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

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Biotechnology

Background:

• Nitrilases are crucial enzymes in pharmaceutical fine chemical synthesis.
• Conventional methods for screening nitrilase activity are time-consuming and labor-intensive.
• A sensitive and direct assay is needed for efficient nitrilase variant screening.

Purpose of the Study:

• To develop a novel, sensitive, and rapid method for detecting nitrilase activity.
• To utilize time-resolved luminescence for enhanced signal detection.
• To optimize fermentation conditions for nitrilase-producing microorganisms.

Main Methods:

• Development of a time-resolved luminescent probe based on o-hydroxybenzonitrile derivatives.
• Enzymatic conversion of derivatives to salicylic acid by nitrilases.
• Luminescence detection of Tb(3+) sensitized by salicylic acid derivatives.
• Application of the assay on 96- or 384-well plates for high-throughput screening.

Main Results:

• The o-hydroxybenzonitrile derivative probe successfully detects nitrilase activity.
• Time-resolved luminescence effectively reduces background noise, increasing assay sensitivity.
• The assay allows for rapid determination of nitrilase activity from microbial sources.
• Optimized fermentation conditions were identified using this novel screening strategy.

Conclusions:

• A novel time-resolved luminescent probe offers a sensitive and rapid method for nitrilase activity screening.
• This assay facilitates efficient enzyme engineering and optimization of bioprocesses.
• The developed method accelerates the discovery of industrially relevant nitrilase variants and production strains.