Comparison of activity indexes for recognizing enzyme mutants of higher activity with uricase as model.

ABSTRACT

BACKGROUND: For screening a library of enzyme mutants, an efficient and cost-effective method for reliable assay of enzyme activity and a decision method for safe recognition of mutants of higher activity are needed. The comparison of activity concentrations of mutants in lysates of transformed Escherichia coli cells against a threshold is unsafe to recognize mutants of higher activity due to variations of both expression levels of mutant proteins and lysis efficiency of transformed cells. Hence, by a spectrophotometric method after verification to measure uricase activity, specific activity calculated from the level of total proteins in a lysate was tested for recognizing a mutant of higher activity. RESULTS: During uricase reaction, the intermediate 5-hydroxyisourate interferes with the assay of uric acid absorbance, but the measurement of absorbance at 293 nm in alkaline borate buffer was reliable for measuring uricase initial rates within a reasonable range. The level of total proteins in a lysate was determined by the Bradford assay. Polyacrylamide gel electrophoresis analysis supported different relative abundance of uricase mutant proteins in their lysates; activity concentrations of uricase in such lysates positively correlated with levels of total proteins. Receiver-operation-curve analysis of activity concentration or specific activity yielded area-under-the-curve close to 1.00 for recognizing a mutant with > 200% improvement of activity. For a mutant with just about 80% improvement of activity, receiver-operation-curve analysis of specific activity gave area-under-the-curve close to 1.00 while the analysis of activity concentration gave smaller area-under-the-curve. With the mean plus 1.4-fold of the standard deviation of specific activity of a starting material as the threshold, uricase mutants whose activities were improved by more than 80% were recognized with higher sensitivity and specificity. CONCLUSION: Specific activity calculated from the level of total proteins is a favorable index for recognizing an enzyme mutant with small improvement of activity.