Molecular dynamics simulations of two GH74 endo-processive xyloglucanases and the mutated variants to understand better the mechanism of the enzyme action.

BACKGROUND: GH74 xyloglucanases are composed of two separate domains connected by two unstructured peptides. Previously, a hypothesis was made that the movement of domains may affect the enzyme mechanism of catalysis. METHODS: The molecular dynamics (MD) simulations of endo-processive xyloglucanases from Paenibacillus odorifer (PoGH74cat) and Myceliophthora thermophila (MtXeg74A) were carried out. RESULTS: MD simulations for both enzymes in complex with XXLG and XGXXLG oligosaccharides confirmed the possibility of domain movement. In the case of MtXeg74A, changes in the distances between Cα atoms of aromatic residues involved in xyloglucan binding in -3 and +3 subsites of the active site cleft and those of selected residues on the opposite side of the cleft reached values up to 10-12 Å. For PoGH74cat the conformational changes were less pronounced. In MtXeg74A variants, the deletion of loop 1, which partially closes the entrance to the cleft, and the additional double mutation of two Trp residues in +3 and +5 subsites caused the enhanced mobility of the XGXXLG and also induced changes in topography of the cleft. CONCLUSIONS: These findings demonstrate the possibility of existence of GH74 xyloglucanases in a more open and more closed enzyme conformation. The enzyme in an open conformation may more easily accommodate the branched polysaccharide, while its transition to the closed conformation, together with loop 1 function, should aid processivity. GENERAL SIGNIFICANCE: Our results provide an insight into a mechanism of action of GH74 xyloglucanases and may be useful for discussing the catalytic mechanisms of glycoside hydrolases from other families.

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.

Disulfide Bond Engineering of an Endoglucanase from Penicillium verruculosum to Improve Its Thermostability.

Endoglucanases (EGLs) are important components of multienzyme cocktails used in the production of a wide variety of fine and bulk chemicals from lignocellulosic feedstocks. However, a low thermostability and the loss of catalytic performance of EGLs at industrially required temperatures limit their commercial applications. A structure-based disulfide bond (DSB) engineering was carried out in order to improve the thermostability of EGLII from Penicillium verruculosum. Based on in silico prediction, two improved enzyme variants, S127C-A165C (DSB2) and Y171C-L201C (DSB3), were obtained. Both engineered enzymes displayed a 15⁻21% increase in specific activity against carboxymethylcellulose and ß-glucan compared to the wild-type EGLII (EGLII-wt). After incubation at 70 °C for 2 h, they retained 52⁻58% of their activity, while EGLII-wt retained only 38% of its activity. At 80 °C, the enzyme-engineered forms retained 15⁻22% of their activity after 2 h, whereas EGLII-wt was completely inactivated after the same incubation time. Molecular dynamics simulations revealed that the introduced DSB rigidified a global structure of DSB2 and DSB3 variants, thus enhancing their thermostability. In conclusion, this work provides an insight into DSB protein engineering as a potential rational design strategy that might be applicable for improving the stability of other enzymes for industrial applications.

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.

Enzymatic hydrolysis of cellulosic materials using synthetic mixtures of purified cellulases bioengineered at N-glycosylation sites.

Mutant forms of recombinant endoglucanase II (EG II, N194A), cellobiohydrolase I (CBH I, N45A) and cellobiohydrolase II (CBH II, N219A) from Penicillium verruculosum with enhanced cellulase activities, achieved by engineering of enzyme N-glycosylation sites in our previous studies, were used as components of the binary and ternary mixtures of cellulases in hydrolysis of Avicel and milled aspen wood. Using the engineered forms of the enzymes at a dosage of 10 mg/g substrate resulted in significant boosting of the glucose release from cellulose in the presence of excess ß-glucosidase relative to the performance of the corresponding wild-type mixtures at the same loading. The boosting effects reached 11-40% depending on the reaction time and substrate type. In hydrolysis of both cellulosic substrates by the binary mixtures of cellulases, all the enzyme pairs exhibited synergism. The magnitude of the synergistic effects (Ks) did not depend notably upon the induced mutations in the enzymes, and they were in the range of 1.3-1.8 for the combinations of EG II with CBH I (or CBH II), and 2.3-2.9 for the CBH I-CBH II pair. The results of this study should provide a basis for the development of a more effective fungal strain capable of producing cellulase cocktails with enhanced hydrolytic performance against lignocellulosic materials.

