Characterization and Cys-directed mutagenesis of urate oxidase from Bacillus subtilis BS04

• Published in: Biológia Vol. 77; no. 1; pp. 291 - 301
• Main Authors: Zhu, Tong-tong, Chen, Hong-na, Yang, Lei, Liu, Ying-bao, Li, Wei, Sun, Wen-xiu
• Format: Journal Article
• Language: English
• Published: Cham Springer International Publishing 2022; Springer Nature B.V
• Subjects: Bacillus subtilis; Biomedical and Life Sciences; Cell Biology; Copper; Enzymatic activity; Enzyme activity; Enzymes; Gout; Life Sciences; Microbiology; Mutagenesis; Mutants; Original Article; Oxidase; pH effects; Plant Sciences; Reagents; Spectrophotometry; Temperature dependence; Thermal resistance; Thermal stability; Urate oxidase; Uric acid; Zoology; Prokaryotic expression; Uricase; Site-directed mutagenesis; Cysteine residues
• ISSN: 0006-3088; 1336-9563
• DOI: 10.1007/s11756-021-00941-4

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 °C with a specific activity of 4.97 U/mg. The enzyme activity increased by 137.12 % by Mn 2+ and inhibited by 95.42 % by Cu 2+ . 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.