title: Study the effect of His-tag on chondroitinase ABC I based on characterization of enzyme.
abstract: Chondroitinase ABC I (ChSase ABC I) which could degrade chondroitin sulfate (CS) to low molecular weight CS was expressed with His-tag in Escherichia coli (E. coli) BL21(DE3). The effect of His-tag on ChSase ABC I was investigated compared with ChSase ABC I which cut His-tag for the first time. After three steps purification, the specific activity of His-ChSase ABC I was 201.9±5.4 IU/mg which was two times lower than ChSase ABC I. Results of multi angle light scattering (MALS) and analytical ultracentrifugation (AUC) showed that the polymeric state of His-ChSase ABC I was not effected by His-tag and it was monomer, and ChSase ABC I was the same. The optimal temperature and pH of His-ChSase ABC I were 37 °C and 7.5, and were almost same with ChSase ABC I. Vmax and kcat/Km of His-ChSase ABC I were 2.4±0.1 μmol/Ls, and 22.2±0.4 L/(μmols) and catalytic efficiency was lower than ChSase ABC I. Generally, His-tag had no effect on polymeric state, optimal temperature and pH, had little negative impact on specific activity, kcat/Km and secondary-structure of ChSase ABC I. This study might guide the application of ChSase ABC I in industrial production.
authors: State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, No.15 East Road of North Third Ring, Chao Yang District, Beijing 100029, China.
authors: Zhenya
authors: Z
authors: Chen
authors: Department of Biotechnology, Beijing Polytechnic, No.1 Taiyanggong Shaoyaoju, Chao Yang District, Beijing 100029, China. Electronic address: liyeyashi@163.com.
authors: Ye
authors: Y
authors: Li
authors: State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, No.15 East Road of North Third Ring, Chao Yang District, Beijing 100029, China. Electronic address: yuanqp@mail.buct.edu.cn.
authors: Qipeng
authors: Q
authors: Yuan
mesh_terms: D002384:Catalysis; D019765:Chondroitin ABC Lyase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins; D013696:Temperature; D013816:Thermodynamics
chemical_list: D011993:Recombinant Fusion Proteins; D019765:Chondroitin ABC Lyase
keywords: Characterization; His-ChSase ABC I; Monomer; Secondary-structure; Thermostability