Xylenol-orange-assay-of-hydrogen-peroxide for Measuring Uricase Activity and Recognizing High-activity Uricase Mutant

Abstract

This study investigated xylenol-orange-assay-of-hydrogen-peroxide(XOAHP) for measuring uricase activities in cell lysates and recognizing mutants of higher activity.Four mutants of the intracellular uricase of Bacillus fastidiosus were combined into pairs so that ratios of their catalytic capacities varied from 1.3 to 4.1.The pET28a vector containing mutant gene was transformed into Escherichia coli BL21(DE3).Each of thirty clones was transferred into 1.0 mL liquid medium for amplification,and subject to induced expression for 16 h at 18 ℃.Cells were then harvested and lyzed via sonication treatment to get the supernatant as lysate.In Tris-HCl buffer at pH 8.9,XOAHP was effective to measure uricase activity with uric acid of no more than 0.33 mmol/L.SDS-PAGE showed small differences in expression efficiency of mutants.The activity concentration was positively related to the level of total proteins in lysates of tested mutants.Receiver-operation-curve(ROC) analysis showed the higher the ratio of catalytic capacities of uricase mutants in pairs the chloser to 1.00 of the area-under-thecurve(AUC),with an AUC 0.95 for a pair of mutants at the ratio of about 1.8 between their catalytic capacities.With a threshold of the difference between the activity concentration of a candidate as 1.4 times that of the standard deviation of the starting material,positive mutants of catalytic capacities 1.8 times that of the starting material could be effectively recognized.Therefore,XOAHP plus a proper threshold is effective for high-throughput-screening of mutant library of uricase.