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

Abstract

BackgroundGH7 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.ResultsThere 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.ConclusionTo 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0752-x) contains supplementary material, which is available to authorized users.

Thermoactivation of a cellobiohydrolase

“…Here, we simply note that the investigated system shows a strong correlation between low affinity and poor thermoactivation, and hence that there appears to be a causal relationship. Several earlier studies of temperature effects on cellulases acting on real cellulose have also shown weak thermoactivation (Ogunmolu et al, ; Sorensen et al, ; Voutilainen et al, ; Ye & Berson, ; Zhang et al, ), and one particularly clear example of this was recently described by Teugjas and Valjamae (). They found that the activity of a cellulase mixture (dominated by Cel7A) decreased when the experimental temperature was raised from 40 °C to 55 °C.…”

“…This is challenging because saccharification relies on a cocktail of synergizing enzymes, which must all be stable, but there has been good progress in this field, and a number of thermostable cellulases have been described (Heinzelman et al, ; Voutilainen, Murray, Tuohy, & Koivula, ). However, the degree of thermoactivation on real cellulose for these thermostable enzymes has generally been moderate (Ogunmolu et al, ; Sorensen et al, ; Voutilainen et al, ; Ye & Berson, ; Zhang et al, ) compared to typical bulk enzyme‐substrate systems (Elias et al, ), including cellulases acting on soluble substrate‐analogs (German, Marcelo, Stone, & Allison, ). In the current work, we suggest that this comparably lower thermoactivation on real cellulose reflects a temperature induced desorption of cellulases from the substrate surface, and show that engineering of a “high‐affinity” variant mitigated this problem and hence improved thermoactivation.…”

pH profiles of cellulases depend on the substrate and architecture of the binding region

“…Earlier investigations of Cel7A's pH profile have mostly used soluble substrate analogs (see Section ), and like the current work on pNPL (Figure ) they have all found the characteristic slope of the acidic limb of the profile. Some earlier studies have reported pH profiles for fungal Cel7As based on activity measurements on Avicel (Colussi et al, ; Marjamaa et al, ; Ogunmolu et al, ). Unlike the results in Figure , however, these reports found sloping acidic limbs although the slope was quite shallow except in one case (Ogunmolu et al, ).…”

“…Some earlier studies have reported pH profiles for fungal Cel7As based on activity measurements on Avicel (Colussi et al, ; Marjamaa et al, ; Ogunmolu et al, ). Unlike the results in Figure , however, these reports found sloping acidic limbs although the slope was quite shallow except in one case (Ogunmolu et al, ). These previous profiles on Avicel were all made at low substrate loads (5–10 g/L), and to test possible effects of this, we repeated the measurements at 40°C with very low load (1 g/L).…”

“…Work with soluble substrate has also identified cellulase wild-types with higher (Cockburn, Vandenende, & Clarke, 2010) or lower (Pellegrini et al, 2015;Texier, Dumon, Neugnot-Roux, Maestracci, & O'Donohue, 2012) pH optima, and engineered enzyme variants with upwards shifts of the optimal value (Becker et al, 2001;Cockburn et al, 2010;Qin, Wei, Song, & Qu, 2008). Some studies have addressed the pH-activity profiles of cellulases acting on pure cellulose (Borisova et al, 2015;Colussi et al, 2012;Marjamaa, Toth, Bromann, Szakacs, & Kruus, 2013;Ogunmolu et al, 2017), and while differences between pH profiles on soluble and insoluble substrates have been identified, no systematic picture has emerged. This could, at least in part, be because most of these pH profiles are based on measurements at a single load of cellulose, although this is known to limit interpretations of pH-activity relationship (Knowles & Jencks, 1976).…”