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Hydrophobins are extracellular proteins produced by filamentous fungi. They show a variety of functions at interfaces that help fungi to adapt to their environment by, for example, adhesion, formation of coatings, and lowering the surface tension of water. Hydrophobins fold into a globular structure and have a distinct hydrophobic patch on their surface that makes these proteins amphiphilic. Their amphiphilicity implies interfacial assembly, but observations indicate that intermolecular interactions also contribute to their functional properties. Here, we used the class II hydrophobin HFBI from Trichoderma reesei as a model to understand the structural basis for the function of hydrophobins. Four different variants were made in which charged residues were mutated. The residues were chosen to probe the role of different regions of the hydrophilic part of the proteins. Effects of the mutations were studied by analyzing the formation and structure of self-assembled layers, multimerization in solution, surface adhesion, binding of secondary layers of proteins on hydrophobins, and the viscoelastic behavior of the air–water interface during formation of protein films; the comparison showed clear differences between variants only in the last two analyses. Surface viscoelasticity behavior suggests that the formation of surface layers is regulated by specific interactions that lead to docking of proteins to each other. One set of mutations led to assemblies with a remarkably high elasticity at the air–water interface (1.44 N/m). The variation of binding of secondary layers of protein on surface-adsorbed hydrophobins suggest a mechanism for a proposed function of hydrophobins, namely, that hydrophobins can act as a specific adhesive layer for the binding of macromolecules to interfaces.

Background During the past few years, the first industrial-scale cellulosic ethanol plants have been inaugurated. Although the performance of the commercial cellulase enzymes used in this process has greatly improved over the past decade, cellulases still represent a very significant operational cost. Depending on the region, transport of cellulases from a central production facility to a biorefinery may significantly add to enzyme cost. The aim of the present study was to develop a simple, cost-efficient cellulase production process that could be employed locally at a Brazilian sugarcane biorefinery. Results Our work focused on two main topics: growth medium formulation and strain improvement. We evaluated several Brazilian low-cost industrial residues for their potential in cellulase production. Among the solid residues evaluated, soybean hulls were found to display clearly the most desirable characteristics. We engineered a Trichoderma reesei strain to secrete cellulase in the presence of repressing sugars, enabling the use of sugarcane molasses as an additional carbon source. In addition, we added a heterologous β-glucosidase to improve the performance of the produced enzymes in hydrolysis. Finally, the addition of an invertase gene from Aspegillus niger into our strain allowed it to consume sucrose from sugarcane molasses directly. Preliminary cost analysis showed that the overall process can provide for very low-cost enzyme with good hydrolysis performance on industrially pre-treated sugarcane straw. Conclusions In this study, we showed that with relatively few genetic modifications and the right growth medium it is possible to produce considerable amounts of well-performing cellulase at very low cost in Brazil using T. reesei. With further enhancements and optimization, such a system could provide a viable alternative to delivered commercial cellulases. Electronic supplementary material The online version of this article (doi:10.1186/s13068-017-0717-0) contains supplementary material, which is available to authorized users.

Two novel GH3 family thermostable β-glucosidases from the filamentous fungus Chaetomium atrobrunneum (CEL3a and CEL3b) were expressed in Trichoderma reesei, purified by two-step ion exchange chromatography, and characterized. Both enzymes were active over a wide range of pH as compared to Neurospora crassa β-glucosidase GH3-3, which was also expressed in T. reesei and purified. The optimum temperature of both C. atrobrunneum enzymes was around 60 °C at pH 5, and both enzymes had better thermal and pH stability and higher resistance to metallic compounds and to glucose inhibition than GH3-3. They also showed higher activity against oligosaccharides composed of glucose units and linked with β-1,4-glycosidic bonds and moreover, had higher affinity for cellotriose over cellobiose. In hydrolysis tests against Avicel cellulose and steam-exploded sugarcane bagasse, performed at 45 °C, particularly the CEL3a enzyme performed similarly to N. crassa GH3-3 β-glucosidase. Taking into account the thermal stability of the C. atrobrunneum β-glucosidases, they both represent promising alternatives as enzyme mixture components for improved cellulose saccharification at elevated temperatures.

The enzymatic hydrolysis and fermentation of reed canary grass, harvested in the spring or autumn, and barley straw were studied. Steam pretreated materials were efficiently hydrolysed by commercial enzymes with a dosage of 10–20 FPU/g d.m. Reed canary grass harvested in the spring was hydrolysed more efficiently than the autumn-harvested reed canary grass. Additional β-glucosidase improved the release of glucose and xylose during the hydrolysis reaction. The hydrolysis rate and level of reed canary grass with a commercial Trichoderma reesei cellulase could be improved by supplementation of purified enzymes. The addition of CBH II improved the hydrolysis level by 10% in 48 hours’ hydrolysis. Efficient mixing was shown to be important for hydrolysis already at 10% dry matter consistency. The highest ethanol concentration (20 g/l) and yield (82%) was obtained with reed canary grass at 10% d.m. consistency.

The adsorption of purified Trichoderma reesei cellulases (TrCel7A, TrCel6A and TrCel5A) and xylanase TrXyn11 and Aspergillus niger β-glucosidase AnCel3A was studied in enzyme mixture during hydrolysis of two pretreated lignocellulosic materials, steam pretreated and catalytically delignified spruce, along with microcrystalline cellulose (Avicel). The enzyme mixture was compiled to resemble the composition of commercial cellulase preparations. The hydrolysis was carried out at 35 °C to mimic the temperature of the simultaneous saccharification and fermentation (SSF). Enzyme adsorption was followed by analyzing the activity and the protein amount of the individual free enzymes in the hydrolysis supernatant. Most enzymes adsorbed quickly at early stages of the hydrolysis and remained bound throughout the hydrolysis, although the conversion reached was fairly high. Only with the catalytically oxidized spruce samples, the bound enzymes started to be released as the hydrolysis degree reached 80%. The results based on enzyme activities and protein assay were in good accordance.

