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

PloS One
Wenjie LiJiong Hong

Abstract

Urate oxidase is a key enzyme in purine metabolism and catalyzes the oxidation of uric acid to allantoin. It is used to treat hyperuricemia and gout, and also in a diagnostic kit. In this study, error-prone polymerase chain reaction and staggered extension process was used to generate a mutant urate oxidase with improved enzyme activity from Bacillus subtilis. After several rounds of mutagenesis and screening, two mutants 6E9 and 8E279 were obtained which exhibited 2.99 and 3.43 times higher catalytic efficiency, respectively. They also exhibited lower optimal reaction temperature and higher thermo-stability. D44V, Q268R and K285Q were identified as the three most beneficial amino acid substitutions introduced by site-directed mutagenesis. D44V/Q268R, which was obtained through random combination of the three mutants, displayed the highest catalytic activity. The Km, kcat/Km and enzyme activity of D44V/Q268R increased by 68%, 83% and 129% respectively, compared with that of wild-type urate oxidase. Structural modeling indicated that mutations far from the active site can have significant effects on activity. For many of them, the underlying mechanisms are still difficult to explain from the static structural model. We also comp...Continue Reading

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Methods Mentioned

BETA
PCR
electrophoresis

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