title: Purification and characterization of an extracellular uricase from a new isolate of Sphingobacterium thalpophilum (VITPCB5).
abstract: An extracellular uricase producing bacterium (VITPCB5) was isolated from soil of the duck farm near Chidambaram, Tamilnadu, India and it was identified based on its 16S rRNA as Sphingobacterium thalpophilum. Uric acid was used as an effective inducer. The enzyme kinetics was studied using uric acid as a substrate. The Km and Vmax for the enzyme was found to be 0.28mM and 0.92μM/minml, respectively. Maximum uricase production was observed when lactose was used as a carbon source. Among the nitrogen sources tested, urea gave the maximum uricase production. The enzyme was successfully purified using a weak cation exchange convective interaction media carboxy methyl (CIM-CM) monolith column with a recovery of 79.7%±0.1 and 14.2±1.8-fold purification. The optimal reaction temperature of the enzyme was observed between 25 and 45°C. The pH optimum of the enzyme was 8.0. The enzyme activity was enhanced by copper and partially inhibited by calcium, iron, zinc and nickel ions. Treatment with ethylene diamine tetraacetic acid completely inhibited the enzyme activity. The in-gel trypsin digested peptides of 48-kDa uricase when analyzed using mass spectrometry, gave 32% sequence coverage with the uricase (30-kDa) from Cyberlindnera jadinii.
authors.affiliation: Centre for Bioseparation Technology, VIT University, Vellore 632014, Tamil Nadu, India.
authors.forename: R
authors.initials: R
authors.lastname: Ravichandran
authors.affiliation: Centre for Bioseparation Technology, VIT University, Vellore 632014, Tamil Nadu, India.
authors.forename: S
authors.initials: S
authors.lastname: Hemaasri
authors.affiliation: Institute of Microbial Technology, Sector 39A, Chandigarh 160036, India.
authors.forename: Swaranjit Singh
authors.initials: SS
authors.lastname: Cameotra
authors.affiliation: Centre for Bioseparation Technology, VIT University, Vellore 632014, Tamil Nadu, India. Electronic address: nsjayaprakash@vit.ac.in.
authors.forename: N S
authors.initials: NS
authors.lastname: Jayaprakash
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D045249:Sphingobacterium / Q000737:chemistry / Q000201:enzymology*; D014503:Urate Oxidase / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D014503:Urate Oxidase
keywords: CIM; Sphingobacterium thalpophilum; Uric acid; Uricase