从巨大芽孢杆菌中鉴定一种新型 GH36 α-半乳糖苷酶及其在棉子糖家族低聚糖降解中的应用。

Characterization of a novel GH36 α-galactosidase from Bacillus megaterium and its application in degradation of raffinose family oligosaccharides.

机构信息

Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, China.

School of Life Science and Technology, China Pharmaceutical University, Nanjing, China.

出版信息

Int J Biol Macromol. 2018 Mar;108:98-104. doi: 10.1016/j.ijbiomac.2017.11.154. Epub 2017 Nov 26.

DOI:10.1016/j.ijbiomac.2017.11.154

PMID:29183739

Abstract

A novel α-galactosidase gene (agaB) from Bacillus megaterium 3-7 was cloned and expressed in Escherichia coli. The gene coded for a protein with 741 amino acids and a calculated molecular mass of 85.4kDa. The native structure of the recombined AgaB was determined to be a homotrimer. AgaB showed the highest identity of 57% with the characterized glycosyl hydrolase family 36 α-galactosidase from Clostridium stercorarium F-9. The enzyme exhibited a specific activity of 362.6U/mg at 37°C and pH 6.8. The enzyme showed strong resistance to proteases and great tolerance to galactose (K=12.5mM). AgaB displayed wide substrate specificity toward pNPGal, melibiose, raffinose and stachyose, with a K of 0.42, 12.1, 17.0 and 25.4mM, respectively. Furthermore, AgaB completely hydrolyzed raffinose and stachyose present in soybean milk at 37°C within 4h when combined with trypsin. These favorable properties make AgaB a potential candidate for applications in the food and feed industries.

摘要

一株巨大芽孢杆菌 3-7 中新型的α-半乳糖苷酶基因（agaB）被克隆并在大肠杆菌中表达。该基因编码一个 741 个氨基酸的蛋白质，分子量为 85.4kDa。重组 AgaB 的天然结构被确定为三聚体。AgaB 与已鉴定的梭状芽胞杆菌 F-9 糖苷水解酶家族 36α-半乳糖苷酶的同源性最高，为 57%。该酶在 37°C 和 pH6.8 时的比活为 362.6U/mg。该酶对蛋白酶具有很强的抗性，对半乳糖的容忍度也很高（K=12.5mM）。AgaB 对 pNPGal、蜜二糖、棉子糖和水苏糖具有广泛的底物特异性，K 值分别为 0.42、12.1、17.0 和 25.4mM。此外，当与胰蛋白酶结合时，AgaB 能在 37°C 下 4 小时内完全水解豆浆中的棉子糖和水苏糖。这些优良的特性使 AgaB 成为食品和饲料工业应用的潜在候选者。