Non-productive binding of cellobiohydrolase i investigated by surface plasmon resonance spectroscopy

Florian Csarman,Lena Wohlschlager,Gijs Van Erven,Mirjam A Kabel,Roland Ludwig,Johannes Konnerth,Antje Potthast,Claudia Gusenbauer

doi:10.1007/s10570-021-04002-6

Abstract

Future biorefineries are facing the challenge to separate and depolymerize biopolymers into their building blocks for the production of biofuels and basic molecules as chemical stock. Fungi have evolved lignocellulolytic enzymes to perform this task specifically and efficiently, but a detailed understanding of their heterogeneous reactions is a prerequisite for the optimization of large-scale enzymatic biomass degradation. Here, we investigate the binding of cellulolytic enzymes onto biopolymers by surface plasmon resonance (SPR) spectroscopy for the fast and precise characterization of enzyme adsorption processes. Using different sensor architectures, SPR probes modified with regenerated cellulose as well as with lignin films were prepared by spin-coating techniques. The modified SPR probes were analyzed by atomic force microscopy and static contact angle measurements to determine physical and surface molecular properties. SPR spectroscopy was used to study the activity and affinity of Trichoderma reesei cellobiohydrolase I (CBHI) glycoforms on the modified SPR probes. N-glycan removal led to no significant change in activity or cellulose binding, while a slightly higher tendency for non-productive binding to SPR probes modified with different lignin fractions was observed. The results suggest that the main role of the N-glycosylation in CBHI is not to prevent non-productive binding to lignin, but probably to increase its stability against proteolytic degradation. The work also demonstrates the suitability of SPR-based techniques for the characterization of the binding of lignocellulolytic enzymes to biomass-derived polymers.Graphic abstract

Highlights

The global climate crisis together with an increasing demand for energy and goods is the driving force of intensified research on sustainable resources
Cellulose films from Microcrystalline cellulose (MCC) dissolved in DMAc/LiCl and lignin films using Milled Wood Lignin (MWL) or organosolv lignin (OSL) dissolved in dioxane/water were prepared by spincoating
While MWL from spruce is almost exclusively composed of G-units and the extraction procedure prevents extensive alterations of the naturally occurring lignin, OSL from beech wood is composed of mainly S and G-units and is substantially modified and condensed during the extraction

Summary

Introduction

The global climate crisis together with an increasing demand for energy and goods is the driving force of intensified research on sustainable resources. The efficient utilization of lignocellulosic biomass is considered to be a key technology for the transition from fossil resources towards a sustainable economy (Ragauskas et al 2006; Chandel et al 2018; De Bhowmick et al 2018). A comprehensive understanding of the structure of plant biomass as well as of the molecular mechanisms, equilibrium constants and kinetic rates of the enzyme– substrate interaction is crucial to optimize enzymatic lignocellulose hydrolysis (Himmel et al 2007; Merino and Cherry 2007; Dos Santos et al 2016). In contrast to the completely amorphous hemicelluloses, cellulose chains are able to interact to regular semi-crystalline fibres and fibrils stabilized by hydrogen bond networks and van der Waals forces

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Results

Conclusion