The role of intracellular ß-glucosidase in cellulolytic response induction in filamentous fungus Penicillium verruculosum.

In this study, CRISPR/Cas9 genome editing was used to knockout the bgl2 gene encoding intracellular ß-glucosidase filamentous fungus Penicillium verruculosum. This resulted in a dramatic reduction of secretion of cellulolytic enzymes. The study of P. verruculosum Δbgl2 found that the transcription of the cbh1 gene, which encodes cellobiohydrolase 1, was impaired when induced by cellobiose and cellotriose. However, the transcription of the cbh1 gene remains at level of the host strain when induced by gentiobiose. This implies that gentiobiose is the true inducer of the cellulolytic response in P. verruculosum, in contrast to Neurospora crassa where cellobiose acts as an inducer.

Combination of 3-O-Levulinoyl and 6-O-Trifluorobenzoyl Groups Ensures α-Selectivity in Glucosylations: Synthesis of the Oligosaccharides Related to Aspergillus fumigatus α-(1 → 3)-d-Glucan.

Stereospecific α-glucosylation of primary and secondary OH-group at carbohydrate acceptors is achieved using glucosyl N-phenyl-trifluoroacetimidate (PTFAI) donor protected with an electron-withdrawing 2,4,5-trifluorobenzoyl (TFB) group at O-6 and the participating levulinoyl (Lev) group at O-3. New factors have been revealed that might explain α-stereoselectivity in the case of TFB and pentafluorobenzoyl (PFB) groups at O-6. They are of conformational nature and confirmed by DFT calculations. The potential of this donor, as well as the orthogonality of TFB and Lev protecting groups, is showcased by the synthesis of α-(1 → 3)-linked pentaglucoside corresponding to Aspergillus fumigatus α-(1 → 3)-d-glucan and of its hexasaccharide derivative, bearing ß-glucosamine residue at the non-reducing end.

Enhancement of thermostability of GH10 xylanase E Penicillium canescens directed by ΔΔG calculations and structure analysis.

Hydrolytic enzymes are highly demanded in the industry. Thermostability is an important property of enzymes that affects the economic costs of the industrial processes. The rational design of GH10 xylanase E (XylE) Penicillium canescens for the thermostability improvement was directed by ΔΔG calculations and structure analysis. Amino acid substitutions with stabilizing values of ΔΔG and providing an increase in side-chain volume of buried residues were performed experimentally. From the six designed substitutions, four substitutions appeared to be stabilizing, one - destabilizing, and one - neutral. For the improved XylE variants, values of Tm were increased by 1.1-3.1 °C, and times of half-life at 70 °C were increased in 1.3-1.7-times. Three of the four stabilizing substitutions were located in the N- or the C-terminus region. This highlights the importance of N- and C-terminus for the thermostability of GH10 xylanases and also enzymes with (ß/α)8 TIM barrel type of structure. The criteria of stabilizing values of ΔΔG and increased side-chain volume of buried residues for selection of substitutions may be applied in the rational design for thermostability improvement.

Expression and Refolding of the Plant Chitinase From Drosera capensis for Applications as a Sustainable and Integrated Pest Management.

Recently, the study of chitinases has become an important target of numerous research projects due to their potential for applications, such as biocontrol pest agents. Plant chitinases from carnivorous plants of the genus Drosera are most aggressive against a wide range of phytopathogens. However, low solubility or insolubility of the target protein hampered application of chitinases as biofungicides. To obtain plant chitinase from carnivorous plants of the genus Drosera in soluble form in E.coli expression strains, three different approaches including dialysis, rapid dilution, and refolding on Ni-NTA agarose to renaturation were tested. The developed « Rapid dilution ¼ protocol with renaturation buffer supplemented by 10% glycerol and 2M arginine in combination with the redox pair of reduced/oxidized glutathione, increased the yield of active soluble protein to 9.5 mg per 1 g of wet biomass. A structure-based removal of free cysteines in the core domain based on homology modeling of the structure was carried out in order to improve the soluble of chitinase. One improved chitinase variant (C191A/C231S/C286T) was identified which shows improved expression and solubility in E. coli expression systems compared to wild type. Computational analyzes of the wild-type and the improved variant revealed overall higher fluctuations of the structure while maintaining a global protein stability. It was shown that free cysteines on the surface of the protein globule which are not involved in the formation of inner disulfide bonds contribute to the insolubility of chitinase from Drosera capensis. The functional characteristics showed that chitinase exhibits high activity against colloidal chitin (360 units/g) and high fungicidal properties of recombinant chitinases against Parastagonospora nodorum. Latter highlights the application of chitinase from D. capensis as a promising enzyme for the control of fungal pathogens in agriculture.

