芽孢杆菌35家族β-半乳糖苷酶的性质及其在合成低聚半乳糖中的应用  

Characterization of GH Family 35β-Galactosidase from Bacillales sp.and Its Application in Synthesis of Galactooligosaccharides

作　　者：王建宇 李婷 李敬 江正强[2] 闫巧娟[1] WANG Jianyu;LI Ting;LI Jing;JIANG Zhengqiang;YAN Qiaojuan(College of Engineering/Key Laboratory of Food Bioengineering(China National Light Industry),China Agricultural University,Beijing 100083,China;College of Food Science and Nutritional Engineering,China Agricultural University,Beijing 100083,China)

机构地区：[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

出　　处：《食品科学技术学报》2023年第6期29-38,共10页Journal of Food Science and Technology

基　　金：国家自然科学基金资助项目(32172159)。

摘　　要：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60 kDa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o NP-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 novelβ-galactosidase from Bacillales sp.were investigated.A glycoside hydrolase(GH)family 35β-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 aβ-galactosidase from Bacillus circulans,and was a novelβ-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 60 kDa.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 o NP-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 35β-galactosidase from Bacillales sp.was potentially suitable for the preparation of galactooligosaccharides.

关 键 词：芽孢杆菌 Β-半乳糖苷酶 功能性低聚糖 酶学性质 低聚半乳糖 

分 类 号：TS245.9[轻工技术与工程—制糖工程]