Interactions Between Deep Eutectic Solvent and Laccase

Jou Chin Chan

Deep eutectic solvents (DESs), a unique class of green solvents, have recently emerged as green and inexpensive alternative solvents to classical ionic liquids after they were unveiled approximately around 17 years ago. The usage of DES as an alternative solvent and co-solvent for biocatalytic reactions has start to grow at an exponential rate, showing attractive and promising potentials in biomass applications. Laccase (EC 1.10.3.2, para-diphenol: dioxygen oxidoreductase), a robust multicopper oxidase that is widely distributed in nature, is capable of catalyzing the oxidation of a broad variety of substrates through the simultaneous reduction of molecular oxygen, producing water as the by product. As enzymatic approaches are currently being pursued to cultivate environmentally friendly processes, laccase is no doubt a promising choice due to its versatility owing to its remarkable substrate specificities. Therefore, this thesis presents a study on the impacts of different DESs on the behavior of laccase. This study demonstrates that different DESs have different activation and inhibition effects on the activity of both Trametes versicolor and Myceliophthora thermophila laccase, while the kinetic studies demonstrates that lactic acid: choline chloride-, glycerol: betaine- and glycerol: choline chloride DES are parabolic mixed type inhibitors, where two or more DES molecules bind onto the enzyme. The crystal structure of M. thermophila laccase revealed that this fungal laccase is a heavily glycosylated three-domain laccase containing four catalytic copper atoms with a type 2 copper and two type 3 coppers close to each other, forming a single trinuclear cluster. The presence of DES induced protein conformational changes at the active site, with the increase in the number of water molecules. The relationship between DES and laccase can be summarized as follows: laccase conformation is dependent on the local environments of the amino acids, where the active site experience slight modifications due to the increase of water molecules upon the addition of DES, thus causing the subsequent changes in the enzymatic activity and stability. This work validates the hypothesis that the presence of extensive hydrogen bond network in DES causes the changes in enzymatic performance, providing new insights toward applicability of DES in biocatalysis processes.

Interactions Between Deep Eutectic Solvent and Laccase

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