Cellobiohydrolase II is the main conidial-bound cellulase in Trichoderma reesei and other Trichoderma strains

Monoclonal antibodies have been used to determine the presence of cellobiohydrolases I and II (CBH I and II), and endoglucanase I (EG I) on the surface of conidia from Trichoderma reesei QM 9414 and RUT C-30, and 8 other Trichoderma species. For this purpose, proteins were released from the conidial surface by treatment with a non-ionic detergent (Triton X-100 and -octylglucoside), followed by SDS-PAGE/Western blotting and immunostaining. Both CBH I and II were clearly present, but — unlike in extracellular culture fluids from Trichoderma — CBH II was the predominant cellulase. In T. reesei EG I could not be detected. The higher producer strain T. reesei RUT C-30 exhibited a higher conidial level of CBH II than T. reesei QM 9414. In order to assess the importance of the conidial CBH II level for cellulase induction by cellulose, multiple copies of the chb2 gene were introduced into the T. reesei genome by cotransformation using PyrG as a marker. Stable multicopy transformants secreted the 2- to 4-fold level of CBH II into the culture medium when grown on lactose as a carbon source, but their CBH I secretion was unaltered. Upon growth on cellulose, both CBH I and CBH II secretion was enhanced. Those strain showing highest cellulase activity on cellulose also appeared to contain the highest level of conidial bound CBH II. CBH II was also the predominant conidial cellulase in various other Trichoderma sp. However, roughly the same amount of conidial bound CBH II was detected in all strains, although their cellulase production differed considerably.     

Synergism of isoenzymes of cellobiohydrolase I and II of trichoderma reesei in hydrolysis of amorphous cellulose

Decompositions of amorphous cellulose induced by cellulases of Trichoderma reesei were evaluated from gradients at zero time of exponential functions which were fitted to nephelometrically measured values of turbidty of incubated solutions of cellulose [turbidity = A × exp (B × t)+ C [A, B, C = constants, t = time]]. Synergistic enhancements of decomposition of amorphous cellulose resulted in the range of 300 p.c. whenever of the two isoenzymes of cellobiohydrolase I of Trichoderma reesei (CBH I, being an exo-glucanase) one was incubated together with one of the isoenzymes of CBH II (being really an endo-glucanase). Accessibility of amorphous cellulose to enzymatic decomposition being calculated from the fitted function by the term (A/(A + C)) × 100 [p.c.] resulted for the CBH I isoenzymes and for the CBH II/1 in the range of 27 to 38 p.c. of the total substrate. Incubations of CBH II/1 in with CBH I/1 and CBH I/2 were followed by increases of accessibility to 85 and 87 p.c., respectively. CBH II/2 by itself caused a substrate accessibility in the range of 80 p.c., which increased to 96 p.c. when it was incubated together with CBH I/1 or CBH I/2. Amorphous cellulose dispersing activity (ACD activity) being evaluated from the fitted function by the term (A + C)/(A_c + C_c) × 100 [p.c.] (A_c + C_c × control turbidity at zero time) was not increased when a CBH I isoenzyme was incubated together with a CBH II isoenzyme. EG I, a convetional endo-glucanase from Tr. reesei proved not to act synergistically in any case when incubated together with one of the CBH isoenzymes. On the contrary, EG I turned out to act antagonistically to CBH II/1 and CBH II/2. Results can be interpreted as an exo-endo-synergism taking place between C₁-specific exo- and endo-glucanases.     

Adsorption of two cellobiohydrolases fromTrichoderma reesei to Avicel: Evidence for “exo-exo” synergism and possible “loose complex” formation

Summary Pure cellobiohydrolases I and II (CBH I & II) fromTrichoderma reesei adsorb strongly (K=10⁴M^–1) onto micro-crystalline Avicel. Equilibration is slow (>40 min) and saturation levels determined from the adsorption isotherms are almost identical:107–110 nmoles enzyme/mg Avicel. In admixture synergistic effects are observed dependent on the ratio of the enzymes. These effects are maximal for non-saturating conditions (1–10 M) and when the enzymes are added in two consecutive steps synergism of binding is only apparent for CBH I.     

Ionic strength effect on adsorption of cellobiohydrolases I and II on microcrystalline cellulose

Cellobiohydrolase I (CBH I) has a higher adsorption affinity (K _ad) and tightness (–H _a) for Avicel than cellobiohydrolase II (CBH II). The adsorption processes of CBH I and II were exothermic, and the degree of exothermy were larger with the increasing ionic strength. Entropy change of CBH I was larger than CBH II with increasing ionic strength. CBH I was more effective than CBH II for binding at a given ionic strength.     

