Cloning,Expression,and Purification Of Uricase Enzyme From Kluyveromyces Marxianus.Using Escherichia Coli

Uricase(UOX)plays an important role in purine metabolism in the human body..Uricase is a key enzyme in purine metabolism,which can catalyze the conversion of uric acid to allantoin and hydrogen peroxide,the solubility of uric acid in the body of is 11 mg / 100 mL H2 O,whereas the solubility of allantoin is about 147 mg / 100 mL H2 O,which is about 13.5 folds of the solubility for uric acid.Therefore,the conversion of uric acid into allantoin can prompt rapid dissolution of allantoin to eliminate the body to reduce the body's uric acid content.Human and primate animals lose the gene for uricase during evolution,so uricase accumulates in the body and causes various conditions such as gout,hyperuricemia,urate nephropathy and the like.So uric acid oxidase is an important medical enzyme.Uricase,as a clinically new drug for treating gout,rapidly catalyzes the breakdown of uric acid and thus reduces the amount of uric acid in the human body,and can degrade the level of uric acid crystals in the human body.Compared with other treatment methods,uric acid oxidase has a rapid effect and no side effects.Serum uric acid is an important indicator of diagnosis of gout and hyperuricemia,uric acid oxidase can also be used for the detection of uric acid in the human body,it's can quickly detect the concentration of uric acid in the human body,uricase can not only for clinical Treatment but also for clinical testing reagent applications.Our study cloned uricase gene from Kluyveromyces marxianus by PCR and constructed the recombinant vector CBM3-INTEIN-UOX.The recombinant plasmid was transformed into Escherichia coli BL21(DE3)expressing cells and induced by IPTG.Successfully expressed the protein.The recombinant strain utilizes the binding of the cellulose binding moiety to the intein,resulting in the cleavage of the intein by a change in temperature and pH to yield pure,unlabeled uricase(KmUOX).This study explored the fragmentation of the peptide under different conditions and the purification efficiency of the protein.Through the exploration of the purification conditions,we finally found that the purification efficiency can reach 77.11% under the conditions of 40 ?for 9 hours.The purity of uricase obtained was measured by the change of UV absorbance at 293 mm.The highest enzyme activity of 50.54 IU / mg was found to be the most active among the uricase tested previously.The Km,Vmax,Kcat were 67.60 ?M,56.35 ?mol / min · mg,and 32.74 S-1,respectively.In the subsequent exploration of the nature found that the optimum ph optimum temperature were 9.0 and 42 ?.After exploring the thermal stability of purified uricase,it was found that it still retains 79.75% of its activity after being incubated at 40 ° C for 90 hours,which has certain thermal stability.Our study found a new uricase,which has higher activity and certain thermal stability than other uricase,which provides a new option for the future study or application of uricase.