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

Chemistry Central Journal

Juan Feng¹  Hongbo Liu¹  Xiaolan Yang¹  Ang Gao¹  Juan Liao¹  Liping Feng¹  Jun Pu¹  Yanling Xie¹  Gaobo Long¹  Yuanli Li¹  Fei Liao¹ 

[1] Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Clinical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China

关键词: Uricase;    Threshold;    Spectrometric methods;    Specific activity;    Positive candidate;    Interference;    Activity concentration;   

Others  :  787924
DOI  :  10.1186/1752-153X-7-69

 received in 2012-12-18, accepted in 2013-03-06,  发布年份 2013

【 摘 要 】

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.

【 授权许可】

   
2013 Feng et al.; licensee Chemistry Central Ltd.

【 图 表 】

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