Purification and characterization of hydroxycinnamate decarboxylase from Brettanomyces anomalus

The yeast, Brettanomyces anomalus, produces a hydroxycinnamic acid decarboxylase which is active toward ferulic acid p-coumaric acid, and caffeic acid The enzyme transforms these hydroxycinnamic acids to hydroxystyrenes by the removal of the carboxyl group from the C3 side chain. We have purified this enzyme 235-fold from B. anomalus NCYC 615 using Mono Q ion exchange, Phenyl Superose, and Superose 12 column chromatography. Enzyme activity was found to be optimal at 40 degrees C and pH 6.0 and was enhanced by EDTA, Mg2+, and Cr3+. Fe3+, Ag+, and SDS completely inhibited the activity. Kinetic studies indicated a K-m of 1.15 mM and a V-max of 13,494 nmol min(-1) mg(-1) for ferulic acid and a K-m of 1.55 mM and a V-max of 22,256 nmol min(-1) mg(-1) for p-coumaric acid. Using gel filtration, an apparent molecular mass of 39.8 kDa was estimated. The decarboxylase was inactive toward both o- and m-coumaric acid and toward cinnamic acid, indicating that the para-hydroxy group is essential for activity. (C) 1998 Elsevier Science Inc.