Phenolic substrates for fluorometric detection of laccase activity

A fluorometric procedure has been developed for detection and estimation of laccase activity in fungal broth cultures. Laccase solution was pretreated with catalase for 1 h at 37°C and pH 5. Homovanillic acid was then added and the reaction mixture incubated for a further hour at 37°C. The fluorescence was then developed by addition of 0.1 M glycine buffer at pH 10. Laccase preparations from Pyricularia oryzae, Coriolus hirsutus and Pycnoporus cinnabarinus catalysed formation of a fluorescent product of HVA but the optimum pH values of enzyme activities varied. The culture fluids of several other fungi also catalysed development of fluorescence in solutions containing HVA. p-Hydroxyphenylacetic acid was a poor substrate for all laccases in vivo except that produced by Perennigora tephropora.