Thermostable carbohydrate binding module increases the thermostability and substrate-binding capacity of Trichoderma reesei xylanase 2

To improve the thermostability of Trichoderma reesei xylanase 2 (Xyn2), the thermostabilizing domain (A2) from Thermotoga maritima XynA were engineered into the N-terminal region of the Xyn2 protein. The xyn2 and hybrid genes were successfully expressed in Pichia pastoris using the strong methanol inducible alcohol oxidase 1 (AOX1) promoter and the secretion signal sequence from S. cerevisiae (α-factor). The transformants expressed the hybrid gene produced clearly increased both the thermostability and substrate-binding capacity compared to the corresponding strains expressed the native Xyn2 gene. The activity of the hybrid enzyme was highest at 65 °C that was 10 °C higher than the native Xyn2. The hybrid enzyme was stable at 60 °C and retained more than 85% of its activity after 30-min incubation at this temperature. The hybrid enzyme was highly specific toward xylan and analysis of the products from birchwood xylan degradation confirmed that the enzyme was an endo-xylanase with xylobiose and xylotriose as the main degradation products. These attributes should make it an attractive applicant for various applications. Our results also suggested that the N-terminal domain A2 is responsible for both the thermostability and substrate-binding capacity of T. maritima XynA.     

Investigation of the function of mutated cellulose-binding domains of Trichoderma reesei cellobiohydrolase I.

The function of the cellulose-binding domain (CBD) of the cellobiohydrolase I of Trichoderma reesei was studied by site-directed mutagenesis of two amino acid residues identified by analyzing the 3D structure of this domain. The mutant enzymes were produced in yeast and tested for binding and activity on crystalline cellulose. Mutagenesis of the tyrosine residue (Y492) located at the tip of the wedge-shaped domain to alanine or aspartate reduced the binding and activity on crystalline cellulose to the level of the core protein lacking the CBD. However, there was no effect on the activity toward small oligosaccharide (4-methylumbelliferyl beta-D-lactoside). The mutation tyrosine to histidine (Y492H) lowered but did not destroy the cellulose binding, suggesting that the interaction of the pyranose ring of the substrate with an aromatic side chain is important. However, the catalytic activity of this mutant on crystalline cellulose was identical to the other two mutants. The mutation P477R on the edge of the other face of the domain reduces both binding and activity of CBHI. These results support the hypothesis that both surfaces of the CBD are involved in the interaction of the binding domain with crystalline cellulose.     

The cellulose-binding domain of cellobiohydrolase Cel7A from Trichoderma reesei is also a thermostabilizing domain

The thermostability of cellobiohydrolase I Cel7A from Trichoderma reesei was investigated using dynamic light scattering. While the whole enzyme displayed a melting point of 59 °C, the catalytic domain obtained via papain-catalyzed proteolysis was shown to denature at 51 °C and the cellulose-binding domain (with linker attached) melted at 65-66 °C. This variation in individual melting temperatures is proposed to account for the full retention of binding capacity of Cel7A at 50 °C, along with a loss of catalytic activity observed for the catalytic domain alone. Thus, the cellulose-binding domain of Cel7A acts as a thermostabilizing domain for the enzyme. The effect of reducing agents on the protein melting behavior was also investigated.     

Three-dimensional structures of three engineered cellulose-binding domains of cellobiohydrolase I from Trichoderma reesei.

Three-dimensional solution structures for three engineered, synthetic CBDs (Y5A, Y31A, and Y32A) of cellobiohydrolase I (CBHI) from Trichoderma reesei were studied with nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. According to CD measurements the antiparallel beta-sheet structure of the CBD fold was preserved in all engineered peptides. The three-dimensional NMR-based structures of Y31A and Y32A revealed only small local changes due to mutations in the flat face of CBD, which is expected to bind to crystalline cellulose. Therefore, the structural roles of Y31 and Y32 are minor, but their functional importance is obvious because these mutants do not bind strongly to cellulose. In the case of Y5A, the disruption of the structural framework at the N-terminus and the complete loss of binding affinity implies that Y5 has both structural and functional significance. The number of aromatic residues and their precise spatial arrangement in the flat face of the type I CBD fold appears to be critical for specific binding. A model for the CBD binding in which the three aligned aromatic rings stack onto every other glucose ring of the cellulose polymer is discussed.     

