Banerjee Anirban, Kaul Praveen, Sharma Rohit, Banerjee U C
Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Sector-67, S. A. S. Nagar, Punjab 160 062, India.
J Biomol Screen. 2003 Oct;8(5):559-65. doi: 10.1177/1087057103256910.
Based on the color change of an indicator due to the release of hydrogen ion from a nitrilase-catalyzed reaction, a rapid colorimetric method was established for the enantioselective screening of nitrilase-producing microorganisms. The formation of acids due to the nitrilase-mediated hydrolysis of nitriles causes a drop in the pH, which in turn results in a change of color of the solution (containing indicator) that can be observed visually. The buffer (0.01 M phosphate, pH 7.2) and indicator (Bromothymol blue, 0.01%) were selected in such a way that both have the same affinity for the released protons. The enantioselectivity of nitrilases was estimated by comparing the hydrolysis of (R)-mandelonitrile with that of racemate under the same conditions. The method was used to screen a library of nitrilase-producing microorganisms, isolated in the authors' laboratory for their ability to enantioselectively hydrolyze mandelonitrile to mandelic acid, an important chiral building block.
基于腈水解酶催化反应中氢离子释放导致指示剂颜色变化，建立了一种用于对产腈水解酶微生物进行对映选择性筛选的快速比色法。腈水解酶介导的腈水解产生的酸会导致pH值下降，进而导致溶液（含指示剂）颜色发生肉眼可见的变化。选择缓冲液（0.01 M磷酸盐，pH 7.2）和指示剂（溴百里酚蓝，0.01%），使其对释放的质子具有相同的亲和力。通过比较相同条件下（R）-扁桃腈与外消旋体的水解情况来评估腈水解酶的对映选择性。该方法用于筛选作者实验室分离的产腈水解酶微生物文库，以评估它们将扁桃腈对映选择性水解为扁桃酸（一种重要的手性结构单元）的能力。
