Gallic acid interference on polyphenolic amperometric biosensing using Trametes versicolor laccase

The present work reports the gallic acid (GA) interference on polyphenolic amperometric biosensing using Trametes versicolor laccase (TvLac). GA′ inhibitory effect on TvLac activity was investigated on the oxidation of caffeic acid (CA) by free TvLac and its immobilised form on modified polyethersulfone membrane (PES/TvLac), using spectrophotometric and amperometric biosensor detection methods. The results have indicated that GA presents inhibitory behaviour on TvLac activity in a concentration-dependent manner. The GA concentration leading to 50% activity lost, IC50, was determined to be 19.15 ± 0.11 μM and 5.11 ± 0.19 μM for free and immobilised enzyme, respectively. The results have also shown that GA exhibited a competitive and a mixed inhibition types on the TvLac activity for spectrophotometric and amperometric biosensor methods, respectively. Further GA′ and CA′ cyclic voltammetry studies have demonstrated that GA′ oxidation products interfered with CA′ redox reaction products. In fact, a decrease of the reduction current was observed at cyclic voltammograms of CA, when mixed with GA. Therefore, the GA′ interference on polyphenolic amperometric biosensing is the result of the combination of two factors: on one hand, we have the inhibitory enzymatic effect, and on the other, the reaction of GA′ oxidation products with the o-quinones obtained by the enzymatic oxidation of CA. Both gave rise to the amperometric signal decreasing effect.

This work investigates the gallic acid (GA) interference on polyphenolic amperometric biosensing using Trametes versicolor laccase (TvLac). (A) Effect of GA on the activity of TvLac. Caffeic acid (CA) was used as substrate; free TvLac (spectrophotometric method) (○) and immobilised (PES/TvLac) (amperometric biosensor method) (●). (B) Cyclic voltammograms of a mixture of CA and GA at fixed concentration of CA (50 μM) and varying the GA concentration from 0 to 500 μM, corresponding to a–g, respectively.Figure optionsDownload as PowerPoint slideHighlights
► Tv Laccase was less sensitive to GA inhibition when free than when immobilised.
► GA decreased peak currents when mixed with CA, as measured by cyclic voltammetry.
► GA should not be used as reference in polyphenolic determinations with PPO's.