title: Expression, purification and characterization of GAPDH-ChSase ABC I from Proteus vulgaris in Escherichia coli.
abstract: Chondroitinases (ChSases) are a family of polysaccharide lyases that can depolymerize high molecular weight chondroitin sulfate (CS) and dermatan sulfate (DS). In this study, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is stably expressed in different cells like normal cells and cancer cells and the expression is relatively insensitive to experimental conditions, was expressed as a fusion protein with ChSase ABC I. Results showed that the expression level and enzyme activity of GAPDH-ChSase ABC I were about 2.2 and 3.0 times higher than those of ChSase ABC I. By optimization of fermentation conditions, higher productivity of ChSase ABC I was achieved as 880 ± 61 IU/g wet cell weight compared with the reported ones. The optimal temperature and pH of GAPDH-ChSase ABC I were 40 °C and 7.5, respectively. GAPDH-ChSase ABC I had a kcat/Km of 131 ± 4.1 L/μmol s and the catalytic efficiency was decreased as compared to ChSase ABC I. The relative activity of GAPDH-ChSase ABC I remained 89% after being incubated at 30 °C for 180 min and the thermostability of ChSase ABC I was enhanced by GAPDH when it was incubated at 30, 35, 40 and 45 °C.
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.
authors: Zhenya
authors: Z
authors: Chen
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: Zhao
authors: Z
authors: Zhou
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: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D002384:Catalysis; D019765:Chondroitin ABC Lyase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D006358:Hot Temperature; D011514:Proteus vulgaris / Q000201:enzymology / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D019765:Chondroitin ABC Lyase
keywords: ChSase ABC I; GAPDH; Productivity; Thermostability