Cloning of a gene encoding thermostable cellobiohydrolase from<Emphasis Type="Italic"> Thermoascus aurantiacus</Emphasis> and its expression in yeast

A gene encoding a cellobiohydrolase (CBH) was isolated from Thermoascus aurantiacus IFO 9748 and designated as cbh1. The deduced amino acid sequence encoded by cbh1 showed high homology with the sequence of glycoside hydrolase family 7. To confirm the sequence of the gene encoding the CBH, the cloned gene was expressed in the yeast Saccharomyces cerevisiae, in which no cellulase activity was found, and the gene product was purified and subjected to enzymatic characterization. The recombinant enzyme was confirmed as a CBH by analysis of the reaction product and designated as CBHI. Recombinant CBHI retained more than 80% of its initial activity after 1 h of incubation at 65 °C and was stable in the pH range 3.0–9.0. The optimal temperature for enzyme activity was about 65 °C and the optimal pH was about 6.0. The recombinant enzyme was found to be highly glycosylated and this glycosylation was shown to contribute to the thermostability of the enzyme. CBHI expression was shown to be induced at higher temperature in T. aurantiacus.     

A novel PCR-based approach for the detection and classification of potential cellulolytic fungal strains isolated from museum items and surrounding indoor environment

Aims: To develop a novel PCR‐based method able to detect potential cellulolytic filamentous fungi and to classify them exploiting the amplification of the cellobiohydrolase gene (cbh‐I) and its polymorphism. Methods and Results: A mixed approach including the combination of (i) fungal cultivation and isolation, (ii) classification of fungal isolates through the amplification of the cbh gene using a fluorescently labelled primer (f‐CBH‐PCR) and (iii) final fungal identification based on amplification and sequencing of the ITS1‐5.8S rDNA‐ITS2 region of the selected fungal strains was developed. By this approach, it was possible to screen 77 fungal strains belonging to 14 genera and 26 species. Conclusions: The f‐CBH‐PCR permitted the discrimination of fungal species, producing typical f‐CBH profiles. Significance and Impact of the Study: In this study, the cbh gene was used as a preliminary classification tool able to differentiate among themselves the fungal members isolated from indoor museum items and surrounding environment. Such mixed approach consented the fast identification of all isolated fungal strains. The f‐CBH‐PCR method demonstrated its discrimination power, and it can be considered as a new molecular system suitable for the classification of fungal strains isolated from different environments.     

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.     

Differential responses of wood-rot fungi cellulases towards polyclonal antibodies against Trichoderma viride cellobiohydrolase I

Cellobiohydrolase (CBH) I, a main component of Trichoderma extracellular protein, was purified to an electrophoretically homogeneous state from a commercial cellulase preparation (Meicelase from T. viride) by column chromatography on anion and cation exchangers. The difference in the cross-reactivity of cellulolytic enzyme systems of brown-rot and white-rot fungi with the polyclonal antibodies to the CBH I was studied by enzyme-linked immunosorbent assay (ELISA). The antibodies were observed to react quantitatively and with great sensitivity with the antigen (CBH I), and at the same time to cross-react to some extent with T. viride cellulase components other than the CBH I. Nevertheless, the intensity of cross-reactivity of wood-rot fungi cellulases with the antibodies was parallel to the activity of exo-1,4-ß-glucanase. The cellulase system from brown-rot fungi, believed to lack exo-1,4-ß-glucanases, gave a negative response towards the antibodies. These results suggested the presence of some homologous sequences and structures with the T. viride CBH I in the enzymes of white-rot fungi and their absence in those of brown-rot fungi. Correspondence to: M. Ishihara     

Cloning of a gene encoding thermostable cellobiohydrolase from the thermophilic fungus Chaetomium thermophilum and its expression in Pichia pastoris

