Rationally designed mutations on recombinant arginine deiminase (ADI) could act as a ‘turn-off’ L-arginine (L-Arg) fluorescent biosensor and provide an alternative method for rapid determination of L-Arg. Double mutations were introduced on the Cys²⁵¹➔Ser²⁵¹ and Thr²⁶⁵➔Cys²⁶⁵ of recombinant ADI, rendering a single cysteine present on the protein surface for the site-specific attachment of a fluorophore, fluorescein-5-maleimide. The double mutations on ADI (265C) and its fluorescein-labelled form (265Cf) conserved the catalytic efficiency of wild-type ADI. Upon binding to L-Arg, 265Cf induced structural conformational changes and rendered the fluorescein moiety to move closer to Trp²⁶⁴, resulting in fluorescence quenching. The duration of fluorescence quenching was dependant on the L-Arg concentration. A linear relationship between the time at the maximum rate of fluorescence change and L-Arg concentrations, which ranged from 2.5 to 100 μM, was found with R² = 0.9988. The measurement time was within 0.15–4 min. Determination of L-Arg concentration in fetal bovine serum could be achieved by the standard addition method and without sample pre-treatment. The result showed a good agreement with the one determined by mass spectrometry, suggesting our biosensor as a promising tool for the detection of L-Arg in biological samples.

重组精氨酸脱亚氨酶（ADI）的合理设计的突变可以充当“关闭”的L-精氨酸（L-Arg）荧光生物传感器，并提供快速测定L-Arg的替代方法。双突变引入上上的Cys ²⁵¹ ➔Ser ²⁵¹和Thr ²⁶⁵ ➔Cys ²⁶⁵重组ADI的，渲染单个半胱氨酸存在用于荧光团的位点特异性附着，荧光素-5-马来酰亚胺蛋白质表面上。ADI的双突变（265C）及其荧光素标记的形式（265Cf）保留了野生型ADI的催化效率。与L-Arg结合后，265Cf诱导结构构象变化，并使荧光素部分更靠近Trp ²⁶⁴，导致荧光猝灭。荧光猝灭的持续时间取决于L-Arg浓度。发现最大荧光变化速率的时间与L-Arg浓度之间的线性关系介于2.5至100μM之间，R ²  = 0.9988。测量时间在0.15–4分钟内。胎牛血清中L-Arg浓度的测定可以通过标准添加方法完成，而无需样品预处理。结果表明与质谱测定的结果吻合良好，表明我们的生物传感器是检测生物样品中L-Arg的有前途的工具。