The presence of lignin is known to reduce the efficiency of the enzymatic hydrolysis of lignocellulosic raw materials. On the other hand, solubilization of hemicellulose, especially of xylan, is known to enhance the hydrolysis of cellulose. The enzymatic hydrolysis of spruce, recognized among the most challenging lignocellulosic substrates, was studied by commercial and purified enzymes from Trichoderma reesei. Previously, the enzymatic hydrolysis of steam pretreated spruce has been studied mainly by using commercial enzymes and no efforts have been taken to clarify the bottlenecks by using purified enzyme components.Steam-pretreated spruce was hydrolyzed with a mixture of Celluclast and Novozym 188 to obtain a hydrolysis residue, expectedly containing the most resistant components. The pretreated raw material and the hydrolysis residue were analyzed for the enrichment of structural bottlenecks during the hydrolysis. Lignin was removed from these two materials with chlorite delignification method in order to eliminate the limitations caused by lignin. Avicel was used for comparison as a known model substrate. Mixtures of purified enzymes were used to investigate the hydrolysis of the individual carbohydrates: cellulose, glucomannan and xylan in the substrates. The results reveal that factors limiting the hydrolysis are mainly due to the lignin, and to a minor extent by the lack of accessory enzymes. Removal of lignin doubled the hydrolysis degree of the raw material and the residue, and reached close to 100% of the theoretical within 2 days. The presence of xylan seems to limit the hydrolysability, especially of the delignified substrates. The hydrolysis results also revealed significant hemicellulose impurities in the commonly used cellulose model substrate, making it questionable to use Avicel as a model cellulose substrate for hydrolysis experiments.

Conversion of lignocellulosic substrates is limited by several factors, in terms of both the enzymes and the substrates. Better understanding of the hydrolysis mechanisms and the factors determining their performance is crucial for commercial lignocelluloses-based processes. Enzymes produced on various carbon sources (Solka Floc 200, lactose and steam-pre-treated corn stover) by Trichoderma reesei Rut C30 were characterised by their enzyme profile and hydrolytic performance. The results showed that there was a clear correlation between the secreted amount of xylanase and mannanase enzymes and that their production was induced by the presence of xylan in the carbon source. Co-secretion of α-arabinosidase and α-galactosidase was also observed. Secretion of β-glucosidase was found to be clearly dependent on the composition of the carbon source, and in the case of lactose, 2-fold higher specific activity was observed compared to Solka Floc and steam-pre-treated corn stover. Hydrolysis experiments showed a clear connection between glucan and xylan conversion and highlighted the importance of β-glucosidase and xylanase activities. When hydrolysis was performed using additional purified β-glucosidase and xylanase, the addition of β-glucosidase was found to significantly improve both the xylan and glucan conversion.

The benefits of applying cellulose selective enzymes as analytical tools for chemical structure characterization of cellulose derivatives have been frequently addressed over the years. In a recent study the high selectivity of cellulase Cel45A from Trichoderma reesei (Tr Cel45A) was utilized for relating the chemical structure to the flow properties of carboxymethyl cellulose (CMC). However, in order to take full advantage of the enzymatic hydrolysis the enzyme selectivity on the cellulose substrate must be further investigated. Therefore, the selectivity of Tr Cel45A on CMC was studied by chemical sample preparation of the enzyme products followed by mass spectrometric chemical structure characterization. The results strongly suggest that, in accordance with recent studies, also this highly selective endoglucanase is able to catalyze hydrolysis of glucosidic bonds adjacent to mono-substituted anhydroglucose units (AGUs). Furthermore, the results also indicate that substituents on the nearby AGUs will affect the hydrolysis.

Three thermostable neutral cellulases from Melanocarpus albomyces, a 20-kDa endoglucanase (Cel45A), a 50-kDa endoglucanase (Cel7A), and a 50-kDa cellobiohydrolase (Cel7B) heterologously produced in a recombinant Trichoderma reesei were purified and studied in hydrolysis (50 °C, pH 6.0) of crystalline and amorphous cellulose. To improve their efficiency, M. albomyces cellulases naturally harboring no cellulose-binding module (CBM) were genetically modified to carry the CBM of T. reesei CBHI/Cel7A, and were studied under similar experimental conditions. Hydrolysis performance and product profiles were used to evaluate hydrolytic features of the investigated enzymes.Each cellulase proved to be active against the tested substrates; the cellobiohydrolase Cel7B had greater activity than the endoglucanases Cel45A and Cel7A against crystalline cellulose, whereas in the case of amorphous substrate the order was reversed. Evidence of synergism was observed when mixtures of the novel enzymes were applied in a constant total protein dosage. Presence of the CBM improved the hydrolytic potential of each enzyme in all experimental configurations; it had a greater effect on the endoglucanases Cel45A and Cel7A than the cellobiohydrolase Cel7B, especially against crystalline substrate. The novel cellobiohydrolase performed comparably to the major cellobiohydrolase of T. reesei (CBHI/Cel7A) under the applied experimental conditions.

Xyloglucan is one of the major components in the primary cell wall of higher plants. It is intimately associated with cellulose chains, forming the load-bearing network of the cell wall. Enzymatic degradation of the xyloglucan polymer by xyloglucanases could potentially improve the overall hydrolysis of lignocellulosic substrates by enabling cellulases to hydrolyze the cellulose polymer more efficiently due to the increased surface area. In order to evaluate the role of xyloglucan in the enzymatic hydrolysis of lignocelluloses, 11 different pretreated cellulosic substrates were hydrolyzed using different combinations of purified cellobiohydrolases I and II, endoglucanase II, xyloglucanases of Trichoderma reesei and Aspergillus β-glucosidase. Xyloglucanase activity improved the total hydrolysis of lignocellulosic substrates in general. These results indicate that this enzyme can be used to enhance total hydrolysis, especially in cases when pretreatment of the lignocellulosic substrate is not optimal.