Widely different off rates of two closely related cellulose-binding domains from Trichoderma reesei.

The filamentous fungus Trichoderma reesei produces two cellobiohydrolases (CBHI and CBHII). These, like most other cellulose-degrading enzymes, have a modular structure consisting of a catalytic domain linked to a cellulose-binding domain (CBD). The isolated catalytic domains bind poorly to cellulose and have a much lower activity towards cellulose than the intact enzymes. For the CBDs, no function other than binding to cellulose has been found. We have previously described the reversibility and exchange rate for the binding of the CBD of CBHI to cellulose. In this work, we studied the binding of the CBD of CBHII and showed that it differs markedly from the behaviour of that of CBHI. The apparent binding affinities were similar, but the CBD of CBHII could not be dissociated from cellulose by buffer dilution and did not show a measurable exchange rate. However, desorption could be triggered by shifting the temperature. The CBD of CBHII bound reversibly to chitin. Two variants of the CBHII CBD were made, in which point mutations increased its similarity to the CBD of CBHI. Both variants were found to bind reversibly to cellulose.     

Identification of functionally important amino acids in the cellulose-binding domain of Trichoderma reesei cellobiohydrolase I.

Cellobiohydrolase I (CBHI) of Trichoderma reesei has two functional domains, a catalytic core domain and a cellulose binding domain (CBD). The structure of the CBD reveals two distinct faces, one of which is flat and the other rough. Several other fungal cellulolytic enzymes have similar two-domain structures, in which the CBDs show a conserved primary structure. Here we have evaluated the contributions of conserved amino acids in CBHI CBD to its binding to cellulose. Binding isotherms were determined for a set of six synthetic analogues in which conserved amino acids were substituted. Two-dimensional NMR spectroscopy was used to assess the structural effects of the substitutions by comparing chemical shifts, coupling constants, and NOEs of the backbone protons between the wild-type CBD and the analogues. In general, the structural effects of the substitutions were minor, although in some cases decreased binding could clearly be ascribed to conformational perturbations. We found that at least two tyrosine residues and a glutamine residue on the flat face were essential for tight binding of the CBD to cellulose. A change on the rough face had only a small effect on the binding and it is unlikely that this face interacts with cellulose directly.     

Cloning and expression in Saccharomyces cerevisiae of a Trichoderma reesei beta-mannanase gene containing a cellulose binding domain.

beta-Mannanase (endo-1,4-beta-mannanase; mannan endo-1,4-beta-mannosidase; EC 3.2.1.78) catalyzes endo-wise hydrolysis of the backbone of mannan and heteromannans, including hemicellulose polysaccharides, which are among the major components of plant cell walls. The gene man1, which encodes beta-mannanase, of the filamentous fungus Trichoderma reesei was isolated from an expression library by using antiserum raised towards the earlier-purified beta-mannanase protein. The deduced beta-mannanase consists of 410 amino acids. On the basis of hydrophobic cluster analysis, the beta-mannanase was assigned to family 5 of glycosyl hydrolases (cellulase family A). The C terminus of the beta-mannanase has strong amino acid sequence similarity to the cellulose binding domains of fungal cellulases and is preceded by a serine-, threonine-, and proline-rich region. Consequently, the beta-mannanase is probably organized similarly to the T. reesei cellulases, having a catalytic core domain separated from the substrate-binding domain by an O-glycosylated linker. Active beta-mannanase was expressed and secreted by using the yeast Saccharomyces cerevisiae as the host. The results indicate that the man1 gene encodes the two beta-mannanases with different isoelectric points (pIs 4.6 and 5.4) purified earlier from T. reesei.     