Quantification and identification of the main components of the Trichoderma cellulase complex with monoclonal antibodies using an enzyme-linked immunosorbent assay (ELISA)

Summary An enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies has been developed to measure the concentration of three main cellulase components from Trichoderma reesei, cellobiohydrolase I (CBH I), cellobiohydrolase II (CBH II) and I (EG I), in both commercial enzyme preparations as well as in samples from laboratory fermentations. The sensitivity of the assay is 1–10 ng protein, depending on the type of cellulase. The coefficient of variability is between 10% and 20%. By a combination of two different domain-specific monoclonals against CBH I or II it is also possible to quantify the concentration of intact and truncated forms of these two enzymes, respectively. The use of the ELISA to quantify the formation of the three cellulase components under different cultivation conditions is described. Offprint requests to: C. P. Kubicek     

Identification of glycan structure and glycosylation sites in cellobiohydrolase II and endoglucanases I and II from Trichoderma reesei

Mass spectrometric techniques combined with enzymatic digestions were applied to determine the glycosylation profiles of cellobiohydrolase (CBH II) and endoglucanases (EG I, II) purified from filamentous fungus Trichoderma reesei. Electrospray mass spectrometry (ESMS) analyses of the intact cellulases revealed the microheterogeneity in glycosylation where glycoforms were spaced by hexose units. These analyses indicated that glycosylation accounted for 12-24% of the molecular mass and that microheterogeneity in both N- and O-linked glycans was observed for each glycoprotein. The identification of N-linked attachment sites was carried out by MALDI-TOF and capillary liquid chromatography-ESMS analyses of tryptic digests from each purified cellulase component with and without PNGase F incubation. Potential tryptic glycopeptide candidates were first detected by stepped orifice-voltage scanning and the glycan structure and attachment site were confirmed by tandem mass spectrometry. For purified CBH II, 74% of glycans found on Asn310 were high mannose, predominantly Hex(7-9)GlcNAc(2), whereas the remaining amount was single GlcNAc; Asn289 had 18% single GlcNAc occupancy, and Asn14 remained unoccupied. EG I presented N-linked glycans at two out of the six potential sites. The Asn56 contained a single GlcNAc residue, and Asn182 showed primarily a high-mannose glycan Hex(8)GlcNAc(2) with only 8% being occupied with a single GlcNAc. Finally, EG II presented a single GlcNAc residue at Asn103. It is noteworthy that the presence of a single GlcNAc in all cellulase enzymes investigated and the variability in site occupancy suggest the secretion of an endogenous endo H enzyme in cultures of T. reesei.     

Effect of different carbon sources on cellulase production by Hypocrea jecorina (Trichoderma reesei) strains

The ascomycete Hypocrea jecorina, an industrial (hemi)cellulase producer, can efficiently degrade plant polysaccharides. At present, the biology underlying cellulase hyperproduction of T. reesei, and the conditions for the enzyme induction, are not completely understood. In the current study, three different strains of T. reesei, including QM6a (wild-type), and mutants QM9414 and RUT-C30, were grown on 7 soluble and 7 insoluble carbon sources, with the later group including 4 pure polysaccharides and 3 lignocelluloses. Time course experiments showed that maximum cellulase activity of QM6a and QM9414 strains, for the majority of tested carbon sources, occurred at 120 hrs, while RUT-C30 had the greatest cellulase activity around 72 hrs. Maximum cellulase production was observed to be 0.035, 0.42 and 0.33 µmol glucose equivalents using microcrystalline celluloses for QM6a, QM9414, and RUTC-30, respectively. Increased cellulase production was positively correlated in QM9414 and negatively correlated in RUT-C30 with ability to grow on microcrystalline cellulose.     

Zum Einfluß der physikalischen Struktur des Substrates auf die Hydrolyse von Cellulose durch verschiedene Enzymsysteme und Enzymkomponenten

