(1.中国农业大学 工学院/中国轻工业食品生物工程重点实验室, 北京 100083;2.中国农业大学 食品科学与营养工程学院, 北京 100083)
(1.College of Engineering/Key Laboratory of Food Bioengineering (China National Light Industry), China Agricultural University, Beijing 100083, China;2.College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China)
低聚半乳糖是一种重要的益生元,芽孢杆菌(Bacillales sp.)来源的β-半乳糖苷酶具有转半乳糖苷活性,能合成低聚半乳糖。研究了一个新的芽孢杆菌来源的β-半乳糖苷酶的表达、酶学性质及其在合成低聚半乳糖中的应用。从芽孢杆菌中克隆了一个糖苷水解酶35家族的β-半乳糖苷酶基因(BABgal35A),并在大肠杆菌BL21(DE3)中表达。多重序列比对结果表明:BABgal35A与环状芽孢杆菌(Bacillus circulans)来源的β-半乳糖苷酶同源性最高,为79.9%,是一个新型的β-半乳糖苷酶(命名为BABgal35A)。粗酶液经Ni-IDA亲和层析纯化得到电泳级纯酶,蛋白电泳分析表明其分子质量为60kDa左右,最适pH值和温度分别为5.0和55℃,并且该酶在pH 值为4.5~8.0,温度为20~45℃时稳定性好。BABgal35A对oNP-β-galactopyranoside具有较高的催化活性。该酶以乳糖为底物合成低聚半乳糖的产率达34%。研究结果表明,芽孢杆菌35家族β-半乳糖苷酶在低聚半乳糖的制备中具有潜在的应用价值。
Galactooligosaccharides are important prebiotics. β-Galactosidases from Bacillales sp. can catalyze the transfer of galactoside, which can be used for the synthesis of galactooligosaccharides. The expression, enzymatic properties and application in the synthesis of galactooligosaccharides of a novel β-galactosidase from Bacillales sp. were investigated. A glycoside hydrolase (GH) family 35 β-galactosidase gene (BABgal35A) from Bacillales sp. was cloned and expressed in E. coli BL21(DE3). The results of multiple sequence alignment showed that BABgal35A shared the highest identity of 79.9% with a β-galactosidase from Bacillus circulans, and was a novel β-galactosidase (named BABgal35A). Crude enzyme was purified through Ni-IDA affinity chromatography to obtain electrophpresis-grade pure enzyme. SDS-PAGE showed that the molecular weight of purified enzyme was about 60kDa. The optimal pH and temperature of BABgal35A were determined to be 5.0 and 55℃, respectively. The enzyme was stable at pH 4.5-8.0 and temperature 20-45℃. BABgal35A exhibited the highest specific activity towards oNP-β-galactopyranoside. A maximum yield of 34% for galactooligosaccharides production was obtained by BABgal35A using lactose as substrate. The study showed that a GH family 35 β-galactosidase from Bacillales sp. was potentially suitable for the preparation of galactooligosaccharides.