Simultaneous Production of Electricity and Galactaric Acid from Pectin with an Enzymatic Biofuel Cell

Abstract

Introduction Enzymes, which are reproducible catalysts, can catalyze selective and environmentally friendly production of platform chemicals from biomass, in simple systems. When the platform chemical-production is through one or more oxidation reactions, the electrons obtained from the substrate are usually just dumped to electron accepters. However, when a biofuel cell anode is used as an electron acceptor, enzymatic, oxidative conversions on the anode can be combined with O2 reduction on the cathode to generate electricity. Hence, the simultaneous production of platform chemicals and electricity is possible through an oxidative conversion with enzymatic biofuel cells. D-Galacturonic acid is included in most land plants as the main component of pectin. Because it is a cheap raw material that can also be extracted in large quantities from food process residues, such as fruit peels and pulp of sugar beets and chicory, the conversion of pectin to fuels and chemicals has been studied. meso-Galactaric acid is a target compound of a pectin biorefinery, and is expected to be both a chelating agent and a precursor for polymers with various applications, a cross-linking agent, and other platform chemicals. Uronate dehydrogenase, uronate oxidase, and glucose oxidase are known to oxidize D-galacturonic acid to produce meso-galactaric acid, so far. However, these enzymes are not suitable for an electrode catalyst. In vitro use of uronate dehydrogenase requires an expensive NAD addition as its cofactor. The oxidases donate electrons to O2 to result in reducing the electric current. Glucose dehydrogenase (GDH) which binds pyrroloquinoline quinone (PQQ) tightly, but not covalently, as its cofactor has superior characteristics as an electrode catalyst. PQQ-GDH contains its cofactor and GDH catalysis does not use O2. To date, there have been no reports on uronic acid oxidation, including D-galactuornic acid, catalyzed by GDHs. However, we expected PQQ-GDH to catalyze the oxidation of D-galacturonic acid into meso-galactaric acid because PQQ-GDH is known to have the broad selectivity for substrates including D-galactose. Hence, we observed the oxidation reaction of D-galacturonic acid catalyzed by PQQ-GDH mainly through NMR spectrometry and constructed a PQQ-GDH bioanode to produce meso-galactaric acid. Experimental NMR measurements. Methylene green (MG: ca. 15 mmol), D-galacturonic acid Na salt (ca. 15 mmol), and 0.13 mg PQQ-GDH were added to 530 μL of deuterated phosphate buffer (pD 7.1). NMR measurements of the mixture were conducted 1 h and 2 days after the mixing at 500 MHz, holding the probe temperature constant at 20 °C. Electrode preparation and electrochemical measurements. A conventional three-electrode cell was used, in which a Pt wire served as the counter electrode and Ag/AgCl (3 M NaCl) served as the reference electrode. PolyMG (PMG) film was formed on the PFC electrode by 5 sets of continuous cyclic sweeps in 0.5 mM MG / 0.1 M phosphate buffer (pH 7.0). Then, 6 μL of 50 μg μL-1 PQQ-GDH / 0.1 M phosphate buffer (pH 7.0) was dropped onto the PMG-modified electrode. The cyclic voltammetry (CV) measurements of the PQQ-GDH-modified electrode were conducted in 0.1 M D-galacturonic acid Na salt / 0.1 M phosphate buffer (pH 7.0). Results & discussion The NMR spectra obtained from the samples with PQQ-GDH showed the production of D-galactaro-1,4-lactone and meso-galactaric acid 1 h and 2 day after reaction started, respectively. The spectra of control samples without PQQ-GDH showed no signal from products. These results clearly showed PQQ-GDH-catalyzed D-galacturonic acid oxidation. The delayed appearance of meso-galactaric acid in the spectra that followed the emergence of D-galactaro-1,4-lactone reflected the non-enzymatic equilibrium among these chemical species as previously reported. To construct an enzymatic anode that produces meso-galactaric acid by using an electrode as the electron acceptor, a PQQ-GDH-modified electrode was fabricated. A redox couple with a half-wave potential (E 1/2) of approximately -0.10 V was observed when CV measurements of the PQQ-GDH-modified electrodes were conducted in the control solution without D-galacturonic acid (Figure 1: broken line). In the D-galacturonic acid Na salt solution, an increase in the anodic current density was observed, which started from around E 1/2 (Figure 1: solid line). In contrast, no catalytic current was observed in the cyclic voltammogram of the PMG-modified electrodes without PQQ-GDH in the D-galacturonic acid Na salt solution. The voltammograms of the PMG-modified electrodes in the D-galacturonic acid Na salt solution and in the control solution without D-galacturonic acid were almost the same. These results demonstrated continuous D-galacturonic acid oxidation, which was catalyzed by PQQ-GDH, and the subsequent oxidation of PQQ-GDH by the electrode. We will also report a biofuel cell with this PQQ-GDH-modified anode, which will produce meso-galactaric acid and electricity simultaneously. Figure 1