Sugar-binding sites on the surface of the carbohydrate-binding module of CBH I from Trichoderma reesei

Molecular dynamics simulations were carried out for a system consisting of the carbohydrate-binding module (CBM) of the cellulase CBH I from Trichoderma reesei (Hypocrea jecorina) in a concentrated solution of β-d-glucopyranose, to determine whether there is any tendency for the sugar molecules to bind to the CBM. In spite of the general tendency of glucose to behave as an osmolyte, a marked tendency for the sugar molecules to bind to the protein was observed. However, the glucose molecules tended to bind only to specific sites on the protein. As expected, the hydrophobic face of the sugar molecules, comprising the axial H1, H3, and H5 aliphatic protons, tended to adhere to the flat faces of the three tyrosine side chains on the planar binding surface of the CBM. However, a significant tendency to bind to a groove-like feature on the upper surface of the CBM was also observed. These results would not be inconsistent with a model of the mechanism for this globular domain in which the cellodextrin chain being removed from the surface of crystalline cellulose passes over the upper surface of the CBM, presumably then available for hydrolysis in the active site tunnel of this processive cellulase.     

Improvement of cellulose-degrading ability of a yeast strain displaying Trichoderma reesei endoglucanase II by recombination of cellulose-binding domains

To improve the cellulolytic activity of a yeast strain displaying endoglucanase II (EGII) from the filamentous fungus Trichoderma reesei QM9414, the genes encoding the cellulose-binding domain (CBD) of EGII, cellobiohydrolase I (CBHI) and cellobiohydrolase II (CBHII) from T. reesei QM9414, were fused with the catalytic domain of EGII and expressed in Saccharomyces cerevisiae. Display of each of the recombinant EGIIs was confirmed using immunofluorescence microscopy. In the case of EGII-displaying yeast strains in which the CBD of EGII was replaced with the CBD of CBHI or CBHII, the binding affinity to Avicel and hydrolytic activity toward phosphoric acid swollen Avicel were similar to that of a yeast strain displaying wild-type EGII. On the other hand, the three yeast strains displaying EGII with two or three tandemly aligned CBDs showed binding affinity and hydrolytic activity higher than that of the yeast strain displaying wild-type EGII. This result indicates that the hydrolytic activity of yeast strains displaying recombinant EGII increases with increased binding ability to cellulose.     

Monoclonal antibodies against core and cellulose-binding domains of Trichoderma reesei cellobiohydrolases I and II and endoglucanase I.

Cellulases from Trichoderma reesei form an enzyme group with a common structural organization. Each cellulase enzyme is composed of two functional domains, the core region containing the active site and the cellulose-binding domain (CBD). To facilitate the specific detection of each domain, monoclonal antibodies (mAb) against cellobiohydrolase I (CBHI), cellobiohydrolase II (CBHII) and endoglucanase I (EGI) were produced. Five mAb were obtained against CBHI, ten against CBHII and eight against EGI. The location of the antigenic epitope for each antibody was mapped by allowing the antibodies to react with truncated cellulases, synthesized from deleted cDNA in Saccharomyces cerevisiae. Proteolytic fragments of Trichoderma cellulases, obtained by papain digestion, were used to confirm the results. Specific antibodies were detected against the core and the CBD epitopes for all three cellulases. Using the truncated enzymes, it was possible to locate the epitopes to a reasonably short region within the protein. To obtain a quantitative assay for each enzyme, a specific mAb against each antigen was chosen, based on the affinity to the corresponding antigen on Western-blot staining and on filter blots of the cellulolytic yeasts. The mAb were used to quantitative the corresponding enzymes in T. reesei culture medium. Specific quantitation of each cellulase enzyme has not been possible by biochemical assays or using polyclonal antibodies, due to their cross-reactions. Now, these mAb can be specifically used to recognize and quantitate different domains of these three important cellulolytic enzymes.     