Aims:  A new cellobiohydrolase (CBH) gene ( cbh3 ) from Chaetomium thermophilum was cloned, sequenced and expressed in Pichia pastoris .
Methods and Results:  Using RACE-PCR, a new thermostable CBH gene ( cbh3 ) was cloned from C. thermophilum . The cDNA of the CBH was 1607 bp and contained a 1356 bp open reading frame encoding a protein CBH precursor of 451 amino acid residues. The mature protein structure of C. thermophilum CBH3 only comprises a catalytic domain and lacks cellulose-binding domain and a hinge region. The gene was expressed in P. pastoris . The recombinant CBH purified was a glycoprotein with a size of about 48·0 kDa, and exhibited optimum catalytic activity at pH 5·0 and 60 °C. The enzyme was more resistant to high temperature. The CBH could hydrolyse microcrystalline cellulose and filter paper.
Conclusions:  A new thermostable CBH gene of C. thermophilum was cloned, sequenced and overexpressed in P. pastoris .
Significance and Impact of the Study:  This CBH offers an interesting potential in saccharification steps in both cellulose enzymatic conversion and alcohol production.     

Cloning of aMicrobispora bispora cellobiohydrolase gene inEscherichia coli

Summary Cellobiohydrolase II was purified from aMicrobispora bispora culture filtrate and a monoclonal antibody to it was prepared. Screening aM. bispora genomic library inEscherichia coli with this antibody yielded three equivalent clones. Subcloning resulted in greater expression, and activity could be monitored using 4-methylumbelliferylcellobioside. Southern analysis provided evidence that there is a single gene coding for CBH II. The original 22-kb fragment was reduced to 4 kb and subcloned into pUC118/119 resulting in a doubling of expression CBH II. The gene was expressed via its own promoter. The optimal pH (6.5) and the optimal temperature (60°C) of the cloned enzyme are similar to that of the native CBH II.     

Synergism between components of the cellulase system of the anaerobic rumen fungus Neocallimastix frontalis and those of the aerobic fungi Penicillium pinophilum and Trichoderma koningii in degrading crystalline cellulose

The cellulase system of Neocallimastix frontalis was separated by differential affinity on cellulose into an adsorbed fraction that could solubilize crystalline cellulose (crystalline-cellulose-solubilizing fraction, CCSF), and a non-adsorbed fraction that contained endoglucanase and -glucosidase activities (non-adsorbed endoglucanase/ -glucosidas, NAE/-G) but which showed no activity to crystalline cellulose. Both fractions were tested for their capacity to act synergistically with the cellobiohydrolase (CBH) components of aerobic fungi in degrading crystalline cellulose. The CCSF acted synergistically with CBH I components of both Penicillium pinophilum and Trichoderma koningii but not with CBH II. The NAE/-G fraction also acted synergistically with the CBH components of P. pinophilum but, remarkably, only when both CBH I and CBH II were present in the reaction mixture. By comparison with previously published studies on the mechanism of action of P. pinophilum cellulase it is speculated that the CCSF of N. frontalis may contain CBH I- and CBH II-type enzymes.     

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.     

Synergistic cooperation of cellulases of trichoderma reesei as distinguished by a new filter-paper destruction assay

WHATMAN 1 CHR filter paper manufactured from macerated cotton fibers was shown to be a soft substrate when broken down by purified cellulases of Trichoderma reesei (CELLUCLAST). Destruction of filter-paper disks was induced by CBH I/1, CBH I/2, CBH II/1, CBH II/2, and EG I in a macroscopic assay. Attack on disks by mixtures of these cellulases (CBH I/1 or CBH I/2 mixed with CBH II/1, CBH II/2, or with EGJ) were followed by synergistically enhanced destructions. SCHLEICHER &SCHUELL filter paper No 595 was shown to be a harder substrate of enzymatical decomposition when induced by cellulases of CELLUCLAST. None of the cellulases could induce macroscopic destruction of filter-paper disks when acting in isolation. However, mixtures of isolated exo and endo-glucanases (CBH I/1 or CBH I/2 mixed with CBH II/1, CBH II/2, or EG I) caused powerful destruction of filter-paper disks. SCHLEICHER &SCHUELL filter paper No 595 incubated first with an endo-glucanase (CBH II/1, CBH II/2, EG I) and treated in a secondary incubation with an exo-glucanase (CBH I/1, CBH I/2) were destroyed to a greater extent than with incubations executed in the reverse order. Results confirm the endo exo concept of explaining cellulose decomposition. The filter-paper destruction assay was performed with filter-paper disks prepared with an office punch. Disks were incubated in 1 ml EPPENDORF reaction tubes filled up beforehand with 0.4 or 0.5 ml of enzyme solution. The degree of synergism of cellulases resulted from the assay in the range of 300 to 1 300 p.c.     

