A practical high-throughput screening system for feruloyl esterases: Substrate design and evaluation

Feruloyl esterases (FAEs) are a group of important industrial enzymes, which could hydrolyze the ester bonds between hydroxycinnamic acids and arabinose or galactose in hemicellulose present in plant cell walls. To establish a practical high-throughput screening system of feruoyl esterases, two new substrates with modified chromo- or fluoro-phore, 2-chloro-4-nitrophenyl ferulate (CNPF; 1b) and umbelliferyl 5-O-feruolyl-α-L-arabinofuranoside (UFA; 2a), were designed and synthesized. Both substrates provided significantly improved signal-to-noise ratio compared with previously known structural analogues. The chromogenic substrate CNPF could be readily adapted to the high-throughput screening of feruloyl esterase A from Aspergillus niger, one of the most studied FAEs, with the coefficient of variance of as low as 10.3% as determined for a single sequence library.