title: [Preparation and identification of recombinant sarcosine oxidase].
abstract: An important index determination for clinical diagnosis of renal function is to assay the creatinine concentration in serum. In the analytical process applied with coupled-enzyme, the quality control of sarcosine oxidase (SOX) as a key enzyme is the first problem to be solved. In order to establish an efficient and laboratory-scale production of SOX, the recombinant sarcosine oxidase (r-SOX) gene was a high-level expression in E. coli induced with lactose on a large-scale fermentation in 300 L fermenter. The results suggested that the biomass concentration reached OD600 of 22 and the expression of recombinant sarcosine oxidase in E. coli accounted for about 25% of total soluble protein in culture after fermentation. The cell-free extract obtained from high pressure homogenizer was processed by selective thermal denaturation and then purified with Ni-Sepharose FF chromatography. The sarcosine oxidase with 97% purity, 25 U/mg specific activity and 92.4% activity recovery was obtained. The molecular weight with single peptide chain of 53 kD and 55 kD of recombinant sarcosine oxidase was assessed by SDS-PAGE in presence or absence of 2-mercaptoehanol and Sephacryl S-200 chromatography. This sarcosine oxidase was found to be a conjugated protein, yellow enzyme, which combined with FAD as prosthetic group by covalent linkage. The contaminant of catalase was not detected in the sample pool of this enzyme. In addition, a further test to the thermal stability of sarcosine oxidase was done. According to the above results, the development and utilization of this enzyme has been set up on a reliable foundation.
authors.forename: Jing
authors.initials: J
authors.lastname: Pu
authors.forename: Rui
authors.initials: R
authors.lastname: Wang
authors.forename: Mingdong
authors.initials: M
authors.lastname: Yao
authors.forename: Zhongjie
authors.initials: Z
authors.lastname: He
authors.forename: Ming
authors.initials: M
authors.lastname: Zhao
authors.forename: Yao
authors.initials: Y
authors.lastname: Meng
mesh_terms: D004926:Escherichia coli; D005285:Fermentation; D011994:Recombinant Proteins / Q000096:biosynthesis; D050879:Sarcosine Oxidase / Q000096:biosynthesis*
chemical_list: D011994:Recombinant Proteins; D050879:Sarcosine Oxidase