As part of the effort to find better cellulases for bioethanol production processes, we were looking for novel GH‐7 family cellobiohydrolases, which would be particularly active on insoluble polymeric substrates and participate in the rate‐limiting step in the hydrolysis of cellulose. The enzymatic properties were studied and are reported here for family 7 cellobiohydrolases from the thermophilic fungi Acremonium thermophilum, Thermoascus aurantiacus, and Chaetomium thermophilum. The Trichoderma reesei Cel7A enzyme was used as a reference in the experiments. As the native T. aurantiacus Cel7A has no carbohydrate‐binding module (CBM), recombinant proteins having the CBM from either the C. thermophilum Cel7A or the T. reesei Cel7A were also constructed. All these novel acidic cellobiohydrolases were more thermostable (by 4–10°C) and more active (two‐ to fourfold) in hydrolysis of microcrystalline cellulose (Avicel) at 45°C than T. reesei Cel7A. The C. thermophilum Cel7A showed the highest specific activity and temperature optimum when measured on soluble substrates. The most effective enzyme for Avicel hydrolysis at 70°C, however, was the 2‐module version of the T. aurantiacus Cel7A, which was also relatively weakly inhibited by cellobiose. These results are discussed from the structural point of view based on the three‐dimensional homology models of these enzymes.

Mixed-linkage β-glucan from barley, with a cellotriosyl to cellotetraosyl ratio of 2.9, was hydrolysed with two endo -1,4-β-glucanases (cellulases) and one non-cellulolytic β-glucanase isolated from Trichoderma reesei . The hydrolysates were precipitated in 90% ethanol and the fragments obtained were further treated with lichenase, followed by analysis of the oligosaccharides released. One of the endo -1,4-β glucanases, Cel 5A (EG II), selectively degraded cellotetraosyl units in the polymer and left the cellotriosyl units unhydrolysed. Blocks of cellotriosyl units were isolated on a larger scale using this enzyme. The isolated blocks were fractionated into four fractions using gel permeation chromatography on Biogel P-6 and the structures of the blocks were analysed by 1 H NMR spectroscopy. The fractions essentially contained cellotriosyl units of different sizes with a 3-linked glucose residue at the non-reducing end and a reducing end linked to two 4-linked glucose residues. The results thus indicated that the enzyme could hydrolyse a 4-linked glucose residue next to the 3-linked residue at the non-reducing terminal but left two 4-linked glucose residues at the reducing end. The isolated blocks of cellotriosyl units had a molecular weight distribution that fits a theoretical model based on random blocks of triosyl units in the mixed-linkage β-glucan.

Cellulase and hemicellulase enzymes were produced by Trichoderma reesei RUT C30 on steam pretreated spruce, willow, corn stover and delignified lignocellulose (Solka Floc), as a reference. The enzymes produced were characterized by protein and various enzyme activity measurements. On steam pretreated corn stover higher cellulolytic enzyme activities were reached than on Solka Floc, while the activities obtained on steam pretreated spruce and willow were considerably lower. The produced and two commercial cellulases (Celluclast 1.5 L and Econase CE) were compared by determining specific activities. There were minor differences among the enzymes corresponding to their specific cellulase activities. In contrast, within hemicellulase and β-glucosidase activities, the differences were found to be more significant. It should be also noted that commercial cellulases had considerably higher specific acetyl xylan esterase activities than the produced enzymes. According to subsequent hydrolysis experiments, performed to characterize the produced enzyme complexes to evaluate their applicability for hydrolysis and enzyme production, it seems that the application of the enzyme that was produced on the same substrate as was used for hydrolysis can be advantageous in the case of some substrates. As a result, these experiments demonstrated that pretreated corn stover is a good substrate both for enzyme production and hydrolysis, since high cellulolytic activities could be reached using it as carbon source. Moreover, high sugar yields could be obtained in the hydrolysis by the enzyme produced on steam pretreated corn stover.

Previous studies onMelanocarpus albomyceslaccase have shown that this enzyme is very interesting for both basic research purposes and industrial applications. In order to obtain a reliable and efficient source for this laccase, it was produced in the filamentous fungusTrichoderma reesei. Two approaches were used: production of a non-fused laccase and a hydrophobin–laccase fusion protein. Both proteins were expressed inT. reeseiunder thecbh1promoter, and significantly higher activities were obtained with the non-fused laccase in shake-flask cultures (corresponding to about 230 mg l−1). Northern blot analyses showed rather similar mRNA levels from both expression constructs. Western analysis indicated intracellular accumulation and degradation of the hydrophobin–laccase fusion protein, showing that production of the fusion was limited at the post-transcriptional level. No induction of the unfolded protein response pathway by laccase production was detected in the transformants by Northern hybridization. The most promising transformant was grown in a fermenter in batch and fed-batch modes. The highest production level obtained in the fed-batch culture was 920 mg l−1. The recombinant laccase was purified from the culture supernatant after cleaving the major contaminating protein, cellobiohydrolase I, by papain. The recombinant and wild-type laccases were compared with regard to substrate kinetics, molecular mass, pH optimum, thermostability, and processing of the N- and C-termini, and they showed very similar properties.

Trichoderma reesei produces five known endoglucanases. The most studied are Cel7B (EG I) and Cel5A (EG II) which are the most abundant of the endoglucanases. We have performed a characterisation of the enzymatic properties of the less well-studied endoglucanases Cel12A (EG III), Cel45A (EG V) and the catalytic core of Cel45A. For comparison, Cel5A and Cel7B were included in the study. Adsorption studies on microcrystalline cellulose (Avicel) and phosphoric acid swollen cellulose (PASC) showed that Cel5A, Cel7B, Cel45A and Cel45Acore adsorbed to these substrates. In contrast, Cel12A adsorbed weakly to both Avicel and PASC. The products formed on Avicel, PASC and carboxymethylcellulose (CMC) were analysed. Cel7B produced glucose and cellobiose from all substrates. Cel5A and Cel12A also produced cellotriose, in addition to glucose and cellobiose, on the substrates.Cel45A showed a clearly different product pattern by having cellotetraose as the main product, with practically no glucose and cellobiose formation. The kinetic constants were determined on cellotriose, cellotetraose and cellopentaose for the enzymes. Cel12A did not hydrolyse cellotriose. The kCat values for Cel12A on cellotetraose and cellopentaose were significantly lower compared with Cel5A and Cel7B. Cel7B was the only endoglucanase which rapidly hydrolysed cellotriose. Cel45Acore did not show activity on any of the three studied cello-oligosaccharides. The four endoglucanases’ capacity to hydrolyse β-glucan and glucomannan were studied. Cel12A hydrolysed β-glucan and glucomannan slightly less compared with Cel5A and Cel7B. Cel45A was able to hydrolyse glucomannan significantly more compared with β-glucan. The capability of Cel45A to hydrolyse glucomannan was higher than that observed for Cel12A, Cel5A and Cel7B. The results indicate that Cel45A is a glucomannanase rather than a strict endoglucanase.