Starting from cellulose samples prepared from cotton lintes and differing in lattice type, crystallinity and fibrillar morphology, enzymatic hydrolysis of fibre cellulose has been studied employing complete enzyme systems from Trichoderma, Sporotrichum, Gliocladium and Penicillium as well as isolated endo- and exo-1,4-β-glucanases from Trichoderma reesei and Sporotorichum pulverulentum. The effect of hydrolysis was characterized by content of reducing sugars (RS) and of glucose in the hydrolyzate as well as by DP and X-ray diffraction pattern of the residues. With all the complete enzyme systems investigated about the same order of degradability was found with a series of substrates differing in physical structure. The hydrolysis effect of cellulase from S. pulverulentum proved to be sensitive to the gas atmosphere above the system (N₂ or O₂), probably due to the interaction of an O₂-atmosphere with the activity of the cellubiose-oxydase existent in the system. Isolated endoglucanase from S. pulverulentum and T.reesei still led to a considerable formation of RS and glucose, a corrosion of the fibre surface and a significant descrease in DP. Influence of substrate physical structure was rather small with regard to RS, but still considerable with regard to residue-DP. The effect of isolated exoglucanases depends largely on the chemical structure of the cellobiohydrolase in question, as demonstrated with the two samples “CBH I” and “CBH II” from T. reesei. With CBH I, rather resembling endo-glucanase behaviour, a considerable formation of RS and a significant corrosion of the fibre surface has been observed. On the other hand, only negligibly small amounts of RS were formed by CBH II. Results are discussed with regard to the complex mechanism of cellulase action on fibrous cellulose and with regard to the relevance of different parameters of physical structure of cellulose in connection with enzymatic hydrolysis. A remarkable acceleration of the Cellulose III → Cellulose I lattice transition due to chain fragmentations in the presence of cellulase can be concluded the experiments.     

Site-specific detection and structural characterization of the glycosylation of human plasma proteins lecithin:cholesterol acyltransferase and apolipoprotein D using HPLC/electrospray mass spectrometry and sequential glycosidase digestion.

Site-specific structural characterization of the glycosylation of human lecithin:cholesterol acyltransferase (LCAT) was carried out using microbore reversed-phase high performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC/ESIMS). A recently described mass spectrometric technique involving monitoring of carbohydrate-specific fragment ions during HPLC/ESIMS was employed to locate eight different groups of glycopeptides in a digest of a human LCAT protein preparation. In addition to the four expected N-linked glycopeptides of LCAT, a di-O-linked glycopeptide was detected, as well as three additional glycopeptides. Structural information on the oligosaccharides from all eight glycopeptides was obtained by sequential glycosidase digestion of the glycopeptides followed by HPLC/ESIMS. All four potential N-linked glycosylation sites (Asn20, Asn84, Asn272, and Asn384) of LCAT were determined to contain sialylated triantennary and/or biantennary complex structures. Two unanticipated O-linked glycosylation sites were identified at Thr407 and Ser409 of the LCAT O-linked glycopeptide, each of which contain sialylated galactose beta 1-->3N-acetylgalactosamine structures. The three additional glycopeptides were determined to be from a copurifying protein, apolipoprotein D, which contains potential N-linked glycosylation sites at Asn45 and Asn78. These glycopeptides were determined to bear sialylated triantennary oligosaccharides or fucosylated sialylated biantennary oligosaccharides. Previous studies of LCAT indicated that removal of the glycosylation site at Asn272 converts this protein to a phospholipase (Francone OL, Evangelista L, Fielding CJ, 1993, Biochim Biophys Acta 1166:301-304). Our results indicate that the carbohydrate structures themselves are not the source of this functional discrimination; rather, it must be mediated by the structural environment around Asn272.     

Protease activity and proteolytic modification of cellulases from a Trichoderma reesei QM 9414 selectant

Summary The secretion of multiple forms of cellulolytic enzymes by a Trichoderma reesei QM 9414 selectant exhibiting high protease activity (T. reesei QM 9414/A 30) was investigated using monoclonal, domain-specific antibodies against cellobiohydrolase (CBH) I, CBH II and -glucosidase, and a polyclonal antibody against endoglucanase I. The pattern of appearance of these proteins was followed during growth of the fungus on Avicel cellulose, using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western blotting/immunostaining. Evidence was obtained that, at late cultivation stages, CBH I and II became partially modified to lower molecular weight components, whereas -glucosidase and endoglucanase I appeared to remain largely intact. Modification of CBH I appeared to commence from the carboxy-terminal AB region, whereas CBH II appeared to become modified both from the amino- (ABB') and the carboxy-terminal. Evidence for a protease activity that modifies the already truncated cellobiohydrolases in the culture filtrate was obtained. These results show that proteolysis at late culture stages may contribute to the multiplicity of cellulases found in T. reesei culture fluids. Initial proteolytic cleavage of CBH I and II may, however, involve an unusual protease not detectable by the azocasein method.Offprint requests to: C. P. Kubicek     

Synergism of Trichoderma reesei cellulases while degrading different celluloses

Summary The synergistic action of purified cellulases from Trichoderma reesei in hydrolysis of cellulose decreased with increasing substrate concentration, depended strongly on the the type of cellulose used, and was maximal on crystalline cellulose. Contrarily, the activity of the individual cellulases was highest on amorphous cellulose. The binary combinations CBH I/EG III and CBH I/CBH II exhibited the greatest degree of synergism on crystalline cellulose.     

