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Insight into Catalytic Features of Native and Engineered Laccases and Synthesis of Added-Value Compounds
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Multicopper oxidases are a family of enzymes that catalyze the oxidation of substrates coupled
to the reduction of molecular oxygen to water. This family can be divided in metallo-oxidases that
preferably oxidize low-valence metal ions and, laccases that oxidize organic compounds more
efficiently. Laccases have a remarkable redox capability in lignin degradation and conversion processes.
In the present study, the catalytic efficiency (kcat/Km) of engineered 15E6-loop5 and 23B3
variants, obtained through laboratory evolution of the Aquifex aeolicus metallo-oxidase McoA, was
evaluated towards a set of lignin-related phenolics. The enzymes were produced, purified and
biochemically characterized. The kinetic parameters for the tested phenolics (syringyl, guaiacyl and p hydroxyphenyl type) were compared with those of model enzyme CotA-laccase. CotA-laccase was able
to oxidize all thirteen substrates while McoA-variants only oxidized eight. However, for these, the McoA
variants showed similar kcat/Km values to CotA. The best substrates were sinapyl alcohol coniferyl
alcohol caffeic acid, showing the importance of ortho and para substituents in the oxidation rates.
Overall, 15E6-loop5 exhibited slightly improved performance than 23B3 variant.
Bioconversion of sinapyl alcohol, coniferyl alcohol and caffeic acid into syringaresinol,
pinoresinol and phenillinsin A, respectively, known to exhibit interesting medical properties, were carried
out. The most promising result was obtained with sinapyl alcohol and this reaction was further optimized,
resulting in a higher yield of conversion to syringaresinol, as assessed by 1H NMR, with CotA than 15E6-
loop. At pH 8, the optimal pH for enzymes´ activity no syringaresinol was observed, pointing out to the
importance of sinapyl alcohol deprotonation (pKa = 9.4) in the target reaction. Beside its potential
medical uses, syringaresinol, with its two phenols and its rigid cis-fused bis-furanic moiety, is an
important substitute of bisphenol A, a petro based compound used as monomer in polycarbonate
plastics and epoxy resins.
Descrição
Palavras-chave
Lignin valorization Phenolics Sinapyl alcohol Syringaresinol Laccases