Journal
BIOLOGIA
Volume 77, Issue 1, Pages 291-301Publisher
SPRINGER
DOI: 10.1007/s11756-021-00941-4
Keywords
Bacillus subtilis; Uricase; Prokaryotic expression; Cysteine residues; Site-directed mutagenesis
Categories
Funding
- National Natural Science Foundation of China [31,701,573]
- Key Projects of Scientific Research Program of Hubei Provincial Department of Education [D20181302]
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Urate oxidase plays a crucial role in the therapy of gout and detection of uric acid, making it significant to produce uricase that meets clinical requirements. A gene encoding extracellular uricase from Bacillus subtilis BS04 was successfully cloned, leading to the purification of a recombinant protein with optimum activity at pH 9.0 and 45 degrees C. Furthermore, mutations involving cysteine substitutions were found to enhance the thermal resistance of uricase, offering insights for enzyme therapeutic development.
Urate oxidase is an important enzyme that is applied as diagnostic reagent in the therapy of gout and detection of uric acid. Therefore, it is of great significance to produce uricase that meets the clinical requirements. A gene that encoded extracellular uricase from Bacillus subtilis BS04 was successfully cloned and inserted into the pET28a (+) expression vector. The recombinant protein was purified to determine its activity by UV spectrophotometry. The purified enzyme of 56.63 kDa was optimally active at pH 9.0 and 45 degrees C with a specific activity of 4.97 U/mg. The enzyme activity increased by 137.12 % by Mn2+ and inhibited by 95.42 % by Cu2+. Furthermore, four Cys-directed mutants were constructed to analyze the effect of Cys on the pH and temperature dependence of the enzyme. Activity measurements revealed that the enzyme activity was impaired in all mutants, but mutant C489A exhibited higher alkali resistance and thermostability than native uricase. Our findings illustrate the relationship between Cys substitution and increased thermal resistance of uricase and provide insights to improve enzyme therapeutic development.
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