Cellulase production by Trichoderma reesei (RUT-C30) in fed-batch culture

Summary Fed-batch cultures of Trichoderma reesei RUT-C30 attained quasi-steady state conditions, in respect of biomass concentration and enzyme production rate, commensurate with a specific cell maintenance coefficient of 0.029 g cellulose.g biomass.^–1h^–1 and specific cellulase production rate of between 9.6 and 11.9 IU (filter paper activity).g biomass.^–1h^–1. A maximum enzyme yield of 57 IU.m1^–1 at an overall productivity of 201 IU.L.^–1h^–1 resulted from a cellulose feed rate of 1.0g.L.^–1h^–1.     

Domain engineering of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> exoglucanases

To illustrate the effect of a cellulose-binding domain (CBD) on the enzymatic characteristics of non-cellulolytic exoglucanases, 10 different recombinant enzymes were constructed combining the Saccharomyces cerevisiae exoglucanases, EXG1 and SSG1, with the CBD2 from the Trichoderma reesei cellobiohydrolase, CBH2, and a linker peptide. The enzymatic activity of the recombinant enzymes increased with the CBD copy number. The recombinant enzymes, CBD2-CBD2-L-EXG1 and CBD2-CBD2-SSG1, exhibited the highest cellobiohydrolase activity (17.5 and 16.3 U mg ^–1 respectively) on Avicel cellulose, which is approximately 1.5- to 2-fold higher than the native enzymes. The molecular organisation of CBD in these recombinant enzymes enhanced substrate affinity, molecular flexibility and synergistic activity, contributing to their elevated action on the recalcitrant substrates as characterised by adsorption, kinetics, thermostability and scanning electron microscopic analysis.     

Ethanol production from cellulosic materials using cellulase-expressing yeast

We demonstrate direct ethanol fermentation from amorphous cellulose using cellulase-co-expressing yeast. Endoglucanases (EG) and cellobiohydrolases (CBH) from Trichoderma reesei, and β-glucosidases (BGL) from Aspergillus aculeatus were integrated into genomes of the yeast strain Saccharomyces cerevisiae MT8-1. BGL was displayed on the yeast cell surface and both EG and CBH were secreted or displayed on the cell surface. All enzymes were successfully expressed on the cell surface or in culture supernatants in their active forms, and cellulose degradation was increased 3- to 5-fold by co-expressing EG and CBH. Direct ethanol fermentation from 10 g/L phosphoric acid swollen cellulose (PASC) was also carried out using EG-, CBH-, and BGL-co-expressing yeast. The ethanol yield was 2.1 g/L for EG-, CBH-, and BGL-displaying yeast, which was higher than that of EG- and CBH-secreting yeast (1.6 g/L ethanol). Our results show that cell surface display is more suitable for direct ethanol fermentation from cellulose.     

Trichoderma reesei cellobiohydrolase II is associated with the outer membrane when overexpressed in Escherichia coli

Cellulose degradation is essential for the future production of many advanced biofuels. Cellulases from the filamentous fungus Trichoderma reesei are among the most efficient enzymes for the hydrolysis of cellulosic materials. One of the cellulases from T. reesei, cellobiohydrolase II (CBH2), was studied because of its industrial relevance and proven enzymatic activity. Using both crude and rigorous membrane fractionation methods we show that full length T. reesei CBH2 is exclusively localized to the outer membrane when expressed recombinantly in Escherichia coli. Even fusing signal sequence-free maltose-binding protein to the N-terminus of CBH2, which has been shown to increase solubility of other proteins, did not prevent the outer membrane localization of CBH2. These results highlight the difficulties in producing fungal cellulases in bacterial hosts and provide a stepping stone for future cellulase engineering efforts.     

Formation of enzymes required for the hydrolysis of plant cell wall polysaccharides byTrichoderma reesei

Summary Enzyme preparations fromTrichoderma reesei RUT-C30, in addition to cellulase, contained various glucanase and glucosidase, acetylxylan esterase, glucuronidase and xylanase activities. These preparations were able to hydrolyse endosperm cell walls of corn and wheat and commercially-available xylans and plant gums having stright chains, but lacked the ability to hydrolyse branched or substituted hemicelluloses.

