微泡菌ALW1重组β-半乳糖苷酶的异源表达和酶学性质

Heterologous Expression and Enzymatic Properties of Recombinant β-Galactosidase from Microbulbifer sp. ALW1

摘要:

β-半乳糖苷酶催化乳糖水解为葡萄糖和半乳糖，是乳制品加工中重要的酶。该研究将微泡菌ALW1菌株的β-半乳糖苷酶在大肠杆菌BL21（DE3）中进行异源表达和纯化，研究其酶学性质。结果表明，微泡菌ALW1的β-半乳糖苷酶属于GH1家族，利用Ni-NTA Agarose亲和层析获得的重组β-半乳糖苷酶分子量约为64 ku。重组酶的最适反应温度为30 ℃，最适pH为4.5。温度低于25 ℃、pH 4.0~5.0条件下，β-半乳糖苷酶具有良好稳定性。重组β-半乳糖苷酶对DTT、吐温20和吐温80具有良好的耐受性；离子型去垢剂SDS和CTAB存在时，β-半乳糖苷酶几乎丧失活性。重组β-半乳糖苷酶的Km和Vmax分别为10.98 mmol/L和7.48 U/mg。结构模拟显示，微泡菌β-半乳糖苷酶的催化酸/碱残基和亲核残基分别为Glu186和Glu370。该研究为来自微泡菌ALW1的β-半乳糖苷酶在食品领域的应用奠定理论基础。

Abstract:

β-Galactosidase, which catalyzes the hydrolysis of lactose into glucose and galactose, is one of the most important enzymes used in dairy processing. In this study, the β-galactosidase from Microbulbifer sp. ALW1 was heterologously expressed and purified in Escherichia coli BL21 (DE3), and the enzymatic properties of the purified enzyme were examined. The results showed that the β-galactosidase from Microbulbifer sp. ALW1 belonged to the glycoside hydrolase (GH) family 1. The recombinant β-galactosidase obtained by Ni-NTA agarose affinity chromatography had a molecular weight of 64 ku. The optimal reaction temperature and pH of the recombinant β-galactosidase were 30 ℃ and 4.5, respectively. The β-galactosidase exhibited good stability when the temperature was lower than 25 ℃ and pH was in the range of 4.0~5.0. The recombinant β-galactosidase had good tolerance to DTT, Tween 20, and Tween 80. In the presence of ionic detergents, SDS and CTAB, β-galactosidase almost lost its activity. The catalytic constants Km and Vmax of the recombinant β-galactosidase were 10.98 mmol/L and 7.48 U/mg, respectively. Structure simulation studies revealed that the catalytic acid/base residue was Glu186 and the nucleophilic residue was Glu370 for the β-galactosidase from Microbulbifer sp. ALW1. The results layed a theoretical foundation for the applications of β-galactosidase from Microbulbifer sp. ALW1 in the food field.