We report on the properties of hydrogel matrix for the immobilization of laccase on conductive supports. The poly(N-isopropylacrylamide) gel is attached firmly to the indium-tin oxide (ITO) electrode, following its silanization with dimethylethoxyvinylsilane. The enzyme entrapped in the gel structure remained active longer than in the solution, and its redox and catalytic properties could be investigated by voltammetric methods. The reduction signals of the active sites, T1 and T2, of the Cerrena unicolor laccase were determined to be 0.79 and 0.38 V, respectively. The laccase catalytic activity toward oxygen in poly(N-isopropylacrylamide) was found to depend strongly on temperature. Reversible swelling/shrinking of the matrix was studied at 30 and 35 degrees C. Shrinking of the gel at higher temperature considerably decreased the efficiency of the catalytic reaction, however, interestingly, did not lead to irreversible changes in the enzyme structure. At temperatures below that corresponding to volume phase transition, the catalytic properties of the film were fully restored. High catalytic efficiency of the gel immobilized enzyme made it possible to employ the gel covered electrode for monitoring oxygen in solutions.

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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.