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Formation d'enzymes requises pour l'hydrolyse des polysaccharides de la paroi de plantes chez Trichoderma reesei

Résumé Les préparations enzymatiques deTrichoderma reesei RUT-C30, contiennent, outre la cellulase, diverses glucanases et glucosidases, acétyl-xylane esterases, glucuronidases et xylanases. Ces préparations demeurent stables pour l'hydrolyse de parois cellulaires de l'endosperme de maïs et de froment ainsi que des xylanes et gommes de plantes à chaînes linéaires, disponibles dans le commerce, mais ne présentent pas le pouvoir d'hydrolyser les hémicelluloses branchées ou substituées.

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Issued as NRCC No. 29855     

Carbon Source Control of Cellobiohydrolase I and II Formation by Trichoderma reesei

Regulation of the formation and secretion of two cellulase components from Trichoderma reesei QM 9414, cellobiohydrolases I and II (CBH I and CBH II, respectively), by the carbon source was investigated. With monoclonal antibodies against CBH I and CBH II it was found that during cultivation on carbon sources which enable fast growth (glucose, glycerol, and fructose), no formation of CBH I occurred, whereas low levels of CBH II were formed. Lactose and cellulose, which allow comparably slower growth, promoted the formation of both CBH I and CBH II. However, noncarbohydrate carbon sources as citrate or acetate, which also enable only slow growth, did not promote the formation of CBH I or CBH II. The addition of glucose or glycerol to lactose- or cellulose-pregrown mycelia, on the other hand, only partially reduced the formation of CBH I. This reduction was also achieved by several other metabolizable and nonmetabolizable carbon compounds, e.g., fructose, galactose, β-methylglucoside, 2-deoxyglucose, and rhamnose, as well as by transfer to no carbon source at all. This result indicates that the control of CBH I synthesis by the carbon source is due to induction and not to repression. The use of cycloheximide and 5-fluorouracil as inhibitors at and before translation, respectively, revealed a half-life for CBH I mRNA of at least several hours, which may, at least in part, account for the prolonged synthesis of some CBH I under these conditions. Northern (RNA) hybridization with full copies of cbh1 and cbh2 genes indicated that the control of CBH I and CBH II biosyntheses by the carbon source operates mainly at the pretranslational level. We conclude that the low rate of cellulase synthesis on glucose and some other carbon sources is due to the lack of an inducer and not to carbon source repression.     

Functional display of fungal cellulases from <Emphasis Type="Italic">Trichoderma </Emphasis><Emphasis Type="Italic">reesei</Emphasis> on phage M13

To test whether the phage display technology could be applied in cellulase engineering, phagemids harboring the genes encoding the mature forms of cellobiohydrolase I (CBH I) and endoglucanase I (EG I) from filamentous fungus Trichoderma reesei were constructed, respectively. CBH I and EG I fused to the phage coat protein encoded by the g3 gene were expressed and displayed on phage M13. The phage-bound cellulases retained their activities as determined by hydrolysis of the corresponding substrates, Also, their binding abilities to insoluble cellulose substrate were confirmed by an ELISA method. Overall, these results demonstrate that cellulases can be displayed on phage surface while maintaining their biological function, thus providing an alternative for directed evolution and high-throughput screening for improved cellulases.     

Enzymatic hydrolysis and simultaneous saccharification and fermentation of steam-pretreated spruce using crude Trichoderma reesei and Trichoderma atroviride enzymes

The aim of this study was to compare the performance of the enzymes produced by Trichoderma reesei Rut C30 and the good extracellular β-glucosidase-producing mutant Trichoderma atroviride TUB F-1663 to that of commercial preparations in the enzymatic hydrolysis and the simultaneous saccharification and fermentation (SSF) of steam-pretreated spruce (SPS).The concentrated TUB F-1663 enzyme was found to be the most efficient in the hydrolysis of washed SPS at 50 g/L water-insoluble solids (WIS) in terms of the glucose produced (18.5 g/L), even in comparison with commercial cellulases (14.1–16.7 g/L). The enzyme preparations were studied at low enzyme loadings (5 FPU/g WIS) in SSF to produce ethanol from SPS. The enzyme supernatant and whole fermentation broth of T. atroviride as well as the whole broth of T. reesei proved to be as efficient in SSF as the commercial cellulase mixtures (ethanol yields of 61–76% of the theoretical were achieved), while low ethanol yields (<40%) were obtained with the β-glucosidase-deficient T. reesei supernatant.Therefore, it seems, that instead of using commercial cellulases, the TUB F-1663 enzymes and the whole broth of Rut C30 may be produced on-site, using a process stream as carbon source, and employed directly in the biomass-to-bioethanol process.