Complex effect of lignocellulosic biomass pretreatment with 1-butyl-3-methylimidazolium chloride ionic liquid on various aspects of ethanol and fumaric acid production by immobilized cells within SSF.

The pretreatment of softwood and hardwood samples (spruce and hornbeam wood) with 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) was undertaken for further simultaneous enzymatic saccharification of renewable non-food lignocellulosic biomass and microbial fermentation of obtained sugars to ethanol and fumaric acid. A multienzyme cocktail based on cellulases and yeast or fungus cells producing ethanol and fumaric acid were the main objects of [Bmim]Cl influence studies. A complex effect of lignocellulosic biomass pretreatment with [Bmim]Cl on various aspects of the process (both action of cellulases and microbial conversion of hydrolysates to target products) was revealed. Positive effects of the pretreatment with [Bmim]Cl included decreasing the lignin content in the biomass, and increasing the effectiveness of enzymatic hydrolysis and microbial transformation of pretreated biomass. Immobilized cells of both yeasts and fungi possessed improved productive characteristics in the biotransformation of biomass pretreated with [Bmim]Cl to ethanol and fumaric acid.

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.

Tissue Specificity of Human Angiotensin I-Converting Enzyme.

BACKGROUND: Angiotensin-converting enzyme (ACE), which metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling, as well as in reproductive functions, is expressed as a type-1 membrane glycoprotein on the surface of endothelial and epithelial cells. ACE also presents as a soluble form in biological fluids, among which seminal fluid being the richest in ACE content - 50-fold more than that in blood. METHODS/PRINCIPAL FINDINGS: We performed conformational fingerprinting of lung and seminal fluid ACEs using a set of monoclonal antibodies (mAbs) to 17 epitopes of human ACE and determined the effects of potential ACE-binding partners on mAbs binding to these two different ACEs. Patterns of mAbs binding to ACEs from lung and from seminal fluid dramatically differed, which reflects difference in the local conformations of these ACEs, likely due to different patterns of ACE glycosylation in the lung endothelial cells and epithelial cells of epididymis/prostate (source of seminal fluid ACE), confirmed by mass-spectrometry of ACEs tryptic digests. CONCLUSIONS: Dramatic differences in the local conformations of seminal fluid and lung ACEs, as well as the effects of ACE-binding partners on mAbs binding to these ACEs, suggest different regulation of ACE functions and shedding from epithelial cells in epididymis and prostate and endothelial cells of lung capillaries. The differences in local conformation of ACE could be the base for the generation of mAbs distingushing tissue-specific ACEs.

Glucoamylases from Penicillium verruculosum and Myceliophthora thermophila: analysis of differences in activity against polymeric substrates based on 3D model structures of the intact enzymes.

Two glucoamylases, a recombinant enzyme from Penicillium verruculosum (PvGla) heterologously expressed in Penicillium canescens RN3-11-7 (niaD-) strain and a native glucoamylase from Myceliophthora thermophila (MtGla), were purified and their properties were studied. MtGla displayed 2-5-fold higher specific activities against soluble starch, amylose and amylopectin than PvGla. MtGla also provided higher glucose yields in extended hydrolysis of the polymeric substrates. Analysis of 3D model structures of the intact PvGla and MtGla, which were built using the 2vn7.pdb crystal structure of the intact Trichoderma reesei glucoamylase (TrGla) as a template, showed that the reason for lower hydrolytic performance of PvGla in comparison to MtGla may be less strong interactions between the enzyme domains as well as a longer (by 17 residues) linker in the first enzyme.

