Protein engineering of laccases towards higher pH

Lignin, a high molecular weight branched polyphenol found in plants, is a source of aromatic compounds. As a by-product in the paper and pulp industry, lignin is traditionally burned and used as fuel. With compounding environmental concerns from climate change to fossil fuel depletion, the need for ecologically friendly, renewable and sustainable fuel alternatives becomes increasingly important. Lignin is a promising candidate with a projected market valuation of €12.2 billion by 2020-2025. Lignin valorization, however, is hampered by its complex chemical structure. Traditional chemical and mechanical lignin depolymerization processes, such as liquefaction, oxidation, solvolysis and hydrocracking are performed under harsh conditions and generate high amount of solid waste. In nature, enzymatic degradation of lignin occurs in water under mild conditions. Laccase-mediated lignin depolymerization has recently gained attention since laccases utilize oxygen from air as electron acceptor and releases water as the only by-product, obviating the need for co-factors or co-substrates for catalysis. One bottleneck, however, is the poor water solubility of lignin itself. Alkaline pHs favor lignin solubility but diminish laccase activity. Therefore, the overall goal of this work was to engineer a laccase with increased alkaline pH tolerance through the knowledge gaining directed evolution (KnowVolution) strategy and concurrently gain molecular understanding of the effect of pH on laccase activity for potential industrial applications. In this work, the laccase from the fungus Melanocarpus albomyces (MaL) was selected as the parent for the directed evolution campaign towards improved alkaline pH tolerance. A suitable high-throughput screening system based on the 4-aminoantipyrine colorimetric method was established and validated for laccase activity. Within the four phase KnowVolution approach, the Melanocarpus albomyces laccase activity at alkaline pH was successfully improved. Seven improved sequence saturation mutagenesis (SeSaM) variants out of 1200 were identified. Out of these variants, nine potential beneficial positions were determined and individually saturated. The most beneficial positions (L365 and L513) were then simultaneously saturated and recombined to generate the final variant MaL-M1 (L365E/L513M). A three-fold improved kcat at pH 9.8 was achieved (MaL-WT: kcat 2.11 ± 0.07 s 1, MaL-M1: kcat 6.0 ± 0.2 s-1) without compromising the catalytic efficiency of the enzyme. The identified substitutions in MaL-M1 are located in close proximity of the T1Cu site. Molecular dynamics simulations studies revealed that the L365E and L513M substitutions work synergistically towards enzyme stability and improved tolerance at higher pH. These results suggest that structural rearrangement surrounding the T1Cu site are key to improve the pH tolerance of laccase. Overall, one round of KnowVolution generated a variant of fungal laccase with the highest specific activity at pH 9.8. This work opens the possibility for an ecologically friendly, renewable and sustainable enzyme-based lignin valorization