Cellulase-poor xylanases produced by Trichoderma reesei RUT C-30 on hemicellulose substrates

Hemicellulose components from industrial viscose fibre production are characterized by a lower cellulose content than commercial xylan and the presence of a carboxylic acid fraction originating from the alkaline degradation of carbohydrates during the process. This substrate, after neutralization, can be used by Trichoderma reesei RUT C-30 for the production of cellulase-poor xylanases, useful for the pulp and paper industry. The yields of xylanase ranged up to almost 400 units/ml, with a ratio of carboxymethylcellulase/xylanase of less than 0.015. This crude xylanase enzyme mixture was shown to be superior to that obtained on beech-wood xylan when used for bleaching and, particularly, upgrading of hard-wood chemical pulp by selective removal of the xylan components. Biochemical studies indicate that the low cellulase production by T. reesei grown on these waste hemicelluloses is the result of a combination of at least three factors: (a) the comparatively low content of cellulose in these hemicellulosic wastes, (b) the inhibitory action of the carboxylic acid fraction present in the hemicellulosic wastes on growth and sporulation of T. reesei, and (c) the use of a mycelial inoculum that is unable to initiate the attack on the cellulose components within the carbon source. Correspondence to: G. Gamerith     

Use of recombinant cellulose-binding domains of Trichoderma reesei cellulase as a selective immunocytochemical marker for cellulose in protozoa

Some unicellular organisms are able to encyst as a protective response to a harmful environment. The cyst wall usually contains chitin as its main structural constituent, but in some cases, as in Acanthamoeba, it consists of cellulose instead. Specific cytochemical differentiation between cellulose and chitin by microscopy has not been possible, due to the similarity of their constituent beta-1,4-linked hexose backbones. Thus, various fluorescent brightening agents and lectins bind to both cellulose and chitin. We have used a recombinant cellulose-binding protein consisting of two cellulose-binding domains (CBDs) from Trichoderma reesei cellulases linked together in combination with monoclonal anticellulase antibodies and anti-mouse immunoglobulin fluorescein conjugate to specifically stain cellulose in the cysts of Acanthamoeba strains for fluorescence microscopy imaging. Staining was observed in ruptured cysts and frozen sections of cysts but not in intact mature cysts. No staining reaction was observed with the chitin-containing cyst walls of Giardia intestinalis, Entamoeba dispar, or Pneumocystis carinii. Thus, the recombinant CBD can be used as a marker to distinguish between cellulose and chitin. Thirteen of 25 environmental or clinical isolates of amoebae reacted in the CBD binding assay. All 13 isolates were identified as Acanthamoeba spp. Five isolates of Hartmannella and seven isolates of Naegleria tested negative in the CBD binding assay. Whether cyst wall cellulose really is a unique property of Acanthamoeba spp. among free-living amoebae, as suggested by our findings, remains to be shown in more extensive studies.     

Three-dimensional crystal structure and enzymic characterization of beta-mannanase Man5A from blue mussel Mytilus edulis

Endo-beta-1,4-d-mannanase is the key depolymerizing enzyme for beta-1,4-mannan polymers present in the cell walls of plants and some algae, as well as in some types of plant seeds. Endo-1,4-beta-mannanase from blue mussel Mytilus edulis (MeMan5A) belongs to the glycoside hydrolase (GH) family 5 enzymes. The MeMan5A structure has been determined to 1.6A resolution using the multiple-wavelength anomalous dispersion method at the selenium K edge with selenomethionyl MeMan5A expressed in the yeast Pichia pastoris. As expected for GH 5 enzymes, the structure showed a (betaalpha)(8)-barrel fold. An unusually large number of histidine side-chains are exposed on the surface, which may relate to its location within the crystalline style of the digestive tract of the mussel. Kinetic analysis of MeMan5A revealed that the enzyme requires at least six subsites for efficient hydrolysis. Mannotetraose (M4) and mannopentaose (M5) were shown to interact with subsites -3 to +1, and -3 to +2, respectively. A clear kinetic threshold was observed when going from M4 to M5, indicating that the +2 subsite provides important interaction in the hydrolysis of short oligomeric mannose substrates. The catalytic centre motif at subsite -1 found in superfamily GH clan A is, as expected, conserved in MeMan5A, but the architecture of the catalytic cleft differs significantly from other GH 5 enzyme structures. We therefore suggest that MeMan5A represents a new subfamily in GH 5.     