There are currently four proteins in family 61 of the glycoside hydrolases, from Trichoderma reesei, Agaricus bisporus, Cryptococcus neoformans and Neurospora crassa. The enzymatic activity of these proteins has not been studied thoroughly. We report here the homologous expression and purification of T. reesei Cel61A [previously named endoglucanase (EG) IV]. The enzyme was expressed in high amounts with a histidine tag on the C-terminus and purified by metal affinity chromatography. This is the first time that a histidine tag has been used as a purification aid in the T. reesei expression system. The enzyme activity was studied on a series of carbohydrate polymers. The only activity exhibited by Cel61A was an endoglucanase activity observed on substrates containing beta-1,4 glycosidic bonds, e.g. carboxymethylcellulose (CMC), hydroxyethylcellulose (HEC) and beta-glucan. The endoglucanase activity on CMC and beta-glucan was determined by viscosity analysis, by measuring the production of reducing ends and by following the degradation of the polymer on a size exclusion chromatography system. The formation of soluble sugars by Cel61A from microcrystalline cellulose (Avicel; Merck), phosphoric acid swollen cellulose (PASC), and CMC were analysed on a HPLC system. Cel61A produced small amounts of oligosaccharides from these substrates. Furthermore, Cel61A showed activity against cellotetraose and cellopentaose. The activity of Cel61A was several orders of magnitude lower compared to Cel7B (previously EG I) of T. reesei on all substrates. One significant difference between Cel61A and Cel7B was that cellotriose was a poor substrate for Cel61A but was readily hydrolysed by Cel7B. The enzyme activity for Cel61A was further studied on a large number of carbohydrate substrates but the enzyme showed no activity towards any of these substrates.

The enzymatic degradation of single crystals of mannan I with the catalytic core domain of a beta-mannanase (EC 3.2.1.78 or Man5A) from Trichoderma reesei was investigated by transmission electron microscopy and electron diffraction. The enzyme attack took place at the edge of the crystals and progressed towards their centres. Quite remarkably the crystalline integrity of the crystals was preserved almost to the end of the digestion process. This behaviour is consistent with an endo-mechanism, where the enzyme interacts with the accessible mannan chains located at the crystal periphery and cleaves one mannan molecule at a time. The endo mode of digestion of the crystals was confirmed by an analysis of the soluble degradation products.

The crystal structure of the catalytic core domain of β-mannanase from the fungus Trichoderma reesei has been determined at a resolution of 1.5 Å. The structure was solved using the anomalous scattering from a single non-isomorphous platinum complex with two heavy-metal sites in space group P21. The map computed with the experimental phases was enhanced by the application of an automated model building and refinement procedure using the amplitudes and experimental phases as observations. This approach is expected to be of more general application. The structure of the native enzyme and complexes with Tris–HCl and mannobiose are also reported: the mannobiose binds in subsites +1 and +2. The structure is briefly compared with that of the homologous β-mannanase from the bacterium Thermomonospora fusca.

The action of monocomponent Trichoderma reesei endoglucanases (EG I, EG II; EC 3.2.1.4) and cellobiohydrolases (CBH I, CBH II; EC 3.2.1.91) and their core proteins was compared using isolated celluloses and bleached chemical pulp. The presence of cellulose binding domain (CBD) in the intact enzymes did not affect their action against soluble substrates. In the case of insoluble isolated celluloses and the chemical pulp the presence of CBD enhanced the enzymatic hydrolysis of cellulose. The effect of CBD was more pronounced in the cellobiohydrolases, hydrolysing mainly crystalline cellulose, than in the endoglucanases which were more efficient in hydrolysing amorphous cellulose. The pulp properties measured, that is, viscosity and strength after PFI refining, were equally affected by the treatment with intact enzymes and corresponding core proteins, suggesting that the presence of CBD in intact cellulases affects mainly the cellulose hydrolysis level and less the mode of action of T. reesei cellulases in pulp. The better beatability of the bleached chemical pulp treated with intact endoglucanases than that treated with the corresponding core proteins suggests that the presence of CBD in endoglucanases could, however, result in beneficial effects on pulp properties.

The presence of a cellulose binding domain was the main factor which influenced the adsorption of two Trichoderma reesei mannanases and a Trichoderma longibrachiatum xylanase onto bleached kraft fibers. The mannanase containing the binding domain adsorbed to a much greater extent than the mannanase and xylanase without binding domains. Once-dried fibers exhibited lower hemicellulase adsorption than the never-dried fibers. The adsorption onto once-dried fibers was only 30–50% the amount adsorbed onto virgin fibers. Adjustment of the ionic strength and pH of treatment was used to modify the charge on the fibers. Ionic strength had opposite effects on mannanase and mannanase core adsorption. Mannanase adsorption increased at high ionic strengths, while the mannanase core adsorption decreased. An increase in pH and fiber charge significantly enhanced the adsorption of xylanase.

The biodegradability of cellulose-based materials was compared in the standard Sturm test and by enzymatic hydrolysis. Trichoderma reesei culture filtrate, the purified enzymes endoglucanase I and II from T. reesei, and β-glucosidase from Aspergillus niger were used in the experiments. The unpurified Trichoderma reesei culture filtrate was found to contain a mixture of enzymes suitable for cellulose degradation. However, when purified enzymes were used the right balance of the individual enzymes was necessary. The addition of β-glucosidase enhanced the enzymatic hydrolysis of cellulose materials when both culture filtrate and purified enzymes were used. In the Sturm test the biodegradability of most of the cellulose materials exceeded 70% carbon dioxide generation, but, in contrast, the biodegradability of the highly substituted aminated cellulose and cellulose acetate was below 10%. The results concerning enzymatic hydrolysis and biodegradability were in good agreement for kraft paper, sausage casing, aminated cellulose, and cellulose acetate. However, diverging results were obtained with cotton fabric, probably as a result of its high crystallinity.