An engineered cellobiohydrolase I for sustainable degradation of lignocellulosic biomass.

This study provides computational-assisted engineering of the cellobiohydrolase I (CBH-I) from Penicillium verruculosum with simultaneous enhanced thermostability and tolerance in ionic liquids, deep eutectic solvent, and concentrated seawater without affecting its wild-type activity. Engineered triple variant CBH-I R1 (A65R-G415R-S181F) showed 2.48-fold higher thermostability in terms of relative activity at 65°C after 1 h of incubation when compared with CBH-I wild type. CBH-I R1 exhibited 1.87-fold, 1.36-fold, and 1.57-fold higher specific activities compared with CBH-I wild type in [Bmim]Cl (50 g/L), [Ch]Cl (50 g/L), and two-fold concentrated seawater, respectively. In the multicellulases mixture, CBH-I R1 showed higher hydrolytic efficiency to hydrolyze aspen wood compared with CBH-I wild type in the buffer, [Bmim]Cl (50 g/L), and two-fold concentrated seawater, respectively. Structural analysis revealed a molecular basis for the higher stability of the CBH-I structure in which A65R and G415R substitutions form salt bridges (D64 … R65, E411 … R415) and S181F forms π-π interaction (Y155 … F181), leading to stabilize surface-exposed flexible α-helixes and loop in the multidomain ß-jelly roll fold structure, respectively. In conclusion, the variant CBH-I R1 could enable efficient lignocellulosic biomass degradation as a cost-effective alternative for the sustainable production of biofuels and value-added chemicals.

Heterologous Expression of Thermogutta terrifontis Endo-Xanthanase in Penicillium verruculosum, Isolation and Primary Characterization of the Enzyme.

Heterologous endo-xanthanase (EX) from the thermophilic planktomycete Thermogutta terrifontis strain was obtained using Penicillium verruculosum 537 (ΔniaD) expression system with the cellobiohydrolase 1 gene promoter. Homogeneous EX with a molecular weight of 23.7 kDa (pI 6.5) was isolated using liquid chromatography methods. This xanthan degrading enzyme also possesses the enzymatic activity towards CM-cellulose, ß-glucan, curdlan, lichenan, laminarin, galactomannan, xyloglucan but not towards p-nitrophenyl derivatives of ß-D-glucose, mannose and cellobiose. The temperature and pH optima of EX were 55°C and 4.0, respectively; the enzyme exhibited 90% of its maximum activity in the temperature range 50-60°C and pH 3-5.

Designing enzyme cocktails from Penicillium and Aspergillus species for the enhanced saccharification of agro-industrial wastes.

The aim of this study was to develop optimized enzyme cocktails, containing native and recombinant purified enzymes from five fungal species, for the saccharification of alkali- and acid-pretreated sugarcane bagasse (SCB), soybean hulls (SBH) and oil palm empty fruit bunches (EFB). Basic cellulases were represented by cellobiohydrolase I (CBH) and endo-glucanase II (EG) from Penicillium verruculosum and ß-glucosidase (BG) from Aspergillus niger. Auxiliary enzymes were represented by endo-xylanase A (Xyl), pectin lyase (PNL) and arabinoxylanhydrolase (AXH) from Penicillium canescens, ß-xylosidase (BX) from Aspergillus japonicus, endo-arabinase (ABN) from A. niger and arabinofuranosidase (Abf) from Aspergillus foetidus. Enzyme loads were 5 mg protein/g dry substrate (basic cellulases) and 1 mg/g (each auxiliary enzyme). The best choice for SCB and EFB saccharification was alkaline pretreatment and addition of Xyl + BX, AXH + BX or ABN + BX + Abf to basic cellulases. For SBH, acid pretreatment and basic cellulases combined with ABN + BX + Abf or Xyl + BX performed better than other enzyme preparations.

Rational design and structure insights for thermostability improvement of Penicillium verruculosum Cel7A cellobiohydrolase.