Evaluation of minimal Trichoderma reesei cellulase mixtures on differently pretreated Barley straw substrates

The commercial cellulase product Celluclast 1.5, derived from Trichoderma reesei (Novozymes A/S, Bagsvaerd, Denmark), is widely employed for hydrolysis of lignocellulosic biomass feedstocks. This enzyme preparation contains a broad spectrum of cellulolytic enzyme activities, most notably cellobiohydrolases (CBHs) and endo-1,4-beta-glucanases (EGs). Since the original T. reesei strain was isolated from decaying canvas, the T. reesei CBH and EG activities might be present in suboptimal ratios for hydrolysis of pretreated lignocellulosic substrates. We employed statistically designed combinations of the four main activities of Celluclast 1.5, CBHI, CBHII, EGI, and EGII, to identify the optimal glucose-releasing combination of these four enzymes to degrade barley straw substrates subjected to three different pretreatments. The data signified that EGII activity is not required for efficient lignocellulose hydrolysis when addition of this activity occurs at the expense of the remaining three activities. The optimal ratios of the remaining three enzymes were similar for the two pretreated barley samples that had been subjeced to different hot water pretreatments, but the relative levels of EGI and CBHII activities required in the enzyme mixture for optimal hydrolysis of the acid-impregnated, steam-exploded barley straw substrate were somewhat different from those required for the other two substrates. The optimal ratios of the cellulolytic activities in all cases differed from that of the cellulases secreted by T. reesei. Hence, the data indicate the feasibility of designing minimal enzyme mixtures for pretreated lignocellulosic biomass by careful combination of monocomponent enzymes. This strategy can promote both a more efficient enzymatic hydrolysis of (ligno)cellulose and a more rational utilization of enzymes.     

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture organisms, such as hyphae-building fungi and anaerobic bacteria, has hastened the use of recombinant technologies in this field. Plant expression methods are a desirable system for large-scale production of enzymes and other industrially useful proteins. Herein, methods for the transient expression of a fungal endoglucanase, Trichoderma reesei Cel5A, in Nicotiana tabacum are demonstrated. Successful protein expression is shown, monitored by fluorescence using an mCherry-enzyme fusion protein. Additionally, a set of basic tests are used to examine the activity of transiently expressed T. reesei Cel5A, including SDS-PAGE, Western blotting, zymography, as well as fluorescence and dye-based substrate degradation assays. The system described here can be used to produce an active cellulase in a short time period, so as to assess the potential for further production in plants through constitutive or inducible expression systems.     

A microfibril-generating factor from the cellulase of Trichoderma reesei

A combination of ionic strength reduction and diafiltration of Trichoderma reesei cellulate complex through a hollow fiber apparatus of 5000 molecular weight (MW) cutoff and subsequent passage of filtrate over a Spherogel-TSK 3000-SW column provided extracts that had the ability to generate microfibrils in filter paper and to disrupt filter paper and corn leaf tissue. Milligram quantities of material obtained from these extracts released small amounts of soluble carbohydrate from filter paper, required ferric iron for increased activity, and contained amino acids. Short fiber formation and disruption of filter paper during interaction with these extracts was enhanced by prior acid treatment and eliminated by prior base treatment. The amount of soluble carbohydrate hydrolyzed in 24 h from filter paper by whole cellulase complex was not changed by first disrupting the substrate with the extracts.     

A high cellulase-producing mutant strain of Trichoderma reesei

A mutant strain with increased production of cellulolytic enzymes was induced from the good cellulase producer Trichoderma reesei QM 9414. Cellulase activities of the mutant in fermenter cultivations were increased two- to three-fold and β-glucosidase activity up to six-fold when compared to the corresponding activities produced by QM 9414.