Cloning, Purification, and Partial Characterization ofBacillus subtilisUrate Oxidase Expressed inEscherichia coli

Abstract

Urate oxidase (EC 1.7.3.3) is an enzyme involved in purine metabolism which is used in the treatment of gout and as diagnostic reagent for detection of uric acid. In order to produce this enzyme in large quantities for biotechnological purposes, the gene coding for the Bacillus subtilis urate oxidase was cloned and heterologously expressed in Escherichia coli. Time course induction in E. coli showed an induced protein with an apparent molecular mass of ∼60 kDa. Soluble recombinant enzyme was purified in a single-step procedure using Ni-NTA column. The enzyme was purified 2.1-fold with a yield of 56% compared to the crude extract. MALDI-TOF analysis revealed an ion with a mass of 58675 Da which is in agreement with the expected mass of the recombinant protein. The purified enzyme showed an optimal pH and temperature of 8.0 and 37°C, respectively, and retained 90% of its activity after 72 hours of incubation at −20°C and 4°C.

PEGylation enhancement of pH stability of uricase via inhibitive tetramer dissociation

“…These results suggest that PEGylation could mitigate the changes in secondary structure, a finding confirmed by the results of the present study. Kinetic studies on native and PEGylated uricase provide compelling evidence that a wrapping effect occurs by showing significantly decreased Km values in PEGylated uricase, confirming a previous work that also reported that the Km value for uricase had decreased from 5.4 × 10 −5 m to 2.7 × 10 −5 m after modifying with mPEG of 5 kDa [31] …”

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

“…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.…”

Efficient purification of a recombinant tag-free thermostable Kluyveromyces marxianus uricase by pH-induced self-cleavage of intein and expression in Escherichia coli

“…Nevertheless, the activity decreases by nearly 50% in 36 h after incubation at 37 °C (Liu et al 2011). The retained activity of uricase from B. subtilis is ~ 50% after incubation at 37 °C for 12 h (Pfrimer et al 2010). Only 15% of uricase activity from A. flavus is preserved after 10-min incubation at 40 °C (Imani and Shahmohamadnejad 2017).…”

“…Furthermore, uricase with a 4-aminoantipyrine peroxidase system is extensively employed as a reagent in the clinical analysis of uric acid in serum (Liu et al 1994). So far, many uricases from various microbes, including Candida utilis (Koyama et al 1996), Bacillus subtilis (Pfrimer et al 2010), Aspergillus flavus (Legoux et al 1992), and Pseudomonas aeruginosa (Shaaban et al 2015), have been recombinantly expressed. Rasburicase mentioned above is the recombinant uricase cloned from A. flavus and expressed in Saccharomyces cerevisiae (Pui et al 2001).…”