Thermostability is a fundamental characteristic of enzymes that is of high importance for industrial implementation of enzymatic catalysis. Cellobiohydrolases are enzymes capable to hydrolyze the most abundant natural polysaccharide - cellulose. These enzymes are widely applied in industry for processing of cellulose containing materials. However, structural and functional engineering of cellobiohydrolases for improving their properties is a challenging task. In this study, the thermostability of Penicillium verruculosum Cel7A cellobiohydrolase was increased through rational design of substitutions with proline. The stabilizing substitution G415P resulted in 3.4-fold increase in half-life time at 60 °C compared to wild-type enzyme. Molecular dynamics simulations indicated a clear effect of the stabilizing substitution G415P and the destabilizing substitutions D62P, S191P, and S273P on the stability of the enzyme tertiary structure. The stabilizing substitution G415P decreased flexibility of the lateral sides of the enzyme active site tunnel, while the considered destabilizing substitutions increased their flexibility.

Engineering Robust Cellulases for Tailored Lignocellulosic Degradation Cocktails.

Lignocellulosic biomass is a most promising feedstock in the production of second-generation biofuels. Efficient degradation of lignocellulosic biomass requires a synergistic action of several cellulases and hemicellulases. Cellulases depolymerize cellulose, the main polymer of the lignocellulosic biomass, to its building blocks. The production of cellulase cocktails has been widely explored, however, there are still some main challenges that enzymes need to overcome in order to develop a sustainable production of bioethanol. The main challenges include low activity, product inhibition, and the need to perform fine-tuning of a cellulase cocktail for each type of biomass. Protein engineering and directed evolution are powerful technologies to improve enzyme properties such as increased activity, decreased product inhibition, increased thermal stability, improved performance in non-conventional media, and pH stability, which will lead to a production of more efficient cocktails. In this review, we focus on recent advances in cellulase cocktail production, its current challenges, protein engineering as an efficient strategy to engineer cellulases, and our view on future prospects in the generation of tailored cellulases for biofuel production.

Cloning, purification and study of recombinant GH3 family ß-glucosidase from Penicillium verruculosum.

A novel bgl1 gene, encoding GH3 family ß-glucosidase from Penicillium verruculosum (PvBGL), was cloned and heterologously expressed in P. canescens RN3-11-7 (niaD-) strain under the control of the strong xylA gene promoter. The recombinant rPvBGL was purified and their properties were studied in comparison with those of rAnBGL from Aspergillus niger expressed previously in the same fungal host. The rPvBGL had an observed molecular mass of 90 kDa (SDS-PAGE data) and displayed the enzyme maximum activity at pH 4.6 and 65 °C. The enzyme half-life time at 60 °C was found to be 87 min. Unlike the rAnBGL, the rPvBGL was not adsorbed on microcrystalline cellulose, which gives the latter enzyme an advantage in cellulose conversion with a longer time of hydrolysis.

Purification and characterization of two forms of the homologously expressed lytic polysaccharide monooxygenase (PvLPMO9A) from Penicillium verruculosum.

Two forms of C1/C4-oxidizing lytic polysaccharide monooxygenase (PvLPMO9A) from Penicillium verruculosum (Talaromyces verruculosus) homologously expressed in P. verruculosum B1-537 auxotrophic strain were isolated in a homogeneous state using two-stage chromatography. The PvLPMO9A-hm form represented a full-size enzyme encoded by the intact lpmo1 gene, while the PvLPMO9A-lm was a truncated enzyme variant consisting of a conserved catalytic core of AA9 family LPMOs and lacking a C-terminal extra peptide sequence that is present in PvLPMO9A-hm. The N-terminal histidine was partially methylated in both enzymes. Most of properties of PvLPMO9A-hm and PvLPMO9A-lm, such as specific activities determined using the 2,6-dimethoxyphenol/H2O2 assay, pH-optima of activity observed at pH 7.5, synergistic effects exhibited with purified cellobiohydrolase I (Cel7A) and/or endoglucanase II (Cel5A) from P. verruculosum in hydrolysis of Avicel and milled aspen wood, were also very similar, except for the higher PvLPMO9A-hm thermostability studied using differential scanning calorimetry (DSC). The DSC profile for the PvLPMO9A-hm holoenzyme demonstrated two overlapping peaks (with maxima at 56.3 and 59.6 °C) due to the presence of two unfolding protein domains, while the PvLPMO9A-lm DSC profile represented one peak with maximum at 48.1 °C. After removing the active site copper with EDTA, the PvLPMO9A-hm and PvLPMO9A-lm melting temperatures decreased by ~10-11 and ~1 °C, respectively. These data show that both active site copper and C-terminal domain present in the PvLPMO9A-hm protect the enzyme from thermal unfolding, while the stabilizing effect of metal is much less pronounced in the truncated PvLPMO9A-lm form.

