Screening,Identification And Functional Gene Study Of Urate Oxidase Producing Strains

Urate oxidase（Uox）is a key enzyme in purine metabolism and plays an important role in purine metabolic pathway.Urate oxidase widely exists in bacteria,fungi and many animals and plants.However,the urate oxidase gene of primates such as humans and apes has mutated in the process of evolution,making the urate oxidase gene a "pseudogene".Therefore,uric acid becomes the end product of purine metabolism in human body.When the concentration is too high,it will cause hyperuricemia,gout and other diseases.At present,urate oxidase has been used in the detection of uric acid and the treatment of some clinical diseases.However,the research on urate oxidase in China lags behind that in foreign countries.Therefore,it is urgent to explore new urate oxidase functional strains and functional genes.The main contents and results of this study are as follows:1.Screening and identification of functional strains:117 strains with the function of degrading uric acid were obtained by enrichment,culture and isolation.By 16 S r RNA gene sequencing and phylogenetic analysis,117 enzyme-producing strains were identified as 23 genera.Eight strains with high activity were selected by hydrolytic circle size and urate oxidase activity,which belonged to Streptomyces sp.（YHBJ-12 and2YHDJ-2）,Bacillus sp.（YHBJ-8 and 2YHBJ-3）,Arthrobacter sp.（CSAJ-12,CSAJ-16 and YHBJ-11）and Microbacterium sp.（YHDJ-3）.2.Amplification and cloning of functional genes:Combining primers were designed based on the sequence of the functional gene of urate oxidase in NCBI database,and urate oxidase genes from YHBJ-12,2YHDJ-2,CSAJ-12,CSAJ-16 and YHBJ-11 were successfully cloned and heterologous expressed using the genome of the functional strain as template.3.Study on enzymatic properties:Determine the influence of temperature,pH,metal ions and inhibitors on the activity of urate oxidase and study its enzymatic properties.The results showed that the optimum reaction temperature of urate oxidase was 20 ℃～25 ℃ and pH 5.0～7.0.The sensitivity of urate oxidase from different sources to metal ions and inhibitors is different.At pH 3.0～9.0,more than 20% enzyme activity could still be maintained for 24 h.Under the optimum temperature and two gradients above and below the optimum temperature（37 ℃ and recombinant enzyme）,the urate oxidase could maintain the activity of more than 60% for a long time（80 min）.These results indicate that there are abundant functional strains producing uric acid oxidase in the soil environment,and the functional strains and genes obtained in this study provide an important enzyme source reserve for the development and utilization of urate oxidase.