Novel sensitive high-throughput screening strategy for nitrilase-producing strains.
Nitrilases have found wide use in the pharmaceutical industry for the production of fine chemicals, and it is important to have a method by which to screen libraries of isolated or engineered nitrilase variants (including bacteria and fungi). The conventional methods, such as high-performance liquid chromatography, liquid chromatography-mass spectrometry, capillary electrophoresis, or gas chromatography, are tedious and time-consuming. Therefore, a direct and sensitive readout of the nitrilase's activity has to be considered. In this paper, we report a novel time-resolved luminescent probe: o-hydroxybenzonitrile derivatives could be applied to detect the activity of the nitrilases. By the action of nitrilases, o-hydroxybenzonitrile derivatives can be transformed to the corresponding salicylic acid derivatives, which, upon binding Tb(3+), serve as a photon antenna and sensitize Tb(3+) luminescence. Because of the time-resolved property of the luminescence, the background from the other proteins (especially in the fermentation system) in the assay could be reduced and, therefore, the sensitivity was increased. Moreover, because the detection was performed on a 96- or 384-well plate, the activity of the nitrilases from microorganisms could be determined quickly. Based on this strategy, the best fermentation conditions for nitrilase-producing strains were obtained.
Zhu
Qing
Q
Institute of Bioengineering, Zhejiang University of Technology (Chaohui Campus), Hangzhou 310014, China. chmzhuq@hotmail.com
Fan
Ao
A
Wang
Yuanshan
Y
Zhu
Xiaoqin
X
Wang
Zhao
Z
Wu
Minghuo
M
Zheng
Yuguo
Y
D000619:Aminohydrolases / Q000032:analysis* / Q000378:metabolism; D001419:Bacteria / Q000201:enzymology*; D005658:Fungi / Q000201:enzymology*; D049449:Luminescence; D015336:Molecular Probe Techniques; D009570:Nitriles / Q000737:chemistry / Q000378:metabolism*; D004364:Pharmaceutical Preparations / Q000032:analysis*
D009570:Nitriles; D004364:Pharmaceutical Preparations; D000619:Aminohydrolases; C021867:nitrilase