硫酸软骨素裂解酶ABCⅡ的高效融合表达及其酶学性质研究  

Study on High-efficiency Fusion Expression and Enzymatic Properties of Chondroitinase ABCⅡ

作　　者：李晔[1] 周朝 袁其朋[2] LI Ye;ZHOU Zhao;YUAN Qipeng(College of Bioengineering,Beijing Polytechnic Institute,Beijing 100176,China;Colllege of Life Science and Technology,Beijing University of Chemical Technology,Beijing 100029,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]北京化工大学生命科学与技术学院,北京100029

出　　处：《食品工业科技》2023年第5期88-97,共10页Science and Technology of Food Industry

基　　金：北京市优秀人才培养资助(拔尖自然科学)(2020Z002-002-KWT);天然产物国家标准样品研制及产品开发技术研究(SG030801)。

摘　　要：目的:本研究旨在实现硫酸软骨素裂解酶ABCⅡ(chondroitinase ABCⅡ,ChSase ABCⅡ)的高效可溶性表达并研究其酶学性质,为ChSase ABCⅡ在药品和保健食品生产中的应用奠定基础。方法:在优化ChSase ABCⅡ基因原始序列的基础上,构建重组质粒pET-28a-His-ChSase ABCⅡ并优化其表达条件;利用亲和层析得到带有多聚组氨酸标签(polyhistidine-tag,His-tag)的ChSase ABCⅡ融合蛋白后,研究了His-ChSase ABCⅡ的部分酶学性质。结果:构建的His-ChSase ABCⅡ融合蛋白表达系统能在大肠杆菌中实现可溶性表达;在表达宿主为E.coli BL21(DE3)和诱导剂(异丙基-β-D-硫代半乳糖苷)浓度为125μmol/L的优化条件下,发酵液酶活力可达到7206.83±184.27 IU/L;纯化后的His-ChSase ABCⅡ酶比活力为22.02±0.39 IU/mg蛋白,最适pH和温度分别为7.5和40℃,其在30~40℃条件下较为稳定,且半衰期在2 h以上。His-ChSase ABCⅡ能特异性有效分解硫酸软骨素,其K_(m)值为10.4±0.8μmol/L,Kcat值为9.4±0.2 s^(-1)。结论:本论文通过基因优化和融合表达等策略实现了His-ChSase ABCⅡ的高效表达和纯化。此外,His-ChSase ABCⅡ的酶学性质可基本满足其在医药和营养产品工业生产中的应用需求。Objectives:The present study aimed to achieve the efficient and soluble expression of chondroitinase ABCⅡ(ChSase ABCⅡ)and investigate its enzymatic properties.The study thus laid the groundwork for the application of ChSase ABCⅡin pharma-and nutraceutical production industries.Methods:Based on the optimization of the original sequence of ChSase ABCⅡgene,the recombinant plasmid pET-28a-His-ChSase ABCⅡwas constructed and the expression conditions of the recombinant plasmid were optimized.The partial enzymatic properties of His-ChSase ABCⅡwere studied after obtaining ChSase ABCⅡfusion protein with a polyhistidine-tag(His-tag)using affinity chromatography.Results:The results showed that the constructed His-ChSase ABC II fusion protein expression system successfully achieved the soluble expression of the protein in Escherichia coli.In the optimal conditions that the expression host was E.coli BL21(DE3)and inducer(isopropyl-β-D-thiogalactoside)concentration was 125μmol/L,the enzyme activity of fermentation broth reached 7206.83±184.27 IU/L.Furthermore,the purified His-ChSase ABCⅡhad an enzyme specific activity of 22.02±0.39 IU/mg protein,with optimal pH and temperature of 7.5 and 40℃,respectively.The enzyme was also found to be stable at 30~40℃with a half-life of over 2 h.Moreover,the His-ChSase ABCⅡwas determined to specifically and effectively decompose chondroitin sulfate,with a K_(m)value of 10.4±0.8μmol/L and a K_(cat)value of 9.4±0.2 s^(-1).Conclusions:Efficient expression and purification of His-ChSase ABCⅡwere achieved using gene optimization and fusion expression strategies in this study.Moreover,the enzymatic properties of the recombinant fusion protein could fundamentally meet the standards essential for its application in the industrial production of pharma-and nutraceutical products.

关 键 词：硫酸软骨素裂解酶ABCⅡ 低分子量硫酸软骨素(LMWCSs) 酶学性质 融合表达 多聚组氨酸标签 

分 类 号：Q814.1[生物学—生物工程]