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     

Turnover of labile and recalcitrant soil carbon differ in response to nitrate and ammonium deposition in an ombrotrophic peatland

The effects of 4 years of simulated nitrogen deposition, as nitrate (NO₃^?) and ammonium (NH₄⁺), on microbial carbon turnover were studied in an ombrotrophic peatland. We investigated the mineralization of simple forms of carbon using MicroResp^? measurements (a multiple substrate induced respiration technique) and the activities of four soil enzymes involved in the decomposition of more complex forms of carbon or in nutrient acquisition: N‐acetyl‐glucosaminidase (NAG), cellobiohydrolase (CBH), acid phosphatase (AP), and phenol oxidase (PO). The potential mineralization of labile forms of carbon was significantly enhanced at the higher N additions, especially with NH₄⁺ amendments, while potential enzyme activities involved in breakdown of more complex forms of carbon or nutrient acquisition decreased slightly (NAG and CBH) or remained unchanged (AP and PO) with N amendments. This study also showed the importance of distinguishing between NO₃^? and NH₄⁺ amendments, as their impact often differed. It is possible that the limited response on potential extracellular enzyme activity is due to other factors, such as limited exposure to the added N in the deeper soil or continued suboptimal functioning of the enzymes due to the low pH, possibly via the inhibitory effect of low phenol oxidase activity.     

Differential induction of apoplastic peroxidase and chitinase activities in susceptible and resistant wheat cultivars by Russian wheat aphid infestation

The intercellular peroxidase and chitinase activities of three wheat cultivars [Triticum aestivum L. cvs `Tugela DN', `Molopo DN' (Gariep) and `Betta DN'] containing the Dn-1 gene for resistance to the Russian wheat aphid (RWA) Diuraphis noxia (Mordvilko) and the corresponding near-isogenic susceptible cultivars (`Tugela', `Molopo' and `Betta') were studied under conditions of infestation and non-infestation. The aim was to gain information on the mechanism of resistance. The resistance response was induced by RWA infestation. Infestation rapidly induced the activities of both enzymes selectively in resistant wheat to levels of magnitudes higher than those in susceptible wheat. The genetic background in which the Dn-1 resistance gene is bred played a role and the level of activity corresponded to the level of resistance. Immunologic studies confirmed that the induction of enzyme activities was due to the induction of higher protein levels. These results indicate that peroxidase and chitinase may have a role in insect resistance. Received: 20 June 1997 / Revision received: 9 April 1998 / Accepted: 5 June 1998     

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.     

Effects of soluble corn bran hemicellulose on lipid metabolism

• 1.1. Since soluble corn bran hemicellulose (CBH) was found to reduce serum cholesterol level in the rat fed with a high cholesterol diet, rats were fed with diets containing orotic acid (OA) to investigate the effect of CBH on lipid metabolism.
• 2.2. Hepatic lipid accumulation induced by OA was reduced by feeding with CBH in rats. The reduction was not due to inhibition of intestinal absorption of OA by CBH.
• 3.3. Administration of acetate or propionate, colonie fermentation products of CBH, tended to alleviate the hepatic lipid accumulation by OA in rats.
• 4.4. OA feeding decreased activities of some hepatic enzymes involved in fatty acid synthesis except for acetyl CoA carboxylase. The decreases were reversed by the concurrent feeding of CBH.