Comparative insights into the saccharification potentials of a relatively unexplored but robust Penicillium funiculosum glycoside hydrolase 7 cellobiohydrolase

Abstract

Background: GH7 cellobiohydrolases (CBH1) are vital for the breakdown of cellulose. We had previously observed the enzyme as the most dominant protein in the active cellulose-hydrolyzing secretome of the hypercellulolytic ascomycete-Penicillium funiculosum (NCIM1228). To understand its contributions to cellulosic biomass saccharification in comparison with GH7 cellobiohydrolase from the industrial workhorse-Trichoderma reesei, we natively purified and functionally characterized the only GH7 cellobiohydrolase identified and present in the genome of the fungus.

Results: There were marginal differences observed in the stability of both enzymes, with P. funiculosum (PfCBH1) showing an optimal thermal midpoint (T_m) of 68 °C at pH 4.4 as against an optimal T_m of 65 °C at pH 4.7 for T. reesei (TrCBH1). Nevertheless, PfCBH1 had an approximate threefold lower binding affinity (K_m), an 18-fold higher turnover rate (k_cat), a sixfold higher catalytic efficiency as well as a 26-fold higher enzyme-inhibitor complex equilibrium dissociation constant (K_i) than TrCBH1 on p-nitrophenyl-β-d-lactopyranoside (pNPL). Although both enzymes hydrolyzed cellooligomers (G2-G6) and microcrystalline cellulose, releasing cellobiose and glucose as the major products, the propensity was more with PfCBH1. We equally observed this trend during the hydrolysis of pretreated wheat straws in tandem with other core cellulases under the same conditions. Molecular dynamic simulations conducted on a homology model built using the TrCBH1 structure (PDB ID: 8CEL) as a template enabled us to directly examine the effects of substrate and products on the protein dynamics. While the catalytic triads-EXDXXE motifs-were conserved between the two enzymes, subtle variations in regions enclosing the catalytic path were observed, and relations to functionality highlighted.

Conclusion: To the best of our knowledge, this is the first report about a comprehensive and comparative description of CBH1 from hypercellulolytic ascomycete-P. funiculosum NCIM1228, against the backdrop of the same enzyme from the industrial workhorse-T. reesei. Our study reveals PfCBH1 as a viable alternative for CBH1 from T. reesei in industrial cellulase cocktails.

Keywords: Biomass degradation; CAZymes: glycoside hydrolases: Penicillium funiculosum; Cellobiohydrolase 1; Molecular dynamics.

Fig. 1

Properties of PfCBH1. a The schematic representation of the amino acid sequence encoded by the PfCBH1 gene. The picture was generated with IBS v1.0 (http://ibs.biocuckoo.org/); signal peptides prediction was made using services of the SignalP 4.1 server (http://www.cbs.dtu.dk/services/SignalP/) and domain prediction with Pfam (http://pfam.xfam.org/). b The SDS-PAGE and Western blot confirmation using anti-PfCBH1 polyclonal antibody. Crude enzyme (lane 1) from the most performing secretome of P. funiculosum was subjected to hydrophobic interaction chromatography (lane 2), followed by anion exchange chromatography separation of active fractions (lane 3), the flow through was further subjected to hydrophobic interaction chromatography (lane 4) yielding pure CBH1 enzyme. M is a protein molecular weight marker. c The thermal stability of purified PfCBH1 under different pH conditions. The T_m optimal and pH are reported as amplitudes and means of the Gaussian fittings, respectively. d The relative Avicelase activity of purified PfCBH1 under different pH and temperature conditions. e The Lineweaver–Burk plot revealing the competitive nature of the inhibition by cellobiose. f The Log(inhibitor) vs. response curve for IC50 determination. Data are expressed as a percentage of uninhibited activity. A Hill slope of −1.6 was obtained implying a reduction in affinity for pNPL in the presence of cellobiose. g The hydrolysis of oligosaccharides by PfCBH1. The oligosaccharides tested are cellobiose (G2), cellotriose (G3), cellotetraose (G4), cellopentaose (G5), and cellohexaose (G6)

Fig. 2

Comparative saccharification potentials of PfCBH1 and TrCBH1 on polymeric cellulosic substrates. a, b The amount of total sugar obtained from the hydrolysis of microcrystalline cellulose (Avicel) using the purified GH7 CBH’s after 1- and 24-h incubation, respectively, while c and d show the hydrolysis potentials of optimized blends on ammonium hydroxide and sodium hydroxide pretreated wheat straws, respectively. In c, cellulase blend C1 contain—PfCBH1 to TrCBH1 ratio—[39:7], C2 is an inversion with—PfCBH1 to TrCBH1 ratio—[7:39]; C3 contains only PfCBH1 at 46%, while C4 contains only TrCBH1 at 46%. In d, cellulase blend D1 contain—PfCBH1 to TrCBH1 ratio [5:34]; D2 is an inversion with-PfCBH1 to TrCBH1 ratio—[34:5]; D3 contains only PfCBH1 at 39%; while D4 contains only TrCBH1 at 39%. All other components were kept as shown in (Additional file 1: Table S3). ****p < 0.0001, while ns: no significant difference at α = 0.05 using Tukey’s multiple comparison test. Error bars represent ±SE

Fig. 3

Analysis of PfCBH1 and TrCBH1 models. a The superposition of the structures of TrCBH1 and PfCBH1. b, c The space-filled structures comparing the substrate tunnel enclosures of CBH1 from P. funiculosum (green colored), and T. reesei (blue colored), respectively. The red-colored regions correspond to the loops along the substrate-binding path, while the catalytic triad region is highlighted in purple. The obviously different regions are highlighted in dotted circles and labeled accordingly. In all frames, the cellononaose ligand from the TrCBH1 Michaelis complex is shown as gray sticks

Fig. 4

MD simulations of PfCBH1 and TrCBH1 catalytic domains. a The energy decomposition comparison between PfCBH1 and TrCBH1 in the presence of cellononaose (G-9), celloheptaose (G-7), and cellobiose (G-2). Binding energies were derived from Molecular Mechanics Generalized Born Surface Area (MMGBSA) calculations. The significance discovery between groups is determined using the Two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. ****p < 0.0001, while ns: no significant difference at α = 0.05. b, d The cluster representations of PfCBH1 and TrCBH1 over a 100-ns trajectory at 5-ns intervals. The enzymes are colored by RMSF, where red represents the highest fluctuations, and blue represents the lowest fluctuations. c The root-mean-square fluctuation (RMSF) of the active site-bound cellononaose by binding subsite. The RMSF values were calculated based on the glucose-heavy atoms over the entire 100-ns MD simulation. The error bars were computed by block averaging. ****p < 0.0001, at α = 0.05

Fig. 5

Histograms of the minimum distance between loops along cellulose binding paths. The minimum distance between loops A1 to B1 (a); loops A3 to B3 (b); loops B2 to B3 (c); and loops A3 to B2 (d) from 100-ns MD simulations of PfCBH1 and TrCBH1 are depicted. The distances have been measured in the presence of a ligand (bound to cellononaose)