摘要
从克隆巴伦葛兹类芽孢杆菌CAU904中克隆得到一个β-葡萄糖苷酶基因,并在大肠杆菌中进行了异源表达,研究了重组酶的酶学性质,进一步解析了该酶的晶体结构。研究结果表明,该酶的最适温度和pH值分别为50℃和7. 5。该酶底物特异性较广,能够水解β-1,2、β-1,3、β-1,4、β-1,6-糖苷键等在内的多种糖苷键,对昆布多糖、大麦β-葡聚糖和地衣多糖等聚糖均有水解作用。晶体结构信息表明,该酶呈现糖苷水解酶(GH) 3家族典型的多结构域结构,其催化口袋由类似(α/β)_5的三明治结构域和(β/α)_8折叠桶结构域中间的loop构成,其中芳香氨基酸残基Trp748侧链提供一1位结合位点,Trp749侧链提供+1位结合位点,Trp131侧链提供+2位结合位点,这些芳香氨基酸残基形成了一个小的口袋和疏水性环境,不利于转糖苷反应。
Aβ-glucosidase gene from Paenibacillus barengoltzii CAU904 was cloned and heterologously expressed in E.coli.The recombinant enzyme was purified and biochemically characterized,and its crystal structure was further resolved.The enzyme had the highest activity at 50℃and pH 7.5.It exhibited broad substrate specificity on various substrates,including laminarin,barleyβ-glucan,lichenan,et al.(containingβ-1,2,β-1,3,β-1,4 andβ-1,6 linkages).Crystal structure analysis revealed that the enzyme exhibited typical glycoside hydrolyase family 3 enzymes,having multiple domain structures.The catalytic packet of the enzyme was composed by a(α/β)5 sandwich domain and a(β/α)8 folding barrel domain linked by a loop region.The side chain aromatic amino acids Trp748 and Trp749 were functioned as-1 and+1 binding site,respectively,while Trp131 was functioned as+2 site.These aromatic amino acids formed a narrow pocked and a hydrophobic environment,which could diminish its transglycosylation activity.
作者
黄平
吴世旺
江正强
杨绍青
HUANG Ping;WU Shiwang;JIANG Zhengqiang;YANG Shaoqing(College of Food Science and Nutritional Engineering,China Agricultural University,Beijing 100083,China)
出处
《食品科学技术学报》
CAS
CSCD
北大核心
2019年第1期20-27,共8页
Journal of Food Science and Technology
基金
国家自然科学基金面上项目(31571774)
关键词
巴伦葛兹类芽孢杆菌
Β-葡萄糖苷酶
异源表达
酶学性质
晶体结构
Paenibacillus barengoltzii
β-glucosidase
heterologous expression
biochemical characterization
crystal structure
