Removal of N -linked glycans in cellobiohydrolase Cel7A from Trichoderma reesei reveals higher activity and binding affinity on crystalline cellulose

• Published in: Biotechnology for biofuels Vol. 13; no. 1; p. 136
• Main Authors: Kołaczkowski, Bartłomiej M, Schaller, Kay S, Sørensen, Trine Holst, Peters, Günther H J, Jensen, Kenneth, Krogh, Kristian B R M, Westh, Peter
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 06.08.2020; BioMed Central; BMC
• Subjects: Analysis; Aspergillus oryzae; Binding; Biodegradation; Biomass; Carbohydrates; Cellobiohydrolase; Cellulase; Cellulose; Computer simulation; Conformation; Crystalline cellulose; Deficient mutant; Density; Enzyme kinetics; Enzymes; Fungi; GH7 cellulase; Glycan; Glycosidases; Glycoside hydrolase; Glycosylation; Heterogeneous interfacial enzyme kinetics; Hydrolase; Kinetics; Lignocellulose; Mass spectrometry; Molecular dynamics; Molecular weight; Mutation; N-glycans; N-Glycosylation; Occupancy; Polysaccharides; Proteins; Reaction kinetics; Scientific imaging; Steric hindrance; Structure-function relationships; Substrates; Trichoderma reesei; Trichoderma reesei Cel7A; Heterogeneous interfacial enzyme kinetics; Trichoderma reesei Cel7A; GH7 cellulase; Aspergillus oryzae; N-Glycosylation; MD
• ISSN: 1754-6834; 1754-6834
• DOI: 10.1186/s13068-020-01779-9

Cellobiohydrolase from glycoside hydrolase family 7 is a major component of commercial enzymatic mixtures for lignocellulosic biomass degradation. For many years, Cel7A ( Cel7A) has served as a model to understand structure-function relationships of processive cellobiohydrolases. The architecture of Cel7A includes an -glycosylated catalytic domain, which is connected to a carbohydrate-binding module through a flexible, -glycosylated linker. Depending on the fungal expression host, glycosylation can vary not only in glycoforms, but also in site occupancy, leading to a complex pattern of glycans, which can affect the enzyme's stability and kinetics. Two expression hosts, and , were utilized to successfully express wild-types Cel7A (WT and WT ) and the triple -glycosylation site deficient mutants Cel7A N45Q, N270Q, N384Q (Δ -glyc and Δ -glyc ). Also, we expressed single -glycosylation site deficient mutants Cel7A (N45Q , N270Q , N384Q ). The Cel7A enzymes were studied by steady-state kinetics under both substrate- and enzyme-saturating conditions using different cellulosic substrates. The Michaelis constant ( ) was consistently found to be lowered for the variants with reduced -glycosylation content, and for the triple deficient mutants, it was less than half of the WTs' value on some substrates. The ability of the enzyme to combine productively with sites on the cellulose surface followed a similar pattern on all tested substrates. Thus, site density (number of sites per gram cellulose) was 30-60% higher for the single deficient variants compared to the WT, and about twofold larger for the triple deficient enzyme. Molecular dynamic simulation of the -glycan mutants Cel7A revealed higher number of contacts between CD and cellulose crystal upon removal of glycans at position N45 and N384. The kinetic changes of Cel7A imposed by removal of -linked glycans reflected modifications of substrate accessibility. The presence of -glycans with extended structures increased and decreased attack site density of Cel7A likely due to steric hindrance effect and distance between the enzyme and the cellulose surface, preventing the enzyme from achieving optimal conformation. This knowledge could be applied to modify enzyme glycosylation to engineer enzyme with higher activity on the insoluble substrates.