Huang Su-Hua, Wu Tung-Kung
Department of Biological Science and Technology, National Chiao Tung University, Hsin-Chu, Taiwan, Republic of China.
Eur J Biochem. 2004 Feb;271(3):517-23. doi: 10.1046/j.1432-1033.2003.03951.x.
This study describes a modified colorimetric assay for uricase activity in flexible 96-well microtiter plates using the uricase/uric acid/horseradish peroxidase/4-aminoantipyrine/3,5-dichloro-2-hydroxybenzene sulfonate colorimetric reaction. The utility of this assay was demonstrated in a screen for mutant uricase enzymes derived from the uricase gene of the thermophilic bacterium Bacillus subtilis by a modified staggered extension process (StEP) mutagenesis. An Escherichia coli library of StEP-derived uricase mutant clones was screened yielding two identical active mutant uricase genes. Two motifs conserved in eukaryotic and prokaryotic uricases are highly conserved in the mutant uricase. The mutant uricase protein was found to exhibit high uricase activity (13.1 U.mg(-1)). Finally, the modified colorimetric method is much more efficient than the conventional ones and greatly reduces assay time from 4 days to less than 20 h.
本研究描述了一种在灵活的96孔微量滴定板中使用尿酸酶/尿酸/辣根过氧化物酶/4-氨基安替比林/3,5-二氯-2-羟基苯磺酸盐比色反应来测定尿酸酶活性的改良比色法。通过改良的交错延伸过程（StEP）诱变，从嗜热细菌枯草芽孢杆菌的尿酸酶基因中筛选突变尿酸酶，证明了该测定方法的实用性。对一个由StEP衍生的尿酸酶突变克隆的大肠杆菌文库进行筛选，得到了两个相同的活性突变尿酸酶基因。在突变尿酸酶中，真核和原核尿酸酶中保守的两个基序高度保守。发现突变尿酸酶蛋白具有高尿酸酶活性（13.1 U.mg(-1)）。最后，改良比色法比传统方法效率更高，大大缩短了测定时间，从4天减少到不到20小时。
