Isothermal titration calorimetric assessment of lignin conversion by laccases.

Shams T A Islam,Fabio Tonin,Vlada B Urlacher,Peter‐Leon Hagedoorn,Yanthi N Deurloo,Jie Zhang,Renske Hinderks

doi:10.1002/bit.27991

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)·103 kJ mol−1, KM = 6.6 ± 1.2 μM and Vmax = 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.

Highlights

Biorefineries create lignin waste streams, the amount of which is expected to exceed 200 megatons by 2022 (Bruijnincx et al, 2016)
Enzyme calorimetry using isothermal titration calorimetry (ITC) and IrCal gives us two parameters: (1) the area under the curve—reflecting the total conversion and the molar enthalpy of the reaction and (2) the power trace over time which reflects the rate of the reaction and which can be further utilized to determine appropriate kinetic parameters of the enzyme‐catalyzed reaction
The multi‐injection method with a versatile peroxidases (VPs)‐ITC instrument (Malvern) was applied as reported previously (Honarmand Ebrahimi et al, 2015) to measure the molar reaction enthalpies of T. versicolor, M. thermophila, and B. subtilis laccases acting upon lignin (ΔHr, lignin)

Summary

| INTRODUCTION

Biorefineries create lignin waste streams, the amount of which is expected to exceed 200 megatons by 2022 (Bruijnincx et al, 2016). LiP generally possesses a very high redox potential of the active site heme cofactor (1.2 V vs NHE at pH 3.0) compared to the other enzymes (Kersten et al, 1990) This property enables it to catalyze the oxidation of non‐phenolic aromatic compounds directly, without the need for a mediator. Under conditions of the same temperature, pH, and enzyme concentration, changes in the rates of the enzyme‐catalyzed reactions can be brought about by changing the concentration of the substrate and these would be reflected in the ITC power measurements. Enzyme calorimetry using ITC and IrCal gives us two parameters: (1) the area under the curve—reflecting the total conversion (and equilibrium) and the molar enthalpy of the reaction and (2) the power trace over time which reflects the rate of the reaction and which can be further utilized to determine appropriate kinetic parameters of the enzyme‐catalyzed reaction. Phosphate‐buffered saline (PBS) was prepared by dissolving 10 mM NaCl in 100 mM potassium phosphate buffer, pH 5.8

| Methods

| RESULTS

| DISCUSSION