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

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

Inhibitory effect of lignin on the hydrolysis of xylan by thermophilic and thermolabile GH11 xylanases

“…The role of enzyme glycosylation on lignin tolerance of enzymes has been studied less. Recently, it has been reported that deglycosylation of T. reesei Cel7A slightly increases the non-productive adsorption of this cellobiohydrolase to lignin [ 37 ]. Furthermore, by changing the glycosylation pattern of the CBM1 of TrCel7A, preferential adsorption to cellulose over lignin could be increased [ 38 ].…”

Cellulases, hemicellulases and ligninolytic enzymes: mechanism of action, optimal processing conditions and obtaining value-added compounds in plant matrices

“…A common reaction that occurs with hemicellulose is acetylation, the acetyl group can interfere with the cellulase active site. 30 Arabinoxylooligomers, specifically the less substituted ones, show a greater affinity to occupy the active site compared to cellulose itself, for which the hydrolysis of arabinoxylan to xylose and arabinose is recommended. 31 There are different soluble toxins in the hydrolytic medium that can cause enzymatic deactivation or cause enzyme precipitation, one of them corresponds to the generation of compounds during the process, such as phenolic compounds derived from lignin or the generation of acetyl groups previously described, the same glucose production can generate the inhibition of β-glucosidase.…”

Fluorescent Imaging of Extracellular Fungal Enzymes Bound onto Plant Cell Walls

“…Different cellulose preparations, including Avicel or α-cellulose, as well as lignin isolated by different extraction procedures, are commonly used as model substrates to study the adsorption of enzymes on lignocellulosic biomass [33][34][35]. In this study, commercial α cellulose generated by alkali treatment of cotton linters was selected to investigate enzyme localization and distribution by confocal laser scanning microscopy (CLSM) using two different approaches: (i) directly labeled enzymes; or (ii) applying native enzymes and specific conjugated antibodies.…”