Novel sensitive high-throughput screening strategy for nitrilase-producing strains

Abstract

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.

FIG. 1.

Design principle of the time-resolved luminescent nitrilase probes.

FIG. 2.

Synthesis of the fluorogenic nitrilase probes.

FIG. 3.

Proposed mechanisms for luminescence quenching.

FIG. 4.

Luminescence spectrum of the four probes after the treatment of nitrilase (20 μg/ml in 50 mM Tris buffer, pH 8, at 30°C). The increase in luminescence was monitored with a λ_ex of 328 nm in Spectrum M2 96-well black flat-bottom plates after 1 h.

FIG. 5.

Luminescence spectra of probe 1 (50 μM) against different enzymes. The increase in luminescence was monitored with a λ_ex of 328 nm in Spectrum M2 96-well black flat-bottom plates after 1 h of incubation at 30°C.

FIG. 6.

Fluorescence responses of Rhodococcus equi (20 μg/ml in 50 mM Tris buffer, pH 8, at 30°C) to various concentrations of probes 1 to 3. Data were collected after incubation for 1 h.

FIG. 7.

Michaelis-Menten plots of probe 2 with nitrilases from Rhodococcus equi.

FIG. 8.

Effect of carbon source on the activity of Bacillus subtilis E9 by HPLC (A) and the fluorescent method (probe 2) (B).