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

• Published in: Talanta (Oxford) 2024-04, Vol.270, p.125501-125501, Article 125501
• Main Authors: Romero-Soto, Itzel Celeste, Rodríguez, Jorge A, Armenta-Pérez, Vicente Paúl, Martínez-Pérez, Raúl Balam, Camacho-Ruiz, Rosa María, Alencar Menezes, Leociley Rocha, Sassaki, Guilherme Lanzi, Santana-Filho, Arquimedes, Camacho-Ruiz, María Angeles
• Format: Article
• Language: English
• ISSN: 0039-9140; 1873-3573
• DOI: 10.1016/j.talanta.2023.125501

Biocatalytic processes play a crucial role in the valorization of lignin; therefore, methods enabling the monitoring of enzymes such as β-etherases, capable of breaking β-O-4 aryl-ether bonds, are of significant biotechnological interest. A novel method for quantifying β-etherase activity was developed based on the β-ester bond formation between a chromophore and acetovainillone. The chromogenic substrate β-(ρ-nitrophenoxy)-α-acetovanillone (PNPAV), was chemically synthesized. Kintetic monitoring of ρ-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.