Screening of urate oxidase-producing Streptomyces strains, gene cloning and heterogeneous expression, and characterization of the recombinant enzyme

Urate oxidase catalyzes the decomposition of uric acid into hydrogen peroxide and the more highly soluble allantoin. It is commonly used for uric acid detection and the treatment of diseases related to hyperuricemia. In this study, a high-yielding urate oxidase strain, 2YHDJ-2, was isolated from the lake sediments of Chaka Salt Lake in Qinghai Province. Through 16S rRNA gene sequencing and phylogenetic analysis, it was identified as Streptomyces sp. Degenerate primers were designed based on the conserved sequences of known urate oxidase genes in GenBank for PCR amplification, yielding a 952 bp urate oxidase gene. This gene shares up to 99% homology with the reported urate oxidase gene from Streptomyces sp. strain NSC9. The gene was cloned into the pET-15b vector and transformed into Escherichia coli BL21(DE3) for heterologous expression, resulting in a recombinant enzyme with an approximate molecular weight of 38 kDa. Enzymatic characterization revealed that the recombinant enzyme exhibited optimal activity at pH 5.0 and 20 ℃. Furthermore, the enzyme demonstrated broad pH and temperature tolerance, retaining over 60% of its relative activity after incubation at pH 3.0–8.0 for 12 h, or at 15, 20, 25, and 37 ℃ for 100 min. Notably, it exhibited robust stability near the human physiological temperature (37 ℃). The addition of K⁺, Mg²⁺, and Zn²⁺ significantly enhanced the enzyme's activity to over 120% of its initial level. These findings indicate that the urate oxidase derived from Streptomyces sp. 2YHDJ-2 possesses broad pH and temperature adaptability alongside high stability. This study provides a promising microbial resource and a strong experimental basis for developing highly sensitive uric acid diagnostic reagents and novel urate oxidase preparations.