Background: Endoglucanase has been extensively employed in industrial processes as a key biocatalyst for lignocellulosic biomass degradation. Thermostable endoglucanases with high catalytic activity at elevated temperatures are preferred in industrial use. To improve the activity and thermostability, site-directed mutagenesis was conducted to modify the N-glycosylation sites of the thermostable β-1,4-endoglucanase CTendo45 from Chaetomium thermophilum. Results: In this study, structure-based rational design was performed based on the modification of N-glycosylation sites in CTendo45. Eight single mutants and one double mutant were constructed and successfully expressed in Pichia pastoris. When the unique N-glycosylation site of N88 was eliminated, a T90A variant was active, and its specific activity towards CMC-Na and β-d-glucan was increased 1.85- and 1.64-fold, respectively. The mutant R67S with an additional N-glycosylation site of N65 showed a distinct enhancement in catalytic efficiency. Moreover, T90A and R67S were endowed with extraordinary heat endurance after 200 min of incubation at different temperatures ranging from 30 to 90 °C. Likewise, the half-lives (t_1/2) indicated that T90A and R67S exhibited improved enzyme thermostability at 80 °C and 90 °C. Notably, the double-mutant T90A/R67S possessed better hydrolysis activity and thermal stability than its single-mutant counterparts and the wild type. Conclusions: This study provides initial insight into the biochemical function of N-glycosylation in thermostable endoglucanases. Moreover, the design approach to the optimization of N-glycosylation sites presents an effective and feasible strategy to improve enzymatic activity and thermostability.

Endoglucanases are increasingly applied in agricultural and industrial applications as a key biocatalyst for cellulose biodegradation. However, the low performance in extreme conditions seriously challenges the enzyme’s commercial utilization. To obtain endoglucanases with substantially improved activity and thermostability, structure-based rational design was carried out based on the Chaetomium thermophilum β-1,4-endoglucanase CTendo45. In this study, five mutant enzymes were constructed by substitution of conserved and noncatalytic residues using site-directed mutagenesis. Mutants were constitutively expressed in Pichia pastoris, purified, and ultimately tested for enzymatic characteristics. Two single mutants, Y30F and Y173F, increased the enzyme’s specific activity 1.35- and 1.87-fold using carboxymethylcellulose sodium (CMC-Na) as a substrate, respectively. Furthermore, CTendo45 and mutants exhibited higher activity towards β-D-glucan than that of CMC-Na, and activities of Y173F and Y30F were also increased obviously against β-D-glucan. In addition, Y173F significantly improved the enzyme’s heat resistance at 80 °C and 90 °C. More interestingly, the double mutant Y30F/Y173F obtained considerably higher stability at elevated temperatures but failed to inherit the increased catalytic efficiency of its single mutant counterparts. This work gives an initial insight into the biological function of conserved and noncatalytic residues of thermostable endoglucanases and proposes a feasible path for the improvement of enzyme redesign proposals.

Phytopathogens deploy glycoside hydrolases (GHs) to disintegrate plant cell walls for nutrition and invasion. However, the pathogenic mechanisms of the majority of GHs in virulence remain unknown, especially in oomycetes. In this study, a Phytophthora sojae gene encodes a GH7 family cellobiohydrolase, named PsGH7a, was identified. PsGH7a was highly induced during the cyst germination and infection stages. PsGH7a is conserved in oomycetes, and shares a high amino acid sequence identity (>85%) within Phytophthora genus. The recombinant PsGH7a catalyzes the hydrolysis of β-1,4-glucan and avicel, which represent the major components of cellulose in plant cell wall. The mutation of catalytic residue Glu236 to alanine resulted in a lower catalytic activity. In addition, the PsGH7a promotes Phytophthora invasion, while the mutant can not. Notably, PsGH7a protein triggers hypersensitive cell death in diverse plants. PsGH7a knockout mutants were generated via CRISPR/Cas9 system, to investigate its biological function. Compared to wild-type strain P6497, the mutants showed reduced virulence on susceptible soybean, indicates PsGH7a is indispensable to P. sojae virulence.

The novel cellobiohydrolase gene ctcel7 was identified from Chaetomium thermophilum, and its recombinant protein CtCel7, a member of glycoside hydrolase family 7, was heterologously expressed in Pichia pastoris and biochemically characterized. Compared with commercial hydrolases, purified CtCel7 exhibited superior bifunctional cellobiohydrolase and xylanase activities against microcrystalline cellulose and xylan, respectively, under optimal conditions of 60°C and pH 4.0. Moreover, CtCel7 displayed remarkable thermostability with over 90% residual activity after heat (60°C) treatment for 180 min. CtCel7 was insensitive to most detected cations and reagents and preferentially cleaved the β-1,4-glycosidic bond to generate oligosaccharides through the continuous saccharification of lignocellulosic substrates, which are crucial for various practical applications. Notably, the hydrolysis effect of a commercial cellulase cocktail on pretreated wheat straw was substantively improved by its combination with CtCel7. Taken together, these excellent properties distinguish CtCel7 as a robust candidate for the biotechnological production of biofuels and biobased chemicals.