Protein surface engineering of endoglucanase Penicillium verruculosum for improvement in thermostability and stability in the presence of 1-butyl-3-methylimidazolium chloride ionic liquid.

Thermostability and stability in ionic liquids are essential properties of cellulases that are applied in industrial processes of bioconversion. Engineering of protein surface of endoglucanase II from Penicillium verruculosum was used to improve the enzyme thermostability and stability in 1-butyl-3-methylimidazolium chloride ([Bmim]Cl). The engineering was based on analysis of the protein surface topography and enhanced by multiple sequence alignment and ΔΔG calculations. In the case of the thermostability, half-life time was improved in 1.3-1.6 times at 70 °C and 1.2-1.4 times at 80 °C. In the case of the stability in [Bmim]Cl, the residual activity after 72 h of incubation in the presence of [Bmim]Cl (50 g/L, 50 °C, pH 4.5) was 1.7-1.9 times greater for the tailored enzyme. The yield of reducing sugars after enzymatic hydrolysis of aspen wood pretreated with [Bmim]Cl was 10-20% higher with the tailored endoglucanase.

Enhancement of the enzymatic cellulose saccharification by Penicillium verruculosum multienzyme cocktails containing homologously overexpressed lytic polysaccharide monooxygenase.

The gene lpmo1 encoding Penicillium verruculosum lytic polysaccharide monooxygenase (PvLPMO9A) was sequenced and homologously overexpressed in P. verruculosum B1-537 (ΔniaD) auxotrophic strain under the control of the cbh1 gene promoter in combination with either the cbh1 signal sequence (sCBH1-X series of samples) or the native lpmo1 signal sequence (sLPMO1-X series). Three enzyme samples of the sCBH1-X series were characterized by a lower overall content of cellobiohydrolases (CBHs: 26-45%) but slightly higher content of endoglucanases (EGs: 17-23%) relative to the reference B1-537 preparation (60% of CBHs and 14% of EGs), while the PvLPMO9A content in them made up 9-21% of the total secreted protein. The PvLPMO9A content in four enzyme preparations of the sLPMO1-X series was much higher (30-57%), however the portion of CBHs in most of them (except for sLPMO1-8) decreased even to a greater extent (to 21-42%) than in the samples of the sCBH1-X series. Two enzyme preparations (sCBH1-8 and sLPMO1-8), in which the content of cellulases was substantially retained and the portion of PvLPMO9A was 9-30%, demonstrated the increased yields of reducing sugars in 48-h saccharification of Avicel and milled aspen wood: 19-31 and 11-26%, respectively, compared to the reference cellulase cocktail.

Properties of a recombinant GH49 family dextranase heterologously expressed in two recipient strains of Penicillium species.

The dexA gene encoding Penicillium funiculosum dextranase (GenBank accession MH581385) belonging to family 49 of glycoside hydrolases (GH49) was cloned and heterologously expressed in two recipient strains, P. canescens RN3-11-7 and P. verruculosum B1-537. Crude enzyme preparations with the recombinant dextranase content of 8-36% of the total secreted protein were obtained on the basis of new Penicillium strains. Both recombinant forms of the dextranase were isolated in a homogeneous state using chromatographic techniques. The purified enzymes displayed very similar properties, that is, pI 4.55, activity optima at pH 4.5-5.0 and 55-60 °C and a melting temperature of 60.7-60.9 °C. They were characterized by similar specific activities (1020-1340 U/mg) against dextrans with a mean molecular mass of 20, 70 and 500 kDa, as well as similar kinetic parameters in the hydrolysis of 70 kDa dextran (Km = 1.10-1.11 g/L, kcat = 640-680 s-1). However, the recombinant dextranases expressed in P. canescens and P. verruculosum had different molecular masses according to the data of SDS-PAGE (∼63 and ∼60 kDa, respectively); this was the result of different N-glycosylation patterns as MALDI-TOF mass spectrometry analysis showed. The main products of dextran hydrolysis at its initial phase were isomaltooligosaccharides, while after the prolonged time (24 h) the reaction system contained isomaltose and glucose as the major products and minor amounts of other oligosaccharides.

Monitoring of reactions catalyzed by lytic polysaccharide monooxygenases using highly-sensitive fluorimetric assay of the oxygen consumption rate.

