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Yuto Nakano, Speaker at Speaker at CCT 2022 Conference
National Institute of Technology, Oyama College, Japan
Title : How an industrial laccase and anionic vesicles work on the oxidation of a mixture of the two substrates aniline and p-aminodiphenylamine in order to synthesize polyaniline in its conductive emeraldine salt form


Industrial laccase-catalyzed oxidation of the miscible substrates consisting of aniline and p-aminodiphenylamine (PADPA) in the presence of vesicles from sodium bis (2-ethylhexyl) sulfosuccinate (AOT) as templates was multilaterally investigated. Reaction products with their kinetic development were analyzed with in situ ultraviolet/visible/near infrared spectroscopy (UV/Vis/NIR), in situ electron paramagnetic resonance (EPR), and ex situ liquid chromatography with photodiode array detector/mass spectrometry (LC-PDA/MS). In addition, the oxidizing activity of industrial laccase was evaluated by 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS).

The reaction condition was preliminarily optimized based on in situ UV/Vis/NIR measurement. The reaction mixture obtained from [aniline] + [PADPA] = 0.6 mM + 0.7 mM, [AOT vesicles] = 1.5 mM, [laccase Y120] = 17 nM colored to dark green, and notably developed absorption peaks at λ ≈ 420, 1000 nm corresponding to formation of polaron (radical cations) until 24 h. In situ EPR measurement revealed that the reaction products contained significant radicals, which confirms polyaniline in its conductive emeraldine salt form (PANI-ES)-like products obtained. For ex situ LC-PDA/MS measurements of the extracted, deprotonated, and reduced products after 1 week, the main products were aniline tetramer while aniline and PADPA were almost consumed.

In contrast, the reaction products without AOT vesicles only colored to reddish-purple with small peak at λ ≈ 800 nm and no signal was observed in EPR. The reaction without laccase remained colorless and polaron was not detected UV/Vis/NIR and EPR. The comparative evaluation indicates both laccase and vesicles lead to the selective synthesis of PANI-ES.

Since the kinetic evaluation of the oxidizing activity of industrial laccase on ABTS, the presence of AOT vesicles decreased the maximum reaction rate Vmax, the turnover number (kcat) and Michaelis coefficient Km, however increased Hill coefficient n slightly. It was predicted that the protonated substrates in the acidic solution were loosely constrained on anionic interface of AOT vesicles. Therefore, the compatibility between substrates and laccase was advanced apparently.

The oxidation of aniline and PADPA was carried out at room temperature (T ≈ 25?C) in a phosphate solution of pH = 3.5  (0.1 M NaH2PO4 + H3PO4). This process is environmentally friendlier than the traditional chemical synthesis of PANI-ES, which occurs at strongly acidic conditions and a strong chemical oxidant, such as potassium dichromate or ammonium peroxodisulfate. Industrial laccase-catalyzed synthesis of PANI-ES in the presence of AOT vesicles as templates is promising to green chemical engineering environmentally as well as economically.

Audience Take Away:

  • Green chemical synthesis of electro-conductive polyaniline
  • Details of characterization for enzymatic oxidation of aniline and its dimer PADPA
  • Information of soft interfaces, especially vesicles, guiding the oxidative reaction
  • Utilization of industrial laccase as an oxidant for material production


Financial support for this study was provided by the Grants-in-Aid for Scientific Research (KAKENHI) 19K15353. Laccase Y120 was provided from Amano Enzyme CO., Ltd., Japan.


Mr. Yuto Nakano have studied chemical engineering, organic chemistry, inorganic chemistry, physical chemistry, and bioengineering at Department of Materials Chemistry and Bioengineering in National Institute of Technology, Oyama College (Oyama KOSEN) since 2016 for 5years. Afterward, he proceeded the advanced course of National Institute of Technology, Oyama College 2021. Mr. Nakano currently works on the enzymatic synthesis of conductive polyaniline in Laboratory of Chemical Engineering.