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. 2022 Jul 11:13:876466.
doi: 10.3389/fmicb.2022.876466. eCollection 2022.

Recombinant Family 1 Carbohydrate-Binding Modules Derived From Fungal Cellulase Enhance Enzymatic Degradation of Lignocellulose as Novel Effective Accessory Protein

Hexue Jia  1 , Xiaoting Feng  1 , Jiamin Huang  1 , Yingjie Guo  1 , Daolei Zhang  2 , Xuezhi Li  1 , Jian Zhao  1
Affiliations

Recombinant Family 1 Carbohydrate-Binding Modules Derived From Fungal Cellulase Enhance Enzymatic Degradation of Lignocellulose as Novel Effective Accessory Protein

Hexue Jia et al. Front Microbiol. .

Abstract

Fungal cellulases usually contain a family 1 carbohydrate-binding module (CBM1), and its role was considered to recognize the substrate specifically. This study testified that the CBM1s derived from cellobiohydrolase I of Trichoderma reesei, Penicillium oxalicum, and Penicillium funiculosum could be used as an effective accessory protein in cellulase cocktails to enhance the saccharification of lignocellulose, and its enhancement effect was significantly superior to some reported accessory proteins, such as bovine serum albumin (BSA). The promoting effects of the CBM1s were related to not only the CBM1 sources and protein dosages, but also the substrate characteristics and solid consistency during enzymatic hydrolysis. The adsorption capacity of the CBM1s, the adsorption kinetic of TrCBM from T. reesei and cellobiohydrolase, endoglucanase, and β-glucosidase from P. oxalicum, and the effect of adding TrCBM on enzyme activities of free cellulases in the hydrolysis system were investigated, and the binding conformations and affinities of CBM1s to cellulose and lignin were predicted by molecular docking. It was speculated that the higher affinity of the CBM1s to lignin than cellulases could potentially enable the CBM1s to displace cellulase adsorbed on lignin or to preferentially adsorb onto lignin to avoid ineffective adsorption of cellulase onto lignin, which enhanced cellulase system efficiency during enzymatic hydrolysis of lignocellulose.

Keywords: accessory protein; enzymatic hydrolysis; family 1 carbohydrate binding module; lignocellulose; promoting mechanism.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
SDS–PAGE analysis of TrCBM, PoCBM, PfCBM, and Tag protein. Lane M, protein marker; Lane 1, Tag; Lane 2, TrCBM; Lane 3, PoCBM; Lane 4, PfCBM.
FIGURE 2
FIGURE 2
Effect of the addition of BSA, Tag, TrCBM, PoCBM, and PfCBM proteins (1.5 mg/g DM) on glucan conversions of FP (A) and LPCS (B) at 72 h of enzymatic hydrolysis. **Represents that there was a significant difference (p < 0.05), and *means that the difference was not significant (p > 0.05).
FIGURE 3
FIGURE 3
Effect of TrCBM enzyme loading (A) and substrate concentrations (B) on glucan conversions at 72 h in enzymatic hydrolysis of FP and LPCS. **Represents that there was a significant difference (p < 0.05).
FIGURE 4
FIGURE 4
Relative activities of pNPCase (A), CMCase (B), and pNPGase (C) in supernatants from enzymatic hydrolysis systems of FP and LPCS with and without TrCBM addition, and the relative activities of FPA, CBH, EG, and BG after 24 h of lignin adsorption (D). In this, the relative activity of cellulase at 0 h of enzymatic hydrolysis was defined as 100%. **Represents that there was a significant difference (p < 0.05), and *means that the difference was not significant (p > 0.05).
FIGURE 5
FIGURE 5
Langmuir isotherm adsorption models of CBHI, EGII, and BGI from P. oxalicum and TrCBM on FP (A) and lignin extracted from LPCS (B).
FIGURE 6
FIGURE 6
Adsorption capacities of TrCBM, PoCBM, and PfCBM onto FP and lignin extracted from LPCS, in which adsorption time was 24 h.
FIGURE 7
FIGURE 7
Binding conformations of cellohexaose to TrCBM (A), PoCBM (B), and PfCBM (C), and the conformations of LGG to TrCBM (D), PoCBM (E), and PfCBM (F). The interacted amino acids are shown in magenta.
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