Modified colorimetric assay for uricase activity and a screen for mutant Bacillus subtilis uricase genes following StEP mutagenesis

Abstract

This study describes a modified colorimetric assay for uricase activity in flexible 96‐well microtiter plates using the uricase/uric acid/horseradish peroxidase/4‐aminoantipyrine/3,5‐dichloro‐2‐hydroxybenzene sulfonate colorimetric reaction. The utility of this assay was demonstrated in a screen for mutant uricase enzymes derived from the uricase gene of the thermophilic bacterium Bacillus subtilis by a modified staggered extension process (StEP) mutagenesis. An Escherichia coli library of StEP‐derived uricase mutant clones was screened yielding two identical active mutant uricase genes. Two motifs conserved in eukaryotic and prokaryotic uricases are highly conserved in the mutant uricase. The mutant uricase protein was found to exhibit high uricase activity (13.1 U·mg−1). Finally, the modified colorimetric method is much more efficient than the conventional ones and greatly reduces assay time from 4 days to less than 20 h.

Purification and characterization of a thermostable uricase from Microbacterium sp. strain ZZJ4-1

“…Since Bongaert et al (Bongaert et al 1978) found that Bacillus fastidiosus could produce uricase and use uric acid as the only carbon source for growth in 1978, many reports on bacterial and yeast uricases, such as those from Arthrobacter globiformis (Nobutoshi et al 2000), Bacillus subtilis (Hunag and Wu 2004), Candida utilis (Liu et al 1994) and Pseudomonas aeruginosa (Ishikawa et al 2004), have been published.…”

Purification and characterization of caprine kidney uricase, possessing novel kinetic and thermodynamic properties

“…The enzyme showed 35% relative activity at 100°C; temperature-dependent properties of this enzyme show resemblance to Bacillus subtilis uricase (Huang & Wu 2004). The protein mid-point inactivation temperature (T m ), activation energy, conformational stability at elevated temperatures, activation parameters for catalytic activity, transition state binding energy, stability of the native state ensemble are potential determinants of thermostable biocatalysts.…”

“…The activity of uricase was measured on the basis of liberated hydrogen peroxide (Huang & Wu 2004) from uric acid and color measured after 20 min incubation.…”

“…It is present in the liver, kidney and brain of vertebrates except some species, which have lost the gene for uricase and are, therefore, unable to degrade urate (Hayashi et al 1989). Uricase has also been produced and purified from different organisms (Huang & Wu 2004). The body's inability to break down uric acid in the blood stream is known as hyperuricaemia and can be cured with complete oral calcium therapy and by increasing the level of uricase (Culleton et al 1999).…”

Purification and characterization of an extracellular uricase from a new isolate of Sphingobacterium thalpophilum (VITPCB5)

“…The uricase preparations from various microbial sources like fungi, yeast and bacteria have been reported [8][9][10][11]. But its increasing importance in treatment and diagnosis, necessitate to screen new sources for production, which are more economical, and may have unique properties to expand its usefulness.…”