High Catalytic Current Density Based on Direct Bioelectrocatalysis of a PQQ Domain of Pyranose Dehydrogenase
Authors: Takeda Kouta , Kusuoka Ryo , Yoshida Makoto , Igarashi Kiyohiko , Samejima Masahiro , Ohno Hiroyuki , Nakamura Nobuhumi 
Published: February 2020

Introduction

A pyranose dehydrogenase from the basidiomycete Coprinopsis cinerea (CcPDH) is an eukaryotic pyrroloquinoline quinone (PQQ)-dependent dehydrogenase consisting three-domains structure [1]. The PQQ and heme b cofactors are located in the 45 kDa and 21 kDa domains, respectively, and a small C-terminal domain is family 1-type carbohydrate-binding module, which these domains are connected by a proline-rich linker region. CcPDH shows the oxidation activity toward monosaccharides in a 1C4 chair conformation such as D-glucosone and L-fucose. The N-terminal cytochrome domain is a 6-coordinated low-spin heme b with Met/His ligands that enables direct electrical contact between the enzyme and the electrode [2]. Electrons are transferred from reduced PQQ in catalytic domain to heme b in cytochrome domain. The previous study reported that intact CcPDH is capable of direct electron transfer (DET)-based bioelectrocatalysis on a glassy carbon (GC) electrode [3]. The catalytic currents were observed at a lower potential than the redox potential of heme b in the cytochrome domain, suggesting that both the PQQ and the cytochrome domains in CcPDH are able to DET with the GC electrode.
In present study, to examine DET reaction of the PQQ domain, an isolated PQQ domain was expressed in Pichia expression system. Here, we demonstrated the direct bioelectrocatalysis for the isolated PQQ domain from C. cinerea pyranose dehydrogenase. The bioelectrocatalytic current density of 1.6 mAcm-2 was achieved on a preformed 2-mercaptoethanol (ME) self-assembled monolayer (SAM) modified gold nanoparticles (AuNPs) electrode under optimized conditions.

Experimental

The direct bioelectrocatalysis of DHPDH was obtained by using glassy carbon (GC) electrode modified with the enzyme as working electrode. An aliquot of 30 μL of 1 μM enzyme solution containing 10 μM PQQ and 1 mM CaCl2 was dropped on the entire surface of the GC electrode and then dried at room temperature. AuNPs-modified and unmodified polycrystalline Au electrodes were used for the direct bioelectrocatalysis of DHPDH. 1 μL of the concentrated AuNPs was dropped onto the surface of the Au electrode, and then air-dried. To prepare electrodes with immobilized DHPDH, AuNPs-modified and unmodified polycrystalline Au electrodes were immersed in 20 mM aqueous solution of 2-mercaptoethanol for 1 h at room temperature (ME-AuNPs electrode and ME-Au electrode). Then they were immersed in 1 μM holo-DHPDH in a 50 mM sodium acetate buffer solution (pH 6.0) at 4 ℃ for over 15 h. Cyclic voltammetry measurements were performed in 50 mM various pH buffer with 100 mM L-fucose. A conventional three-electrode cell was used, in which a platinum wire served as the counter electrode and Ag/AgCl (3 M NaCl) served as the reference electrode.

Results and Discussion

Initially, a clear catalytic current of L-fucose oxidation by the DHPDH on the GC electrode was observed starting at same potential of intact CcPDH. It is demonstrated that the DHPDH can also directly transfer electrons to the electrodes. The catalytic current density of DHPDH (0.9 μA cm-2, at 0.35 V) was an order of magnitude smaller than that of intact CcPDH. To improve the direct bioelectrocatalysis of DHPDH, a thiol-based SAM coated Au electrodes were studied. Accordingly, the optimal SAM for DHPDH was found to be shorter alkyl chain length and hydroxide-functionalized thiols. The catalytic current density of 11 μA cm-2 (at 0.35 V) was obtained when using ME-Au electrode. DHPDH immobilized on ME-Au electrode showed optimum at pH 6.0 and a value of K
m = 35.1 mM, respectively. In order to obtain higher catalytic currents, furthermore, AuNPs were cast on the surface of Au electrode. Increasing the number of AuNP casts from 3 to 15 times on led to increasing catalytic current densities from 125 μA cm-2 to 240 μA cm-2 at 0.35 V. Additionally, we had considered the effects of the electrolyte (ion conductivity) and temperature dependence for catalytic current by AuNPs-Au electrode at the casting cycle three times. The catalytic current at 50 °C was approximately 5-times higher than that at 20 °C. Upon increasing acetate buffer concentration from 50 mM to 500 mM, the current density was enhanced to as 1.7-fold. Figure 1 shows the cyclic voltammogram of L-fucose oxidation on DHPDH-modified ME-AuNPs electrode under optimized conditions of 50 °C, pH 6, 500 mM acetate buffer, and the AuNPs casting cycle 15 times. Finally, the catalytic current reached to a steady-state value of 1.6 mA cm-2.

References

[1] H. Matsumura et al., PloS One
9, e104851 (2014).
[2] K. Takeda et al., PloS One
10, e0115722 (2015).
[3] K. Takeda et al., submitted
.

Figure 1