Development of a rapid assay for -etherase activity using a novel chromogenic substrate

Biocatalytic processes play a crucial role in the valorization of lignin; therefore, methods enabling the monitoring of enzymes such as beta-etherases, capable of breaking beta-O-4 aryl-ether bonds, are of significant biotechnological interest. A novel method for quantifying beta-etherase activity was developed based on the beta-ester bond formation between a chromophore and acetovainillone. The chromogenic substrate beta-(rho-nitrophenoxy)-alpha-acetovanillone (PNPAV), was chemically synthesized. Kintetic monitoring of rho-nitrophenolate release at 410 nm over 10 min, using recombinant LigF from Sphingobium sp SYK-6, LigF-AB and LigE-AB from Althererytrobacter sp B11, yielded enzimatic activities of 404. 3 mU/mg, 72 mU/mg, and 50 mU/mg, respectively. This method is applicable in a pH range of 7.0-9.0, with a sensitivity of up to 50 ng of enzyme, exhibiting no interference with lipolytic, glycolytic, proteolytic, and oxidoreductase enzymes.