Abstract
BackgroundCellobiohydrolase from glycoside hydrolase family 7 is a major component of commercial enzymatic mixtures for lignocellulosic biomass degradation. For many years, Trichoderma reesei Cel7A (TrCel7A) has served as a model to understand structure–function relationships of processive cellobiohydrolases. The architecture of TrCel7A includes an N-glycosylated catalytic domain, which is connected to a carbohydrate-binding module through a flexible, O-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.ResultsTwo expression hosts, Aspergillus oryzae and Trichoderma reesei, were utilized to successfully express wild-types TrCel7A (WTAo and WTTr) and the triple N-glycosylation site deficient mutants TrCel7A N45Q, N270Q, N384Q (ΔN-glycAo and ΔN-glycTr). Also, we expressed single N-glycosylation site deficient mutants TrCel7A (N45QAo, N270QAo, N384QAo). The TrCel7A enzymes were studied by steady-state kinetics under both substrate- and enzyme-saturating conditions using different cellulosic substrates. The Michaelis constant (KM) was consistently found to be lowered for the variants with reduced N-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 N-glycan mutants TrCel7A revealed higher number of contacts between CD and cellulose crystal upon removal of glycans at position N45 and N384.ConclusionsThe kinetic changes of TrCel7A imposed by removal of N-linked glycans reflected modifications of substrate accessibility. The presence of N-glycans with extended structures increased KM and decreased attack site density of TrCel7A 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.
Highlights
Cellobiohydrolase from glycoside hydrolase family 7 is a major component of commercial enzymatic mixtures for lignocellulosic biomass degradation
For the Trichoderma reesei Cel7A (TrCel7A) variants, the successful disruption of the N-glycosylation motifs through N to Q site-directed mutagenesis was verified by a decreased apparent molecular weight as shown by both SDS-PAGE (Fig. 1) and changes in the mass profiles of intact protein mass spectrometry (MS) (Additional file 1: Fig. S1)
Among the TrCel7A variants expressed in A. oryzae, the most pronounced shift in molecular weight compared to the WTAo sequence was observed for the N45QAo, which
Summary
Cellobiohydrolase from glycoside hydrolase family 7 is a major component of commercial enzymatic mixtures for lignocellulosic biomass degradation. Cellulose-degrading enzymes were first discovered in the secretome of the filamentous ascomycete Trichoderma reesei almost 65 years ago [1] Since they have proven essential in commercial enzyme cocktails used in biorefineries that produce fuels and chemicals from lignocellulosic biomass. Extensive research has sought to either engineer catalytically more efficient enzymes or to develop more efficient expression hosts such as Trichoderma reesei [3], Saccharomyces cerevisiae [4] and Aspergillus niger [5] The latter effort has enabled industrial production of cellulases, but usually with a range of isoforms with different apparent molecular weights [6]. This is attributed to the ability of the fungal expression host to decorate proteins with short oligosaccharides. Such post-translational modification called glycosylation can occur at either threonine (T) or serine (S) residues (O-glycosylation), or at asparagine (N) residues (N-glycosylation), which have a general consensus motif N-X-S/T (where X denotes any amino acid residue except proline) [6]
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