一种腈水解酶的高通量筛选方法

One High-Throughput Screening Strategy for Nitrile-Hydrolyzing Enzymes

水解腈的酶类(腈水解酶或腈水合酶/酰胺酶)在制药行业中生产有机羧酸及其衍生物方面有着广泛的应用,为了获得较多的酶源,建立高通量筛选方法是非常必要的。今利用改进的羟肟酸铁分光光度比色法建立了一种简单、快速、高通量的筛选方法,同时利用产酶菌株Rhodococcus sp.CCZU10-1静息细胞催化反应来进行方法的准确性验证。并且通过与液相色谱法检测结果进行对比,结果表明羟肟酸铁比色法对有机羧酸的检测具有较高的准确性。因此,建立的高通量筛选方法在产腈水解酶微生物筛选及应用方面有很大应用前景。

The production of carboxylic acids and their derivatives which are hydrolyzed from nitrile by nitrile-hydrolyzing enzyme（nitrilase or nitrile hydratase/amidase） have been widely used in pharmaceutical.In order to obtain more nitrile-hydrolyzing enzymes（nitrilase or nitrile hydratase/amidase）,it is very important to build a high-throughput screening strategy.In this paper,a modified ferric hydroxamate spectrophotometry was established,which is simple,rapid,and high-throughput.The accuracy of the method established was validated by the bioconversion of nitrile hydrolysis with the resting cells of Rhodococcus sp.CCZU10-1.The results indicate that the accuracy of this method for assaying carboxylic acids is as high as the HPLC-based method.Therefore,the method established can be used for the screening and application of microorganisms with nitrile-hydrolyzing activity.