ORF）编码了一种82.8 kDa的α-半乳糖苷酶（命名为MelA）。部分纯化和特性分析表明，该酶的表观天然分子量约为243 kDa，亚基大小约为85 kDa。该酶属于糖基水解酶36家族，与其他已知的双歧杆菌来源的α-半乳糖苷酶具有高度的氨基酸序列相似性（约73%）。在最佳活性pH条件（pH 6.0）和高蜜二糖浓度（40% w/v）下，该酶能够在高于聚合度（DP）= 2的浓度下形成聚合度（DP）≥ 3的寡糖，总产率为20.5%（w/w）。
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