Lytic polysaccharide monooxygenases (LPMOs) are recently discovered enzymes that catalyze the oxidative deconstruction of polysaccharides. However fast and reliable methods of determination of LPMO activity still need to be developed, especially those based on the initial reaction rates. A method based on the oxygen consumption rate (OCR) measurements, using a Seahorse XFp Analyzer with highly-sensitive fluorimetric sensors, was applied for monitoring the oxidation of amorphous cellulose by three fungal LPMOs: recombinant enzymes from Thielavia terrestris (GH61E), Trichoderma reesei (Cel61A), and a native LPMO9A from Myceliophthora thermophila. The turnover numbers for 4 µM enzymes acting on 4 mg mL-1 cellulose at 37 °C were 0.88, 1.26 and 0.93 min-1, respectively. A possibility of feeding the dissolved reagents into the reaction system during measurements with obtaining a simultaneous response in the OCR allowed in situ monitoring the LPMO inhibition and activation by EDTA and Cu2+ ions as well as studying other effects on the enzymatic reaction.

Site-directed mutagenesis of GH10 xylanase A from Penicillium canescens for determining factors affecting the enzyme thermostability.

In order to investigate factors affecting the thermostability of GH10 xylanase A from Penicillium canescens (PcXylA) and to obtain its more stable variant, the wild-type (wt) enzyme and its mutant forms, carrying single amino acid substitutions, were cloned and expressed in Penicillium verruculosum B1-537 (niaD-) auxotrophic strain under the control of the cbh1 gene promoter. The recombinant PcXylA-wt and I6V, I6L, L18F, N77D, Y125R, H191R, S246P, A293P mutants were successfully expressed and purified for characterization. The mutations did not affect the enzyme specific activity against xylan from wheat as well as its pH-optimum of activity. One mutant (L18F) displayed a higher thermostability relative to the wild-type enzyme; its half-life time at 50-60°C was 2-2.5-fold longer than that for the PcXylA-wt, and the melting temperature was 60.0 and 56.1°C, respectively. Most of other mutations led to decrease in the enzyme thermostability. This study, together with data of other researchers, suggests that multiple mutations should be introduced into GH10 xylanases in order to dramatically improve their stability.

Using an Inducible Promoter of a Gene Encoding Penicillium verruculosum Glucoamylase for Production of Enzyme Preparations with Enhanced Cellulase Performance.

BACKGROUND: Penicillium verruculosum is an efficient producer of highly active cellulase multienzyme system. One of the approaches for enhancing cellulase performance in hydrolysis of cellulosic substrates is to enrich the reaction system with ß -glucosidase and/or accessory enzymes, such as lytic polysaccharide monooxygenases (LPMO) displaying a synergism with cellulases. RESULTS: Genes bglI, encoding ß-glucosidase from Aspergillus niger (AnBGL), and eglIV, encoding LPMO (formerly endoglucanase IV) from Trichoderma reesei (TrLPMO), were cloned and expressed by P. verruculosum B1-537 strain under the control of the inducible gla1 gene promoter. Content of the heterologous AnBGL in the secreted multienzyme cocktails (hBGL1, hBGL2 and hBGL3) varied from 4 to 10% of the total protein, while the content of TrLPMO in the hLPMO sample was ~3%. The glucose yields in 48-h hydrolysis of Avicel and milled aspen wood by the hBGL1, hBGL2 and hBGL3 preparations increased by up to 99 and 80%, respectively, relative to control enzyme preparations without the heterologous AnBGL (at protein loading 5 mg/g substrate for all enzyme samples). The heterologous TrLPMO in the hLPMO preparation boosted the conversion of the lignocellulosic substrate by 10-43%; however, in hydrolysis of Avicel the hLPMO sample was less effective than the control preparations. The highest product yield in hydrolysis of aspen wood was obtained when the hBGL2 and hLPMO preparations were used at the ratio 1:1. CONCLUSIONS: The enzyme preparations produced by recombinant P. verruculosum strains, expressing the heterologous AnBGL or TrLPMO under the control of the gla1 gene promoter in a starch-containing medium, proved to be more effective in hydrolysis of a lignocellulosic substrate than control enzyme preparations without the heterologous enzymes. The enzyme composition containing both AnBGL and TrLPMO demonstrated the highest performance in lignocellulose hydrolysis, providing a background for developing a fungal strain capable to express both heterologous enzymes simultaneously.

N-Linked glycans are an important component of the processive machinery of cellobiohydrolases.

