Improvement of hydrogen peroxide stability of Pleurotus eryngii versatile ligninolytic peroxidase by rational protein engineering.

Peroxide tolerant versatile peroxidases are required for industrial applications. In this study, rational protein engineering was performed to improve the oxidative stability of Pleurotus eryngii versatile ligninolytic peroxidase. Residues which are easily oxidized such as methionine, and close to H2O2-binding pocket and heme were identified for site-directed mutagenesis. Enzyme activity and steady-state kinetics were affected to different extent by different mutations. They were investigated for H2O2 stability, among which mutants A79L, P141A, M247L, M265L, M247L/M265L, A77E/I81L, A77E/A79S/I81L, A77S/A79L/I81L, A77E/A79S/I81L/M265L, A77E/A79S/I81L/M247L/M265L, and A77E/A79S/I81L/S168A showed significantly increased oxidative tolerance, proving that oxidizable residues such as Met247 and Met265, residues close to heme like Pro141 and H2O2-binding pocket such as Ala77, Ala79, and Ile81 exerted important impact on H2O2 stability. Double and triple mutants demonstrated some additive or synergistic effects, which were only inactivated by higher concentration H2O2, whereas multiple mutants A77E/A79S/I81L/M265L, A77E/A79S/I81L/M247L/M265L, and A77E/A79S/I81L/S168A did not. Importantly, mutants I81L, S168A, Met265, M247L/M265L, A77E/A79S/I81L, and A77E/A79S/I81L/M247L/M265L exhibited both improved catalytic efficiencies and H2O2 resistance. The enhanced oxidative stability could result from delayed or suppressed compound III formation and/or heme bleaching caused by replacement of some residues. The identified mutants with higher oxidative tolerance and catalytic efficiencies would be helpful for further improving the oxidative stability of versatile peroxidase.