Two different types of cotton fabrics, i. e. cotton twill and cotton poplin, were treated with purified Trichoderma reesei cellulases. Methods used to evaluate the enzyme effects included weight loss, tear strength, bending hysteresis, wrinkle recovery and pilling tendencies. When the different fabric properties at the same weight loss level were compared, it was apparent that cellobiohydrolase I (CBH I) and endoglucanases had different effects on cotton fabrics. Endoglucanases caused more strength loss than CBH I but had also positive effects on the bending behaviour and pilling properties. The two endoglucanases tested differed in their action. At low hydrolysis levels practically no strength loss was obtained with these endoglucanases whereas at higher dosages EG II caused significantly higher strength losses as compared to EG I. A positive result in the pilling was, however, also obtained with these low enzyme levels, indicating practically no strength loss.

Cellulases can be used to modify pulp fibres. For the development of biotechnical applications, a better understanding of the adsorption of cellulases onto commercial wood fibres is needed. In this work, the adsorption behaviour of purified CBH I and EG II on bleached Kraft fibres was investigated. Three variables were studied with respect to their effect on adsorption: fibre type (hardwood or softwood), fibre history (never-dried or once-dried), and ionic strength. The results showed that fibre history had the largest influence on the extent of adsorption of each enzyme. The effect of ionic strength was shown to be dependent on the enzyme and fibre type. At high ionic strength, CBH I exhibited a higher affinity for both once-dried and never-dried fibres at low enzyme concentrations; however, salt was shown to decrease the extent of adsorption at higher enzyme dosages. In contrast, salt increased the maximum adsorption of EG II, most notably on the once-dried hardwood fibres. Fibre type was also shown to affect adsorption behaviour. CBH I had a higher affinity for softwood fibres than for hardwood fibres at low enzyme concentrations. The maximum adsorption of EG II onto once-dried softwood fibres increased by 80% compared to the once-dried hardwood fibres. Interestingly, this did not correlate to in creased fibre hydrolysis.

A minor xylanase, named XYN IV, was purified from the cellulolytic system of the fungus Trichoderma reesei Rut C30. The enzyme was discovered on the basis of its ability to attack aldotetraohexenuronic acid (HexA-2Xyl-4Xyl-4Xyl, HexA3Xyl3), releasing the reducing-end xylose residue. XYN IV exhibited catalytic properties incompatible with previously described endo-β-1,4-xylanases of this fungus, XYN I, XYN II and XYN III, and the xylan-hydrolyzing endo-β-1,4-glucanase EG I. XYN IV was able to degrade several different β-1,4-xylans, but was inactive on β-1,4-mannans and β-1,4-glucans. It showed both exo-and endo-xylanase activity. Rhodymenan, a linear soluble β-1,3-β-1,4-xylan, was as the best substrate. Linear xylooligosaccharides were attacked exclusively at the first glycosidic linkage from the reducing end. The gene xyn4, encoding XYN IV, was also isolated. It showed clear homology with xylanases classified in glycoside hydrolase family 30, which also includes glucanases and mannanases. The xyn4 gene was expressed slightly when grown on xylose and xylitol, clearly on arabinose, arabitol, sophorose, xylobiose, xylan and cellulose, but not on glucose or sorbitol, resembling induction of other xylanolytic enzymes from T. reesei. A recombinant enzyme prepared in a Pichia pastoris expression system exhibited identical catalytic properties to the enzyme isolated from the T. reesei culture medium. The physiological role of this unique enzyme remains unknown, but it may involve liberation of xylose from the reducing end of branched oligosaccharides that are resistant toward β-xylosidase and other types of endoxylanases. In terms of its catalytic properties, XYN IV differs from bacterial GH family 30 glucuronoxylanases that recognize 4-O-methyl-d-glucuronic acid (MeGlcA) substituents as substrate specificity determinants.

The effects of extraction conditions (pH, temperature, enzymic treatment) on the extractability of rye meal non-starch polysaccharides were studied. The yield of pentosans and extractable matter increased with increases in both temperature and pH during extraction. The extract viscosity was correlated closely with the pentosan content of the extract. The molecular size of the extracted non-starch polysaccharides decreased as the extract pH decreased. Enzymic treatment during extraction increased the amount of pentosan solubilised. Xylanases almost doubled the amount of extractable pentosans, but these were degraded to oligosaccharides. With β-glucanase and protease, the pentosan content of the extract increased by 65% and 40%, respectively. In addition, the β-glucan extractability was increased with protease by 10% and with β-glucanase by 90%. Using size-exclusion HPLC it was observed that the molecular size of extractable rye β-glucans was lower than that of the extractable rye pentosans. β-Glucanase caused considerable hydrolysis of β-glucan and no more β-glucan could be detected by size-exclusion HPLC. Protease treatment decreased the molecular size of extractable β-glucan. All enzymatic treatments caused a disruption of the endosperm cell walls in the extract residue.

The major α-glucuronidase of T. reesei Rut C-30 was purified by chromatographic methods. The molecular and hydrolytic properties of the purified enzyme were studied. The enzyme had a molecular weight of 91,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and a pI of 5.0–6.2 as determined by chromatofocusing. The pH optimum was pH 4.5–6.0 and the enzyme was stable for 24 h at 40°C at pH 4.8–5.5. The purified α-glucuronidase preferred low-molecular-weight xylooligomers as substrate. The enzyme seemed to act almost exclusively on the bond between the terminal xylose at the nonreducing end of a xylose chain and the methyl glucuronic acid attached to it. Minor activity against long-chain glucuronoxylan was also detected. A significant enhancing effect of α-glucuronidase on the hydrolysis of glucuronoxylan by pure xylanases was observed.

In this work the effects of individual purified cellulases of Trichoderma reesei were studied in the enzyme-aided bleaching of kraft pulps. The cellobiohydrolases I and II, when used alone, had no positive effect on the bleachability of kraft pulps. The endoglucanase I (EG I), however, acted on pulp similarly to xylanases and with an enzyme dosage of 0.1 mg/g a clear increase in pulp brightness could be observed. Due to the unspecificity of this enzyme, the viscosity of the pulp was simultaneously decreased. Of the cellulases, EG II was clearly most detrimental in reducing the pulp viscosity. Hence, the action of purified cellulases of T. reesei on pulp as a substrate differs profoundly, and all cellulases are not detrimental to the pulp properties.

