High-yield expression, purification, characterization, and structure determination of tag-free Candida utilis uricase

Abstract

We report the successful high-yield expression of Candida utilis uricase in Escherichia coli and the establishment of an efficient three-step protein purification protocol. The purity of the recombinant protein, which was confirmed to be C. utilis uricase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer analysis, was >98% and the specific activity was 38.4 IU/mg. Crystals of C. utilis uricase were grown at 18°C using 25% polyethylene glycol 3350 as precipitant. Diffraction by the crystals extends to 1.93 Å resolution, and the crystals belong to the space group P2(1)2(1)2(1) with unit cell parameters a = 69.16 Å, b = 139.31 Å, c = 256.33 Å, and α = β = γ = 90°. The crystal structure of C. utilis uricase shares a high similarity with other reported structures of the homologous uricases from other species in protein database, demonstrating that the three-dimensional structure of the protein defines critically to the catalytic activities.

<b><i>Arxula adeninivorans</i></b> Recombinant Urate Oxidase and Its Application in the Production of Food with Low Uric Acid Content

“…However, Auox6hp showed an increase in pH optimum to pH 9.5. The K m values indicate a high affinity of urate oxidase for uric acid as substrate and are comparable with C. utilis (33.7 μ M ) and Bacillus fastidiosus (180 μ M ) [Bongaerts et al, 1978;Liu et al, 2011]. The reason for differences in pH, temperature profiles and kinetic parameters of Auox6hp may be caused by the C-terminal HisTag which can have influence on the protein structure and protein charge [Votchitseva et al, 2006].…”

“…Partially purified endogenous Auoxp showed optimum activity at pH 8.0 and 40 ° C which are properties shared with urate oxidases from Aspergillus flavus [Li et al, 2006], Bacillus subtilis [Pfrimer et al, 2010] and Candida utilis [Liu et al, 2011]. However, Auox6hp showed an increase in pH optimum to pH 9.5.…”

Directed evolution to improve the catalytic efficiency of urate oxidase from Bacillus subtilis

“…It catalyzes uric acid degradation and produces allantoin (Fig 1), which is much more soluble than uric acid [1]. Urate oxidase is present in various organisms such as animals, plants, fungi, yeasts, and bacteria [2].…”

“…However, during the evolutionary process, urate oxidase activity seems to have been lost in some higher primates including humans [3, 4]. In these species, the end product of purine metabolism is uric acid rather than allantoin [1]. …”

“…The accumulation of uric acid in the body can result in hyperuricemia. In some cases, its crystallization and precipitation lead to gout [1]. During chemotherapy of leukemia and lymphoma, the excretion of uric acid increased sharply because of the degradation of nucleic acid from malignant cells.…”