Fermentation, Purification, Characterization And Gene Studies Of Arthrobacter Xanthine Oxidase

Xanthine oxidase (XOD, EC 1.2.3.22) is a complex molybdoflavoenzyme that exists in as an available oxidoreductase in all kinds of organisms. It is also a key enzyme in the catabolism of purines path way, which mainly catalyzes the conversion of hypoxanthine and xanthine to xanthine and uric acid, respectively. The enzyme has broad substrate specificity, except catalyzing the oxidation of purine, pyrimidine, pterine, and aldehyde, it catalyses 7,8-dihydropterin to 7,8-dihydroxanthopterin, xanthopterin to leucopterin, and pterin to isoxanthopterin or drosopterin as well. It has been clinically used as anti-tumor and clinical diagnosis of reperfusion injury.A novel method to determine the activity of xanthine oxidase through the chromogenic reaction of 4 -aminoantipyrine (AAP), phenicacid (PA) and hydrogen peroxide, which was produced via the oxidation of xanthine catalyzed by XOD, under the help of horseradish peroxidase (HRP), was proposed. The influences of temperature,pH and amount of substrate on the activity of XOD were investigated. The optimal conditions for XOD activity assay were obtained as follows: HRP (7000U·L^-1), AAP (1mmol·L^-1), PA (6mmol·L^-1) and xanthine (1mmol·L^-1) were dissolved in 50 mmol·L^-1 Tris-HCl buffer solution (pH8.4), the reaction temperature was 37℃, the reaction time was 20 min, the detective wavelength was 508nm. Under the conditions mentioned above, the linear range of calibration was between 5.0 U·L^-1 and 100.0 U·L^-1, and the detection limit was 1.3 U·L^-1. The above system is a potential alternative method to determine the activity of XOD in the areas either for laboratory assay or clinical diagnosis.Using xanthine as sole carbon, nitrogen, energy sources and inductor, a xanthine oxidase producer was isolated from the soil. The strain was characterized not only by morphologic and biochemiccal properties, but also by clone and sequence of its 16S rDNA. As a result, the organism has been identified as Arthrobacter and named Arthrobacter sp.XL26 whose GenBank acceptor number is EF532600.The effects of inductor, carbon and nitrogen sources on XOD production by Arthrobacter sp. XL26 were investigated. The medium required for XOD production was optimized with statistics based experimental designs. The important medium components, identified by initial screening method of Plackett-Burman, were xanthine, yeast extract and CaCl₂. Box-Behnken and response surface methodology were then employed to further optimize XOD production. The optimal compositions for higher production of XOD were (g·L^-1): glucose 12.0, yeast extract 2.13, xanthine 2.57, (NH₄)₂SO₄ 4.0, KH₂PO₄.3H₂O 0.94, Na₂HPO₄ 5.92; CaCl₂ 5.33×10^-3, MgSO₄.7H₂O 0.2×10^-3, FeSO₄.7H₂O 0.5×10^-3. Using this statistical optimization method, the XOD was found to increase from 27.4 U·L^-1 to 409.6 U·L^-1. XOD has been purified to a single protein band from cell-free extracts of Arthrobacter sp. XL26 via procedures of ultrasonication, ammonium sulfate fractionation, Butyl-Sepharose 4B chromatography, DEAE Sepharose CL-6B FF chromatography, Phenyl Sepharose CL-4B chromatography and Sephacryl S-200 HR gel filtration. The enzyme specific activity was found to increase to 270-fold and reache 24 U·mg^-1 with a final recovery rate of 11%. The native PAGE and SDS-PAGE show that the enzyme has a molecular weight of 260,000 and consists of two heterogeneous subunits, xodA and xodB, with a molecular weight of 55,480 and 85,670, repectively.The enzyme shows the highest activity at 55℃and pH 8.0. And it is still stable between pH 7 and 9 at 25℃for 16h or below 35℃at pH 8 for 30 min. K_m values of the enzyme for hypoxanthine and xanthine are0.133 and 0.032 mM, respectively. The activity can be stimulated by Mn²⁺, Co²⁺and Ba²⁺, and inhibited by Fe²⁺, Cu²⁺, Pb²⁺, Ag⁺and Hg²⁺ at a concentration of 2mM or more. Among the compounds tested, sodium salicylate, histamine, triton, tween 80, rochelle salt, mannitol, sorbitol, glycerine, PVP, DTT and PEG6000 are activity stimulator. The activity can be strongly inhibited by NaN₃ and urea as well.The xodA and xodB from Arthrobacter sp. XL26, encoding the heterodimeric molybdo-iron-sulfur-protein xanthine oxidase, were cloned and sequenced by the technology of inverse PCR and nest PCR. The results show these two genes consist of 1542 bp and 2355 bp and encode two peptides with 513 and 784 amino acids, whose sequences acceptor number in GenBank are EF648005 and EF648204 respectively. The deduced amino acid sequences of xodA and xodB show homologies of 92% and 84% comparing to the small and large subunits of Acinetobacter baumannii ATCC 17978 xanthine dehydrogenases, respectively. Two-dimensional structure models indicate its [2Fe-2S] and FAD-binding domains locate in xodA, whereas Mo-binding and a/b hammer-head domain of aldehyde oxidase or xanthine dehydrogenase seat in xodB.