Background Due to the complexity of lignocellulosic materials, a complete enzymatic hydrolysis into fermentable sugars requires a variety of cellulolytic and xylanolytic enzymes. Addition of xylanases has been shown to significantly improve the performance of cellulases and to increase cellulose hydrolysis by solubilizing xylans in lignocellulosic materials. The goal of this work was to investigate the effect of acetyl xylan esterase (AXE) originating from Trichoderma reesei on xylan solubilization and enzymatic hydrolysis of cellulose. Results The solubilization of xylan in pretreated wheat straw and giant reed (Arundo donax) by xylanolytic enzymes and the impact of the sequential or simultaneous solubilization of xylan on the hydrolysis of cellulose by purified enzymes were investigated. The results showed that the removal of acetyl groups in xylan by AXE increased the accessibility of xylan to xylanase and improved the hydrolysis of xylan in pretreated wheat straw and giant reed. Solubilization of xylan led to an increased accessibility of cellulose to cellulases and thereby increased the hydrolysis extent of cellulose. A clear synergistic effect between cellulases and xylanolytic enzymes was observed. The highest hydrolysis yield of cellulose was obtained with a simultaneous use of cellulases, xylanase and AXE, indicating the presence of acetylated xylan within the cellulose matrix. Acetylated xylobiose and acetylated xylotriose were produced from xylan without AXE, as confirmed by atmospheric pressure matrix-assisted laser desorption/ionization ion trap mass spectrometry. Conclusions The results in this paper demonstrate that supplementation of xylanase with AXE enhances the solubilization of xylan to some extent and, consequently, increases the subsequent hydrolysis of cellulose. The highest hydrolysis yield was, however, obtained by simultaneous hydrolysis of xylan and cellulose, indicating a layered structure of cellulose and xylan chains in the cell wall substrate. AXE has an important role in the hydrolysis of lignocellulosic materials containing acetylated xylan.

Xylanase treatment improved the bleachability of softwood kraft pulp both in conventional chlorine bleaching and in peroxide delignification. The mannanase isolated from Trichoderma reesei acted differently and improved the bleachability in the peroxide delignification significantly more than in the chlorine bleaching. When mannanase and xylanase were dosed together a positive effect was detected in both bleaching sequences. The reason for the different efficiencies of these enzymatic treatments in the bleaching may be the selectivities of the bleaching chemicals. The mannanase could hydrolyze amounts of glucomannan comparable to the xylan hydrolyzed by xylanase, suggesting that at least part of both hemicelluloses are located on the surface of the fibres.

Five enzymes with mannanase activity were separated from Trichoderma reesei culture filtrate using analytical isoelectric focusing and subsequently detected with the zymogram technique. The crude enzymes had isoelectric points in the range of 3.6–6.5. Two of the mannanases with pI values of 4.6 and 5.4 were purified using ion-exchange chromatography, affinity chromatography and chromatofocusing. The molecular weights determined with SDS-PAGE were 51 000 (mannanase pI 4.6) and 53 000 (mannanase pI 5.4). The two enzymes had similar properties with respect to pH optimae and pH stabilities. Both mannanases hydrolyzed ivory nut mannan mainly to mannotriose and mannobiose. The specific activities (against locust bean gum) of the purified enzymes were 1860 and 1430 nkat mg−1 for the pI 4.6 and pI 5.4 mannanases, respectively.

To understand the limitations occurring during enzymatic hydrolysis of cellulosic materials in renewable energy production, we used wide-angle X-ray scattering (WAXS), small-angle X-ray scattering (SAXS), X-ray microtomography, and transmission electron microscopy (TEM) to characterize submicrometer changes in the structure of microcrystalline cellulose (Avicel) digested with the Trichoderma reesei enzyme system. The microtomography measurements showed a clear decrease in particle size in scale of tens of micrometers. In all the TEM pictures, similar elongated and partly ramified structures were observed, independent of the hydrolysis time. The SAXS results of rewetted samples suggested a slight change in the structure in scale of 10−20 nm, whereas the WAXS results confirmed that the degree of crystallinity and the crystal sizes remained unchanged. This indicates that the enzymes act on the surface of cellulose bundles and are unable to penetrate into the nanopores of wet cellulose.

Enzymatic liquefaction was studied by measuring continuously the flowability change of high-solids lignocellulose substrates using a real time viscometric method. Hydrolysis experiments of hydrothermally pretreated wheat straw were carried out with purified enzymes from Trichoderma reesei; Cel7A, Cel6A, Cel7B, Cel5A, Cel12A and Xyn11A. Results obtained at 15% (w/w) solids revealed that endoglucanases, in particular Cel5A, are the key enzymes to rapidly reduce the viscosity of lignocellulose substrate. Cellobiohydrolases had only minor and the xylanase practically no effect on the viscosity. Efficient, fast liquefaction was obtained already at a dosage of 1.5 mg of Cel5A/g dry solids. Partial replacement or supplementation of Cel5A by the other major hydrolytic enzymes did not improve the liquefaction. The reduction of viscosity did not correlate with the saccharification obtained in the same reaction, suggesting that efficient liquefaction is rather dependent on the site than the frequency of enzymatic cleavages.

Endoglucanases are useful tools in the chemical structure analysis of cellulose derivatives. However, knowledge on the endoglucanase selectivity, which is of central importance for data interpretation, is still limited. In this study, new reverse-phase liquid chromatography mass spectrometry (LC–MS) methods were developed to investigate the selectivity of the endoglucanases Cel5A, Cel7B, Cel45A, and Cel74A from the filamentous fungus Trichoderma reesei. The aim was to improve the identification of the regioisomers in the complex mixtures that are obtained after enzymatic hydrolysis. Reduction followed by per-O-methylation was performed in order to improve the separation in reverse-phase LC, increase MS sensitivity, and to facilitate structure analysis by MS/MS of O-carboxymethyl glucose and cellooligosaccharides. The cellulose selective enzymes that were investigated displayed interesting differences in enzyme selectivity on CMC substrates.

