Cold generation of smoke flavour by the first phenolic acid decarboxylase from a filamentous ascomycete - Isaria farinosa.

A decarboxylase (IfPAD) from the ascomycete Isaria farinosa converted ferulic acid to 4-vinylguaiacol (4-VG), a volatile which imparts the distinct "smoke flavor" of pyrolized wood. The activity was enhanced by adding (E)-ferulic acid to the culture medium and peaked with 3.6 U g-1 mycelium (1 μmol 4-VG min-1 ). The coding sequence of 543 bp was translated into a 25 kDa protein with a homology of 91 % to putative phenolic acid decarboxylases of its teleomorph, Cordyceps militaris, and Beauveria bassiana, the anamorph of Cordyceps bassiana. Cold shock expression in Escherichia coli yielded 411 U g-1 wet mass. Substrate conversion required a hydroxyl substituent para to a trans-unsaturated C3-side chain of the aromatic ring. Km and kcat /Km values were determined to 0.3 mM and 78.4 mM-1 s-1 for p-coumaric acid and 1.9 mM and 45.1 mM-1 s-1 for (E)-ferulic acid, respectively. The native enzyme and its recombinant counterpart showed pH-optima at pH 6.0 and pH 5.5, and low temperature optima of 19 °C and 14 °C, respectively. IfPAD produced 4-VG from destarched wheat bran and sugar beet fiber, confirming activity on complex plant biomass. This is the first report on the biochemical characterization of a phenolic acid decarboxylase from a filamentous ascomycete.