Immobilization of the Pyrroloquinoline Quinone Dependent Alcohol Dehydrogenase with Polyelectrolyte Membranes on the Glassy Carbon Electrode
Authors: Sakurada Yuki , Sugimoto Chikanobu , Takeda Kouta , Ohno Hiroyuki , Nakamura Nobuhumi 
Published: February 2020

Introduction

　Quinoproteins containig pyrroloquinoline quinone (PQQ) catalyze a dehydrogenation of the primary and the secondly hydroxyl group in alcohols or sugars. These are interesting not only in their role in physiological function but also in biotechnological approaches in the fields of bioelectronics, such as biofuel cells and biosensors. In a previous study, PQQ alcohol dehydrogenase from Pseudomonas putida KT2440 (PpADH) was overexpressed in the methylotrophic yeast Pichia pastoris for a bioanode [1]. This enzyme has broad substrate selectivity towards various alcohols and aldehydes, and can catalyze the two-step oxidation of alcohols. Thus, it is expected to develop a new bioanode that the two-step oxidation of ethanol to acetate using only PpADH. Immobilization of enzymes on electrodes is critical for the development of the enzymatic bioanode. Some polymeric materials were utilized for the modification of the enzyme electrodes so far. In this study, we have optimized the immobilization of PpADH on the glassy carbon electrodes using polyelectrolyte membranes.

 Experimental

 Recombinant PpADH was heterologously expressed in P. pastoris and purified as described previously [1]. Cationic polymers; polyethyleneimine (PEI) and poly (diallyldimethylammonium chloride) (PDDA) and an anionic polymer; poly(sodium 4-styrenesulfonate) (PSS) were used to immobilize purified PpADH on the glassy carbon electrodes (GCE). Three types of single polyelectrolyte modified electrodes were prepared; PEI (A), PDDA (B), and PSS (C). 0.5 wt % PEI, PDDA, or PSS and 9 µM the enzyme aqueous solution were dropped onto a pretreated GCE. After drying at least an hour at room temperature, the single polyelectrolyte modified electrodes with PpADH were obtained. Further, four types of multi polyelectrolyte modified electrodes were prepared by combining different polyelectrolytes; electrode (A) with PSS (D), electrode (B) with PSS (E), electrode (C) with PEI (F), and electrode (C) with PDDA (G). All electrochemical measurements were performed using a conventional three-electrode cell containing GCEs modified with PpADH and polyelectrolytes as working electrodes, a platinum wire as a counter electrode, and an Ag/AgCl (3 M NaCl) electrode as a reference electrode. Cyclic voltammetry (CV) was performed in the 50 mM CHES buffer (pH 9.0) containing 1 mM CaCl2, 30 mM ethylamine, and 100 µM aminoferrocene as an electron mediator (Fig 1).

 Results and Discussion

 The cyclic voltammograms of aminoferrocene exhibited reversible responses at -80 mV (E1/2) without substrate for all polyelectrolyte modified electrodes. The catalytic currents of ethanol oxidation by PpADH were observed for electrodes (A), (B), and (C). However, when the number of the cycles was increased, the catalytic currents were decreased. This result suggested that PpADH was removed from the electrodes. To prevent the release of PpADH, the modified electrodes with PpADH were prepared by combining different polyelectrolytes. Catalytic currents of ethanol oxidation of PpADH were also observed in electrodes (D), (E), (F), and (G). Figure 2 shows the current densities of these electrodes. While the current density of electrode (F) was the lowest in all electrodes, the current densities of the other electrodes were about the same (18 µA/cm2) at 0.3 V of the first cycle. However, the catalytic current of electrode (D) at the fifth cycle was considerably decreased to 35 % (6.4 µA/cm2). The catalytic currents of (E) and (G) electrodes slightly decrease to 73 % (13.2 µA/cm2) and 89 % (17.2 µA/cm2), respectively. The current density of electrode (G) was the highest in all electrodes at the fifth cycle. These results indicated that the electrostatic interaction between PSS and PDDA is effective to immobilize PpADH on the electrodes. The order to add PSS and PDDA is also important. PSS and PpADH should be added on the electrode firstly and then PDDA should be added after drying.

 Reference [1] K. Takeda et al., Bioelectrochemistry
94 (2013) 75-78

Figure 1