Thermostable enzymes offer potential benefits in the hydrolysis of lignocellulosic substrates; higher specific activity decreasing the amount of enzymes, enhanced stability allowing improved hydrolysis performance and increased flexibility with respect to process configurations, all leading to improvement of the overall economy of the process. New thermostable cellulase mixtures were composed of cloned fungal enzymes for hydrolysis experiments. Three thermostable cellulases, identified as the most promising enzymes in their categories (cellobiohydrolase, endoglucanase and β-glucosidase), were cloned and produced in Trichoderma reesei and mixed to compose a novel mixture of thermostable cellulases. Thermostable xylanase was added to enzyme preparations used on substrates containing residual hemicellulose. The new optimised thermostable enzyme mixtures were evaluated in high temperature hydrolysis experiments on technical steam pretreated raw materials: spruce and corn stover. The hydrolysis temperature could be increased by about 10–15 °C, as compared with present commercial Trichoderma enzymes. The same degree of hydrolysis, about 90% of theoretical, measured as individual sugars, could be obtained with the thermostable enzymes at 60 °C as with the commercial enzymes at 45 °C. Clearly more efficient hydrolysis per assayed FPU unit or per amount of cellobiohydrolase I protein used was obtained. The maximum FPU activity of the novel enzyme mixture was about 25% higher at the optimum temperature at 65 °C, as compared with the highest activity of the commercial reference enzyme at 60 °C. The results provide a promising basis to produce and formulate improved enzyme products. These products can have high temperature stability in process conditions in the range of 55–60 °C (with present industrial products at 45–50 °C) and clearly improved specific activity, essentially decreasing the protein dosage required for an efficient hydrolysis of lignocellulosic substrates. New types of process configurations based on thermostable enzymes are discussed.

Xylanase IV (XYN IV) is a new recently characterized xylanase from Trichoderma reesei. It is able to degrade several different xylans, mainly producing xylose. XYN IV has been crystallized by the hanging-drop vapour-diffusion method, using PEG 6000 as a precipitant. The crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 86.3, b = 137.5, c = 196.1 Å, = = = 90°. Assuming a molecular weight of 50.3 kDa, the VM values indicate there to be four XYN IV monomers in an asymmetric unit and the solvent content of the crystals to be 57%. Based on dynamic light-scattering measurements, XYN IV is a dimer in solution. A native data set to 2.8 Å resolution has been collected at a home laboratory and a data set to 2.2 Å resolution has been collected using synchrotron radiation.

Two minor xylanases present in Trichoderma reesei Rut C30 cultivation broth were purified as a mixture using ion‐exchange, hydrophobic‐interaction and gel chromatography. The purified enzyme preparation contained two active xylanases with pI values of 7.1 and 8.1. Both components had a molecular mass of 20 kDa. The purified xylanase preparation exhibited properties very similar to those of the previously isolated XYL II (pI 9.0) of T. reesei Rut C30. The activity and stability properties, apparent kinetic parameters as well as the titration curve forms were similar. The major difference in enzymic properties was the significantly lower specific activity of the pI‐7.1+8.1 xylanase mixture (3350 nkat/mg) compared with the specific activity of XYL II (13500 nkat/mg). Amino acid sequences of tryptic peptides (34% of the total amino acid sequence was determined) were identical to the amino acid sequence of XYL II. Furthermore, in vitro modification of the pI‐9.0 form of XYL II to pI‐8.1 and pI‐7.1 forms was demonstrated. Thus the purified xylanase preparation most probably contained two modified forms of XYL II. The primary amino acid sequence of XYL II contains 28 glutamine and asparagine residues and theoretically deamination of one of them lowers the pI to 8.06 and deamination of two amino acids lowers the pI to 7.02.

Crystals of the catalytic core domain of a Trichoderma reesei β-mannanase belonging to glycoside hydrolase family 5 have been grown by the sitting-drop method at room temperature using ammonium sulfate as precipitant. The crystals grow as thin colourless plates and belong to space group P21, with unit-cell parameters a = 50.0, b = 54.3, c = 60.2 Å, β = 111.3°, and have a single monomer of mannanase in the asymmetric unit. Native data to 2.0 Å resolution have been collected at room temperature using synchrotron radiation. Data for a platinum derivative have been collected to 1.65 Å at 110 K in a very short time at the CCLRC Daresbury synchrotron source, using a charge-coupled device (CCD) as detector.

The adsorption and the hydrolytic action of purified cellulases of Trichoderma reesei, namely, cellobiohydrolase I (CBH I), endoglucanase II (EG II), and their core proteins, on steam-pretreated willow were compared. The two enzymes differed clearly in their adsorption and hydrolytic behavior. CBH I required the cellulose-binding domain (CBD) for efficient adsorption and hydrolysis, whereas EG II was able to adsorb to steam pretreated willow without its CBD. Absence of the CBD decreased the hydrolysis of cellulose by EG II, but the decrease was less pronounced than with CBH I. A linear relationship was observed between the amount of enzyme adsorbed and the degree of hydrolysis of cellulose only for CBHI. EG II and EG II core appeared to be able to hydrolyze only 1 to 2% of the substrate regardless of the amount of protein adsorbed.

HPLC, MALDI-TOF MS and NMR spectroscopy were used to investigate the hydrolysis of cello- and manno-oligosaccharides by Cel7A and Man5A from Trichoderma reesei. The experimental progress curves were analysed by fitting the numerically integrated kinetic equations, which provided cleavage patterns for oligosaccharides. This data evaluation procedure accounts for product inhibition and avoids the initial slope approximation. In addition, a transglycosylation step had to be included in the model to reproduce the experimental progress curves. For the hydrolysis of manno-oligosaccharides, Man4–6, by Man5A no mannose was detected at the beginning of the reaction showing that only the internal linkages are hydrolysed. For cellotriose and cellotetraose hydrolysis by Cel7A, the main product is cellobiose and glucose is released from the non-reducing end of the substrate. Intermediary products longer than the substrates were detected by MALDI-TOF MS when oligosaccharides (Glc4–6 or Man4–6) were hydrolysed by either Cel7A or Man5A. Interestingly, two distinct transglycosylation pathways could be observed. Cel7A produced intermediates that are one unit longer than the substrate, whereas Man5A produced intermediates that are two units longer than the substrate.

