An efficient assay for ferulic acid esterase and para-coumaric acid esterases in plants at physiologically relevant activities

4-Nitrophenyl ferulate and 4-nitrophenyl coumarate have been synthesised as chromogenic substrates for measurement of the activity of feruloyl esterases. These enzymes have been isolated from a number of fungi and are found in micro-organisms involved in the degradation of plant material. Ferulic and para-coumaric acid were coupled with 4-nitrophenol using water-soluble carbodiimide to yield 4-nitrophenyl ferulate and 4-nitrophenyl coumarate, respectively. Maize, tall fescue and Miscanthus varieties, transformed with an Aspergillus niger ferulic acid esterase (FAE) gene, were analysed for activity using these synthetic substrates. This technique gave a rapid measurement of enzymic activity when compared with the conventional HPLC assay. This colorimetric assay was also more sensitive and accurate for determining rates of activity. A rapid high-throughput assay, based on substrates described here, would greatly facilitate identification of enzymes with potential to increase the efficiency of sugar release from lignocellulosic biomass and thereby reduce costs of processing.