A biochemical comparison of fungal GH6 cellobiohydrolases

Abstract

Cellobiohydrolases (CBHs) from glycoside hydrolase family 6 (GH6) make up an important part of the secretome in many cellulolytic fungi. They are also of technical interest, particularly because they are part of the enzyme cocktails that are used for the industrial breakdown of lignocellulosic biomass. Nevertheless, functional studies of GH6 CBHs are scarce and focused on a few model enzymes. To elucidate functional breadth among GH6 CBHs, we conducted a comparative biochemical study of seven GH6 CBHs originating from fungi living in different habitats, in addition to one enzyme variant. The enzyme sequences were investigated by phylogenetic analyses to ensure that they were not closely related phylogenetically. The selected enzymes were all heterologously expressed in Aspergillus oryzae, purified and thoroughly characterized biochemically. This approach allowed direct comparisons of functional data, and the results revealed substantial variability. For example, the adsorption capacity on cellulose spanned two orders of magnitude and kinetic parameters, derived from two independent steady-state methods also varied significantly. While the different functional parameters covered wide ranges, they were not independent since they changed in parallel between two poles. One pole was characterized by strong substrate interactions, high adsorption capacity and low turnover number while the other showed weak substrate interactions, poor adsorption and high turnover. The investigated enzymes essentially defined a continuum between these two opposites, and this scaling of functional parameters raises interesting questions regarding functional plasticity and evolution of GH6 CBHs.

Activity of fungal β-glucosidases on cellulose

“…This is moderately lower than specificity constants reported for the cellulases Cel6A, Cel7A and Cel7B on the same substrate. Values for these latter enzymes fall in the range (10-45) × 10 −3 (g/l) −1 s −1 [13][14][15][16]. We conclude that the AfBG has kinetic hallmarks of an EG with high turnover and weak substrate binding, but that its specificity constant on Avicel is lower than for typical EGs and CBHs.…”

“…The purity of the Af BG sample was evaluated before and after the SEC step by two independent methods. First, mass spectrometry was used to analyze tryptic digests by filter-aided sample preparation (FASP) as described previously [ 14 ]. The second purity analysis was LabChip GXII microfluidic CE‐SDS electrophoresis (Perkin Elmer) with the Protein Express assay according to the instructions by the manufacturer.…”

LPMO AfAA9_B and Cellobiohydrolase AfCel6A from A. fumigatus Bost Enzymatic Saccharification Activity of Cellulase Cocktail

“…LPMO (AfAA9_B) and Cellobiohydrolase GH6 (AfCel6A) from A. fumigatus and expressed in E. coli and A. oryzae, respectively, have been described [33,35]. However, to evaluate the action of the combined enzymes, we characterized and analyzed their biochemical properties after expressing them in P. pastoris, and we detected some differences.…”

“…These results showed that AfCel6A was stable at high temperatures, especially at 50 ºC. In another study, after expression in A. oryzae, AfCel6A was stable at 60 °C, but it completely lost its activity at 70 °C [33]. Therefore, AfCel6A was more stable after expression in P. pastoris than in A. oryzae.…”

“…Enzymes from thermophilic microorganisms offer several advantages for industrial applications. For example, A. fumigatus produces thermophilic CAZymes, which have high cellulolytic activity and stability in a wide range of pH and at elevated temperatures, unlike commercial fungal cellulases [30][31][32][33].…”