Isothermal titration calorimetric assessment of lignin conversion by laccases

Abstract

Lignin valorization may offer a sustainable approach to achieve a chemical industry that is not completely dependent on fossil resources for the production of aromatics.However, lignin is a recalcitrant, heterogeneous, and complex polymeric compound for which only very few catalysts can act in a predictable and reproducible manner.Laccase is one of those catalysts and has often been referred to as an ideal "green" catalyst, as it is able to oxidize various linkages within lignin to release aromatic products, with the use of molecular oxygen and formation of water as the only side product. The extent and rate of laccase-catalyzed lignin conversion were measured using the label-free analytical technique isothermal titration calorimetry (ITC). IITC provides the molar enthalpy of the reaction, which reflects the extent of conversion and the time-dependent power trace, which reflects the rate of the reaction. Calorimetric assessment of the lignin conversion brought about by various fungal and bacterial laccases in the absence of mediators showed marked differences in the extent and rate of conversion for the different enzymes. Kraft lignin conversion by Trametes versicolor laccase followed Michaelis-Menten kinetics and was characterized by the following thermodynamic and kinetic parameters ΔH ITC = −(2.06 ± 0.06)•10 3 kJ mol −1 , K M = 6.6 ± 1.2 μM and V max = 0.30 ± 0.02 U/mg at 25°C and pH 6.5. We envision calorimetric techniques as important tools for the development of enzymatic lignin valorization strategies.

Isothermal Titration Calorimetry in Biocatalysis

“…The calorimetric assessment of lignin oxidation by different bacterial and fungal laccases was performed using the IrCal method (Figure 1) (A. Islam et al, 2022). This showed that the extent of lignin conversion, as measured from the apparent molar enthalpy, and the rate of that conversion, obtained from the power, showed large differences among the different enzymes.…”

“…Here ITC can offer a pragmatic solution by measuring the overall conversion of lignin by an enzyme. This has been done for the lignin conversion by different laccase variants and showed that the extent of conversion and the rate of that conversion can be determined (Figure 1), although the precise molecular nature of the products needs to be determined independently (Islam et al, 2022).…”

“…Large differences in the extent and kinetics of lignin conversion among the different bacterial and fungal laccases were found. Reprinted with permission from(Islam et al, 2022).…”

Laccase-catalyzed lignin depolymerization in deep eutectic solvents: challenges and prospects

“…Solution-based spectrophotometric assays are unsuitable for measuring the oxidative rates of non-soluble lignin by laccase (Valles et al 2020 ). Recently, advanced techniques such as 2D NMR, nanostructure-initiator mass spectrometry (NIMS), electron paramagnetic resonance (EPR) spectroscopy, and isothermal titration calorimetric (ITC) have been developed to study lignin depolymerization by laccase (Deng et al 2018 ; Perna et al 2019 ; ST et al 2022 ). These methods present distinct advantages over solution-based spectrophotometric assays: NIMS offers abundant information on bond cleavage and quantitative analysis of product generation along with kinetics, EPR enables monitoring the kinetics of radical formation during the laccase-catalyzed conversion of lignin, and ITC can gain independent information on the extent and rate of lignin conversion.…”