Comparative Spectroelectrochemical Studies of Lyophilized and Nonlyophilized Laccases from Cerrena unicolor Basidiomycete

Abstract

The electrochemical, spectroelectrochemical, and kinetic investigations of two preparations of Cerrena unicolor laccase, lyophilized (LLAC) and nonlyophilized frozen enzymes (FLAC), were performed. It was found that the value of the redox potential of the T1 site of C. unicolor laccase is ca. 750 vs. NHE. It was also shown that one of the redox potentials of the T2/T3 cluster of C. unicolor laccase is close to 400 mV, as was previously confirmed for other blue multicopper oxidases, such as trees and fungal laccases, ascorbate oxidase, and bilirubin oxidase. Furthermore, the poor stability of both preparations, but especially of LLAC, in their reduced state was confirmed using mediated and mediatorless spectroelectrochemical studies. DET-based biocatalytic reduction of O 2 by C. unicolor laccase was only obtained, when FLAC was directly adsorbed on a spectrographic graphite electrode. Moreover, only low values of the steady-state potentials of gold and graphite electrodes modified by C. unicolor laccase were also found. Heterogeneity of the 3-D structures of laccase molecules, conformational changes, and partial denaturation of the enzyme, which appeared after enzyme isolation, purification, and especially lyophilization, were found to be the reasons for the low bioelectrocatalytic current, the high K M -value towards O 2 , and the unusual electrochemical behavior of C. unicolor laccase used in the present study. In spite of the comparable specific activity and long-term stability of both preparations in homogeneous solution, the stability of immobilized LLAC was found to be inadmissibly low for both fundamental studies and possible electrochemical applications. Indeed, FLAC is a much better source of enzyme than its lyophilized counterpart.

Direct Heterogeneous Electron Transfer Reactions of Bacillus halodurans Bacterial Blue Multicopper Oxidase

“…590 mM O 2 ) at 458C. The apparent Michaelis constant (K M ) for the adsorbed enzyme was found to be very low compared to previously reported values of other BMCOs; approximately 100 mM for enzymes in solution up to 300 mM for adsorbed enzymes [31,46,54,59]. In order to measure a K Mvalue for the non-adsorbed BMCO additional experiments were performed with the enzyme dissolved in buffer Fig.…”

“…For the formation of the MUA-modified gold acid monolayer, a similar procedure was used except that the thiol solution was 1 mM MUA in ethanol, which contained 2% (v/v) trifluoroacetic acid to avoid the formation of multilayers of the enzyme [45,46].…”

Biocathodes for Dioxygen Reduction in Biofuel Cells

“…Obviously, the latter group is most interesting for application in biocathodes. The redox potential of the T 1 center of laccase from Cerrena unicolor used by our groups is equal 0.75 V versus NHE [30][31][32].…”

“…The first of the proposed strategies involves irreversible adsorption of the enzyme [30,33,50,52, or its covalent bonding to the electrode surface [30,33,34,[84][85][86][87][88][89][90][91][92]. Two types of materials, carbon-based and bare and modified gold, were used to prepare biocathode with adsorbed laccase, bilirubin oxidase, copper efflux oxidase, microperoxidase-11, and Fe-Fe dehydrogenase.…”

“…The first two enzymes were found to weakly adsorb on smooth carbon material-glassy carbon (GC) [56]. The other carbon-based materials for adsorption of these enzymes include graphite [30,55,76,77], carbon surface with high crystal graphite edge density [69], and edge plane of pyrolytic graphite [57,79,80]. A significant enhancement of laccase adsorption was found when pyrolytic graphite was covered by anthracene-based units immobilized by diazonium coupling [33].…”