Stability Increase of Phenolic Acid Decarboxylase by a Combination of Protein and Solvent Engineering Unlocks Applications at Elevated Temperatures

• DOI: 10.1021/acssuschemeng.3c06513
• Publication Date: 2024-02-21T10:39:10Z
• Authors: Kamela Myrtollari; Elia Calderini; Daniel Kracher; Tobias Schöngaßner; Stela Galušić; Anita Slavica; Andreas Taden; Daniel Mokos; Anna Schrüfer; Gregor Wirnsberger; Karl Gruber; Bastian Daniel; Robert Kourist
• Keywords: Decarboxylation; Thermal Stability; Biocatalysis

Enzymatic decarboxylation of biobased hydroxycinnamic acids gives access to phenolic styrenes for adhesive production. Phenolic acid decarboxylases are proficient enzymes that have been applied in aqueous systems, organic solvents, biphasic and deep eutectic which makes stability a key feature. Stabilization the enzyme would increase total turnover number thus reduce energy consumption waste accumulation associated with biocatalyst In this study, we used ancestral sequence reconstruction generate thermostable decarboxylases. Investigation set 16 ancestors resulted identification variant an unfolding temperature 78.1 °C half-life time 45 h at 60 °C. Crystal structures were determined three selected ancestors. Structural attributes calculated fit different regression models predicting thermal variants not yet experimentally explored. The rely on hydrophobic clusters, salt bridges, hydrogen bonds, surface properties can identify more stable proteins out pool candidates. Further stabilization was achieved by application mixtures natural solvents buffers. Our approach is straightforward option enhancing industrial process.