Bioconversion of phenolic acids from hydrolyzed sugarcane bagasse into vinyl derivatives by recombinant phenolic acid...

Several commercially important vinyl phenolic compounds can be produced by enzymatic decarboxylation of phenolic acids such as ferulic and p-coumaric acids which can be extracted from agro-industrial waste. Phenolic acid decarboxylase is an enzyme that acts in the decarboxylation of these acids to 4-vinylguaiacol and 4-vinylphenol, respectively. In this study, the gene encoding phenolic acid decarboxylase from Klebsiella pneumoniae TD 4.7 was isolated and identified as a 504 bp fragment, encoding a polypeptide of 167 amino acid residues. A 98 % predicted amino acid sequence identity between ferulic acid decarboxylase from other bacteria of the same genus was determined. The gene was successfully expressed in Escherichia coli BL21 (DE3), and the recombinant enzyme was purified as active in absence of cofactor. The protein had a mass of 22-kDa protein, with greater activity at pH 5.5 and 40 °C. The decarboxylase activity was inhibited by Hg^2 + , Zn²⁺, Cu²⁺, and Cd²⁺ ions and increased by 20 % in the presence of Co²⁺. The K_m and V_max values for the recombinant enzyme were estimated at 2.95 mM and 102.10 µmol min^-1 mg^-1, respectively. The enzyme's structure was modelled using the structural prediction programs AlphaFold Multimer and SWISS-MODEL, with an RMSD of just 0.7 Å, demonstrating the absence of cysteine and disulfide bonds in the homodimer, with the presence of a high number of lysine residues. The amino acids involved in the catalytic site were Tyr27, Glu134, and Asn23. The Enzyme activity on substrates ferulic and p-coumaric acids extracted from sugarcane bagasse, resulted in 4-vinylguaiacol and 4-vinylphenol, respectively, with conversion yields of 43 % for ferulic acid and 55 % for p-coumaric acid. These data are important in terms of obtaining an enzyme that decarboxylates ferulic and p-coumaric acids obtained from sugarcane bagasse hydrolyzed with similar efficiency, in a single step and without the need for a cofactor, making it an excellent option for bioprocesses using lignocellulosic biomass derivatives.