硫酸软骨素ABCⅠ酶的融合表达及传感器应用研究  

Fusion expression of chondroitinase ABCⅠ and biosensor application

作　　者：李晔[1] 金丽华[1] 李栋 辛秀兰[1] 陈振娅 LI Ye;JIN Lihua;LI Dong;XIN Xiulan;CHEN Zhenya(College of Biotechnology,Beijing Polytechnic,Beijing 100176,China;College of Life Science,Beijing Institute of Technology,Beijing 100081,China)

机构地区：[1]北京电子科技职业学院生物工程学院,北京100176 [2]北京理工大学生命学院,北京100081

出　　处：《中国酿造》2020年第11期153-157,共5页China Brewing

基　　金：北京市自然基金面上项目(2182019);北京市优秀人才培养资助(拔尖自然科学)(2020Z002-002-KWT);北京电子科技职业学院院内科技类重点课题(2019Z002-033-KXZ)。

摘　　要：硫酸软骨素酶(ChS ase)是一类能将硫酸软骨素(CS)催化裂解为小分子多糖的酶,根据结合的底物种类可分为ChSase ABC,ChSase AC,ChSase B和ChSase C。将普通变形杆菌(Proteus vulgaris)KCTC 2579的硫酸软骨素酶ABC (IChSase ABCⅠ)与麦芽糖结合蛋白(MBP)融合表达,构建了pMAL-c2x-ChSase ABCⅠ重组载体,并成功在大肠杆菌(Escherichia coli)BL21(DE3)宿主中高效表达。将麦芽糖结合蛋白-硫酸软骨素酶(MBP-ChSase)ABCⅠ固定在聚苯胺载体上,制备生物传感器,并利用循环伏安法对其进行检测。结果表明,MBP-ChSase ABCⅠ酶活和比酶活分别为3 180 IU/L发酵液和76 IU/mg蛋白。选择硫酸软骨素A(CS A)作为酶的反应底物,在最适反应条件下,反应的峰值电流与CS A在浓度0.1 nmol/L^0.1μmol/L范围内呈线性关系。该生物传感器对底物反应迅速,具有较高的灵敏度,可以进行有效检测。Chondroitinase(ChSase) is a class of enzymes that can degrade chondroitin sulfate(CS) to small molecular polysaccharides, which can be classified as ChSase ABC, ChSase AC, ChSase B and ChSase C according to the kind of substrates. In this study, the ChSase ABCⅠ derived from Proteus vulgaris KCTC 2579 was integrated and expressed with maltose-binding protein(MBP). The recombinant vector p MAL-c2x-ChSase ABCⅠ was constructed and successfully expressed in Escherichia coli BL21(DE3). The maltose-binding protein-chondroitinase(MBP-ChSase) ABCⅠ enzyme was immobilized on a polyaniline carrier to prepare a biosensor, which was detected by cyclic voltammetry. The results showed that the enzyme activity and specific activity of MBP-ChSase ABCⅠ were 3 180 IU/L fermentation broth and 76 IU/mg protein, respectively. Using chondroitin sulfate A(CS A) as the reaction substrate of the enzyme, under the optimized conditions, the peak current of the reaction had a linear relationship with the concentration of CS A of 0.1 nmol/L-0.1 μmol/L.The biosensor reacted quickly to the substrate, with high sensitivity, and could perform effective detection.

关 键 词：普通变形杆菌 硫酸软骨素ABCⅠ 麦芽糖结合蛋白 表达 生物传感器 

分 类 号：R651.2[医药卫生—外科学]