Cellobiohydrolases (CBHs), belonging to glycoside hydrolase families 6 and 7 (GH6 and GH7), are the major components of cellulase systems of filamentous fungi involved in biodegradation of cellulose in nature. Previous studies demonstrated that N-linked glycans in the catalytic domains of GH7 CBHs significantly affect the enzyme activity against cellulosic substrates. The influence of N-linked glycans on the activity and processivity of recombinant GH6 CBH II from Penicillium verruculosum (PvCel6A) was studied using site-directed mutagenesis of the respective Asn residues. Depending on the position of N-glycans on the surface of a protein globule, they affected the enzyme activity against cellulose either negatively or positively. The decrease or increase in the degree of processivity of recombinant forms of PvCel6A generally correlated with activity changes against Avicel. The mechanism of the N-glycan influence seems to be universal for GH6 and GH7 CBHs. The observed effects for CBHs from both families are explained in terms of a mechanistic model that also makes clear our previously published data on the highly active CBH IIb from Myceliophthora thermophila (MtCel6B). This study, together with data of other researchers, strongly suggests that the N-linked glycans in the catalytic domains of GH6 and GH7 CBHs are involved in processive catalytic machinery of these enzymes. Data obtained should be taken into account during development of new and more effective biocatalysts by protein engineering techniques.

Effect of N-linked glycosylation on the activity and other properties of recombinant endoglucanase IIa (Cel5A) from Penicillium verruculosum.

Endoglucanase IIa from Penicillium verruculosum (PvCel5A) has three potential N-glycosylation sites: Asn19, Asn42 and Asn194. In order to study the role of N-glycosylation, the wild type (wt) PvCel5A and its mutant forms, carrying Asn to Ala substitutions, were cloned into Penicillium canescens. All forms of the rPvCel5A were successfully expressed and purified for characterization. The MALDI-TOF mass spectrometry peptide fingerprinting showed that N-glycans linked to Asn42 and Asn194 represent variable oligosaccharides, according to the formula (Man)1-9(GlcNAc)2. No evidence for Asn19 glycosylation was found. Mutations had no notable effect on the enzyme thermostability; however, the N-linked glycans stabilized the enzyme against proteolytic attack. For N42A and N194A mutants, a slight shift of pH-optimum to pH 5.0 was observed (from pH-optimum of 4.5 for the native enzyme, rPvCel5A-wt and N19A mutant). The N19A mutation led to a notable decrease in the specific activity against carboxymethylcellulose and barley ß-glucan (by 26% and 12% relative to the rPvCel5A-wt), while the N42A and N194A mutants displayed 12-13% and 32-35% increase in the activities. Similar effects of the mutations were observed in prolonged hydrolysis of ß-glucan and milled aspen wood by rPvCel5A forms in the presence of purified ß-glucosidase.

N-linked glycosylation of recombinant cellobiohydrolase I (Cel7A) from Penicillium verruculosum and its effect on the enzyme activity.

Cellobiohydrolase I from Penicillium verruculosum (PvCel7A) has four potential N-glycosylation sites at its catalytic module: Asn45, Asn194, Asn388, and Asn430. In order to investigate how the N-glycosylation influences the activity and other properties of the enzyme, the wild type (wt) PvCel7A and its mutant forms, carrying Asn to Ala substitutions, were cloned into Penicillium canescens PCA10 (niaD-) strain, a fungal host for production of heterologous proteins. The rPvCel7A-wt and N45A, N194A, N388A mutants were successfully expressed and purified for characterization, whereas the expression of N430A mutant was not achieved. The MALDI-TOF mass spectrometry fingerprinting of peptides, obtained as a result of digestion of rPvCel7A forms with specific proteases, showed that the N-linked glycans represent variable high-mannose oligosaccharides and the products of their sequential enzymatic trimming, according to the formula (Man)0-13 (GlcNAc)2 , or a single GlcNAc residue. Mutations had no notable effect on pH-optimum of PvCel7A activity and enzyme thermostability. However, the mutations influenced both the enzyme adsorption ability on Avicel and its activity against natural and synthetic substrates. In particular, the N45A mutation led to a significant increase in the rate of Avicel and milled aspen wood hydrolysis, while the substrate digestion rates in the case of N194A and N388A mutants were notably lower relative to rPvCel7A-wt. These data, together with data of 3D structural modeling of the PvCel7A catalytic module, indicate that the N-linked glycans are an important part of the processive catalytic machinery of PvCel7A.