Softwood and hardwood dissolving pulps were treated with purified Trichoderma reesei endoglucanases, cellobiohydrolases and hemicellulases. Endoglucanase II decreased the viscosity and increased the alkaline solubility of the pulps most efficiently. The effect of EG II was not clearly improved by the action of hemicellulases or other cellulases. Only limited removal of the residual hemicelluloses of the pulps could be obtained using hemicellulases. No synergy was observed between the cellulases and hemicellulases nor between the different endoglucanases in a combined treatment. As expected, a synergistic effect in the hydrolysis of cellulose was obtained in the combined treatment with endoglucanases and cellobiohydrolases. However, this was not visualised in either the viscosity or the alkaline solubility.

The Trichoderma reesei cDNA coding for α-glucuronidase (GLRI), which releases glucuronic acid attached to xylose units of xylan, was cloned and sequenced. The deduced N-terminal amino acid (aa) sequence of the protein was verified by sequencing of the purified GLRI. The aa sequence of the GLRI displayed no similarity with any aa sequence available in the data bases.

The separation of a β-xylanase by ultrafiltration from a mixture of xylanases and cellulases produced by Trichoderma reesei was investigated. The effects of the sizes and the pH dependent charges of the proteins and the membranes on separation were studied. A stirred cell device with hydrophobic polysulphone membranes of different cut-off values and manufacturers was employed and there were distinct differences in the pore charges of the virgin membranes from different manufacturers. The flux reductions for the membranes were high and the permeate fluxes dropped to the same flux level in spite of the large differences in the initial water fluxes. β-Xylanase (isoelectric point 9·0) selectivity increased with increasing pH and high selectivity values were obtained with PM30 membranes at pH 7·0.

The two β‐mannanases from Trichoderma reesei with pI of 4.6 and 5.4, respectively, have been characterised by NMR spectroscopy. Following the kinetics of manno‐oligosaccharide degradation with complete progress‐curve analysis the stereospecificity and degradation pattern have been delineated. It was found that degradation of mannotriose and mannopentaose proceeds with retention of the anomeric configuration. Mannotriose degradation proceeds by almost random release of mannose. For mannopentaose there is initially no mannose formed showing that only the two middle mannosidic linkages are attacked. Progress‐curve analysis shows that there is preference (70%) for cleavage of mannopentaose in such a way that mannobiose is released from the reducing end. The final product composition from the mannotriose degradation showed that transglycosylation has to be taken into account. Model calculation and progress‐curve analysis showed that the transglycosylation rate is the fastest of all the rates in this system, 15 s‐1 compared with mannohexaose and mannotetraose hydrolysis rates of 2 s‐1 and mannotriose hydrolysis rate of 0.03 s‐1 at 50°C.

A routinely employed and specific affinity chromatography purification method was used to isolate cellobiohydrolase II (CBHII) from a Trichoderma reesei culture supernatant. Two different identically purified batches of CBHII were found to have distinctly different properties in barley β-glucan hydrolysis. The abilities of the two preparations to produce small oligosaccharides and reduce the viscosity of β-glucan were substantially different, but no contamination or heterogeneity was detected in sodium dodecyl sulfate-electrophoresis used to assess the protein purity. Furthermore, the specific activities of the preparations on cellotetraose substrate were similar. Careful analysis with specific chromogenic oligosaccharide substrates showed that the considerable variation in β-glucan hydrolysis was caused by a minor endoglucanase contamination consisting of only 0.4% of the total protein. The contamination is possibly caused by nonspecific interactions between the proteins and chromatographic column material or by unfavorable protein-protein interactions during the chromatographic separation. The data demonstrate clearly the caution needed in the interpretation of hydrolysis studies on polymeric substrates with cellulolytic enzyme preparations.

Background The main technological impediment to widespread utilization of lignocellulose for the production of fuels and chemicals is the lack of low-cost technologies to overcome its recalcitrance. Organisms that hydrolyze lignocellulose and produce a valuable product such as ethanol at a high rate and titer could significantly reduce the costs of biomass conversion technologies, and will allow separate conversion steps to be combined in a consolidated bioprocess (CBP). Development of Saccharomyces cerevisiae for CBP requires the high level secretion of cellulases, particularly cellobiohydrolases. Results We expressed various cellobiohydrolases to identify enzymes that were efficiently secreted by S. cerevisiae. For enhanced cellulose hydrolysis, we engineered bimodular derivatives of a well secreted enzyme that naturally lacks the carbohydrate-binding module, and constructed strains expressing combinations of cbh1 and cbh2 genes. Though there was significant variability in the enzyme levels produced, up to approximately 0.3 g/L CBH1 and approximately 1 g/L CBH2 could be produced in high cell density fermentations. Furthermore, we could show activation of the unfolded protein response as a result of cellobiohydrolase production. Finally, we report fermentation of microcrystalline cellulose (Avicel™) to ethanol by CBH-producing S. cerevisiae strains with the addition of beta-glucosidase. Conclusions Gene or protein specific features and compatibility with the host are important for efficient cellobiohydrolase secretion in yeast. The present work demonstrated that production of both CBH1 and CBH2 could be improved to levels where the barrier to CBH sufficiency in the hydrolysis of cellulose was overcome.

Fibre-bound and isolated galactoglumanans from pine-wood and pine kraft pulp were hydrolysed with purified mannanases from Trichoderma reesei and Bacillus subtilis. The isolated galactoglucomannans from both wood and pulp could be hydrolysed fairly extensively with both enzymes. In addition to mixed oligomers, the fungal mannase produced mannobiose as the main hydrolysis product whereas the bacterial mannanase produced mannobiose, mannotriose and mannotetraose. Both enzymes hydrolysed the native galactoglucomannan in finely ground pinewood, whereas galactoglucomannan in pine kraft pulp was only hydrolysed by the T. ressei mannanase. Thus, mannanases exhibit different specificities on fibre-bound, modified substrates. In spite of the high enzyme loading, the degree of hydrolysis of fibre-bound substrates did not exceed 10% of the theoretical, probably due to poor accessibility of the substrates.