Homologous cloning, purification and characterization of highly active cellobiohydrolase I (Cel7A) from Penicillium canescens.

Penicillium canescens is a filamentous fungus that normally does not secrete notable levels of cellulase activity. Cellobiohydrolase I of P. canescens (PcCel7A) was homologously cloned into a host strain RN3-11-7 (niaD-) and then expressed under the control of a strong xylA promoter. Using three steps of chromatography, PcCel7A was purified. The enzyme displayed maximum activity at pH 4.0-4.5. PcCel7A was stable at 50°C and pH 4.5 at least for 3h, while at 60°C it lost 45% of activity after 30min of incubation. When equalized by protein concentration, PcCel7A demonstrated a higher performance in prolonged hydrolysis of Avicel and milled aspen wood than CBH I (Cel7A) from Trichoderma reesei, the most industrially utilized cellulase at this moment. The high catalytic efficiency of the PcCel7A makes it a potential candidate for industrial applications.

Comment on "Revealing nature's cellulase diversity: the digestion mechanism of Caldicellulosiruptor bescii CelA".

Brunecky et al. (Reports, 20 December 2013, p. 1513) compared the cellulolytic activity of bacterial multimodular cellulase CelA with fungal Cel7A (cellobiohydrolase I from Trichoderma reesei). If more active Cel7A from another fungus were used as a reference enzyme under optimal conditions with ß-glucosidase added, the reported difference between bacterial and fungal enzymes would be less dramatic.

N-glycosylation patterns in two α-l-arabinofuranosidases from Penicillium canescens belonging to the glycoside hydrolase families 51 and 54.

Using MALDI-TOF mass spectrometry (MS) peptide fingerprinting procedure followed by the analysis of MS data with the GlycoMod tool from the ExPASy proteomic site, N-glycosylation of two GH51 and GH54 family α-l-arabinofuranosidases (Abf51A and Abf54A) from Penicillium canescens was studied. Variable N-linked glycans were identified at five out of eight potential N-glycosylation sites in the Abf51A and one out of three potential N-glycosylation sites in the Abf54A. The discriminated glycans represented high-mannose oligosaccharides (Man)x(GlcNAc)2 with a number of Man residues up to 7 or the products of sequential enzymatic trimming of a high-mannose glycan with α-mannosidases and ß-N-acetylhexosaminidases. The Abf54A peptide, containing the Asn254 glycosylation site, and one peptide from the Abf51A, containing the Asn163 glycosylation site, were found to exist not only in glycosylated, but also in a native non-modified form.

Engineering the pH-optimum of activity of the GH12 family endoglucanase by site-directed mutagenesis.

Endo-1,4-ß-glucanase from Penicillium verruculosum (PvEGIII) belongs to family 12 of glycoside hydrolases (GH12). Analysis of the enzyme 3D model structure showed that the amino acid residue Asp98 may directly affect the pH-profile of enzyme activity since it is located at the distance of hydrogen bond formation from Glu203 that plays the role of a general acid in catalysis. The gene encoding the PvEGIII was cloned into Escherichia coli. After the deletion of two introns, a plasmid construction was obtained allowing the PvEGIII expression in E. coli. Using site-directed mutagenesis, the Asp98Asn mutant of the PvEGIII was obtained. Both the wild type and mutant PvEGIIIs were expressed in E. coli with a yield of up to 1 g/L and then isolated in a highly purified form. The enzyme specific activity against soluble carboxymethylcellulose was not changed after a single amino acid substitution. However, the pH-optimum of activity of the mutant PvEGIII was shifted from pH 4.0 to 5.1, compared to the wild type enzyme. The shift in the enzyme pH-optimum to more neutral pH was also observed on insoluble cellulose, in the process of enzymatic depigmentation of denim fabric. Similar situation featuring the effect of the Asp/Asn residue, located near the Glu catalytic residue, on the enzyme activity pH-profile has previously been described for xylanases of the GH11 family. Thus, the glycoside hydrolases belonging to the GH11 and GH12 families function by a rather similar mechanism of catalysis.