Thermostable Peroxidase−Polylysine Films for Biocatalysis at 90 °C

Connecticut, Storrs, Connecticut 06269, and Department of Pharmacology, UniVersity of Connecticut Health. Center, Farmington, Connecticut 06032. Recei...
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J. Phys. Chem. B 2007, 111, 9125-9131

9125

Thermostable Peroxidase-Polylysine Films for Biocatalysis at 90 °C Peterson M. Guto,† Challa V. Kumar,† and James F. Rusling*,†,§,‡ Department of Chemistry and Institute of Materials Science, 55 North EagleVille Road, UniVersity of Connecticut, Storrs, Connecticut 06269, and Department of Pharmacology, UniVersity of Connecticut Health Center, Farmington, Connecticut 06032 ReceiVed: February 23, 2007; In Final Form: May 10, 2007

Cross-linked films of poly(L-lysine) (PLL) and enzymes covalently linked to surfaces provided remarkable thermostability, enabling biocatalysis at 90 °C. Soret spectra, circular dichroism, and voltammetry showed that PLL films containing peroxidases or myoglobin were stable for up to 9 h at 90 °C, while the same enzymes in solution denatured completely within 20 min. Biocatalytic reduction of t-BuOOH with enzymePLL films, using rotating disk voltammetry, provided Michaelis kcat/Km values. Results showed that horseradish peroxidase (HRP)-PLL is 3-fold more active than soybean peroxidase (SBP)-PLL at 25 °C, but SBP-PLL is slightly more active at 90 °C. SBP-PLL films had 8-fold larger kcat/Km values at 90 °C compared to 25 °C. Oxidation of o-methoxyphenol to 3,3′-dimethoxy-4,4′-biphenoquinone by peroxidase-PLL-coated silica colloids gave better yields at 90 °C than 25 °C, suggesting increasing catalytic efficiency and selectivity at the higher temperature. These biocolloids were reusable with little loss of activity at 90 °C.

Introduction Biocatalysis is predicted to have a major impact on future industrial chemical processes, especially in regio- and stereoselective syntheses.1 Significant effort has been expended to discover enzymes that operate at high temperatures to improve catalytic efficiency. For example, enzymes isolated from thermophilic bacteria have been used in industrial processes.2 Improved thermal stability of enzymes has also been achieved by chemical modification and directed evolution,3 and by immobilization on specialized solid supports.4 While these approaches have been effective, they rarely produce biocatalysts that can be used near the boiling point of water, and typically operate between 45 and 70 °C. We successfully stabilized redox proteins for use in potentially denaturing media such as acidic and basic solutions and microemulsions by incorporating them into polyelectrolyte films. Specifically, films grown a layer at a time by alternate deposition of proteins and oppositely charged polyions stabilized the secondary structure of myoglobin (Mb) in acidic and basic media in which the dissolved protein is rapidly and completely denatured.5 When we later wanted to use biocatalyst films in microemuslions, we found that enhancing enzyme stability in these media required covalent cross-linking between proteins, polyions, and solid surfaces. Excellent stability and catalytic efficiency in buffers and microemuslions at room temperature was obtained for films made by linking Mb to poly(L-lysine) (PLL) covalently bound to graphite electrodes.6,7 We focus here on enzymes with peroxidase activity, specifically soybean peroxidase (SBP), an inherent thermostable enzyme, horseradish peroxidase (HRP), and Mb. Peroxidases have produced excellent product yields with high stereo- and enantioselectivities for reactions catalyzed at ordinary temperatures.8,9 * Address correspondence to this author. E-mail: [email protected]. † Department of Chemistry, University of Connecticut. § Department of Pharmacology, University of Connecticut Health Center. ‡ Institute of Material Science, University of Connecticut.

Scheme 1 illustrates peroxidase-catalyzed pathways for organic reactant (R) involving oxygenation6,7 and carboncarbon bond formation8 via radical dimerization. Compound I is the •PFeIVdO radical form of the enzyme obtained by reaction with peroxides that can transfer oxygen to or accept electrons from substrate R. Compound II, nonradical PFeIVdO, can also oxidize substrate.8,9 Scheme 1 can be driven by electrochemical catalytic reduction of oxygen to H2O2 (on right) or by adding H2O2 (on left), a cheap source of oxidizing power. In this paper, we demonstrate for the first time remarkable stability for up to 9 h at 90 °C for HRP, SBP, and Mb afforded by cross-linking them into films of PLL attached to oxidized carbon electrodes or carboxylated silica surfaces. In contrast, the dissolved enzymes are completely denatured in