Urate oxidase (Uox) is the enzyme involved in purine metabolism. Pseudogenization of Uox gene is the underlying mechanism of hyperuricemia and gout in human. Although Uox from various microorganisms has been used in clinical practice for many years, its application is limited by potential immunogenicity. In order to develop a more "human like" uricase, DNA shuffling was used to create chimeric uricase with both improved enzymatic activity and increased homology with deduced human uricase (dHU) gene. By using wild porcine uricase (wPU) gene and dhu as parental genes, a diverse chimeric library was generated. After preliminary screening by a "homebrew" high throughput protocol, approximately 100 chimeras with relatively high enzymatic activity were obtained. By further activity comparison of the purified enzymes, chimera-62 with increase in both activity and homology with dHU compared with wPU was selected. Its Km and catalytic efficiency were determined as 9.43±0.04μM and 2.67s(-1)μM(-1) respectively. There were 33 amino acid substitutions in chimera-62 when compared with dHU and 5 substitutions when compared with wPU. By homology modeling and 3-D structure analysis, it was speculated that mutations G248S and L266F contributed to the increased activity of chimera-62 by increasing the stability of α-helix and surface polarity respectively.
Keywords: DNA shuffling; High-throughput screening; Urate oxidase.
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