title: New extracellular thermostable oxalate oxidase produced from endophytic Ochrobactrum intermedium CL6: Purification and biochemical characterization.
abstract: Oxalate oxidase (EC 1.2.3.4) catalyzes the oxidative cleavage of oxalate to carbon dioxide with the reduction of molecular oxygen to hydrogen peroxide. Oxalate oxidase found its application in clinical assay for oxalate in blood and urine. This study describes the purification and biochemical characterization of an oxalate oxidase produced from an endophytic bacterium, Ochrobactrum intermedium CL6. The cell-free fermentation broth was subjected to two-step enzyme purification, which resulted in a 58.74-fold purification with 83% recovery. Specific activity of the final purified enzyme was 26.78 U mg(-1) protein. The enzyme displayed an optimum pH and temperature of 3.8 and 80°C, respectively, and high stability at 4-80°C for 6 h. The enzymatic activity was not influenced by metal ions and chemical agents (K(+), Na(+), Zn(2+), Fe(3+), Mn(2+), Mg(2+), glucose, urea, lactate) commonly found in serum and urine, with Cu(2+) being the exception. The enzyme appears to be a metalloprotein stimulated by Ca(2+) and Fe(2+). Its Km and Kcat for oxalate were found to be 0.45 mM and 85 s(-1), respectively. This enzyme is the only known oxalate oxidase which did not show substrate inhibition up to a substrate concentration of 50 mM. Thermostability, kinetic properties, and the absence of substrate inhibition make this enzyme an ideal candidate for clinical applications.
authors.affiliation: a Department of Chemical Engineering , National Institute of Technology Karnataka , Mangalore , Karnataka , India.
authors.forename: Kunal
authors.initials: K
authors.lastname: Kumar
authors.affiliation: a Department of Chemical Engineering , National Institute of Technology Karnataka , Mangalore , Karnataka , India.
authors.forename: Prasanna D
authors.initials: PD
authors.lastname: Belur
mesh_terms: D002474:Cell-Free System; D004795:Enzyme Stability; D005285:Fermentation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D008970:Molecular Weight; D042041:Ochrobactrum / Q000201:enzymology*; D010088:Oxidoreductases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature
chemical_list: D008670:Metals; D010088:Oxidoreductases; C020598:oxalate oxidase
keywords: Biochemical characterization; Ochrobactrum intermedium; enzyme purification; oxalate oxidase