Application of cellulases from Acrophialophora nainiana and Penicillium echinulatum in textile processing of cellulosic fibres

New cellulases from the fungi Acrophialophora nainiana and Penicillium echinulatum were used in the finishing of knitted cotton fabrics (biopolishing) and compared with the well established enzymes from Trichoderma reesei. Both cellulases reduced the pilling tendency with a lower weight loss than T. reesei cellulases. Cellulases from P. echinulatum were also studied in stonewashing of denim fabrics to obtain the fashionable aged look in indigo dyed jeans ware and were found to remove more colour from denim fabrics and produce less indigo dye redeposition (back-staining) than commercial acid or neutral cellulases under the test conditions. Efficiency was found to be influenced by pH during textile processing and the substrate used for the production of cellulases. Cellulases produced by P. echinulatum grown on cellulose showed better stonewashing results (higher colour removal and less back-staining) than cellulases produced on sugar cane bagasse. The substrate used during enzyme production of P. echinulatum cellulases seems to have a significant influence on cellulose composition, which affects textile processing results.     

Trichoderma reesei cellulases in the bleaching of kraft pulp

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. Correspondence to: J. Buchert     

Induction and production of cellulases by l-sorbose in Trichoderma reesei

Summary Cellulase production in Trichoderma reesei mutants was induced by l-sorbose, known to be an inhibitor of -1,3-glucan synthesis. In the experiments the washed mycelia were used as resting cells. For CMCase induction over 24 h using T. reesei PC-3-7, the most effective pH, temperature and l-sorbose concentration were 2.8, 28° C and 0.3 mg/ml, respectively. Comparison with other cellulase inducers showed that the inductive level of CMCase by l-sorbose was similar to that by sophorose, known to be the most potent inducer of cellulases. Since the induction of CMCase was inhibited completely by 10 g of cycloheximide per ml, the induction process was considered to involve de novo synthesis. Although l-sorbose had the effective inducibility of CMCase, the assimilation rate of l-sorbose was very low in T. reesei PC-3-7.Production of Ethanol from Biomasses. Part III.Production of Ethanol from Biomasses. Part III.     

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     

Expression of Trichoderma reesei cellulases CBHI and EGI in Ashbya gossypii

To explore the potential of Ashbya gossypii as a host for the expression of recombinant proteins and to assess whether protein secretion would be more similar to the closely related Saccharomyces cerevisiae or to other filamentous fungi, endoglucanase I (EGI) and cellobiohydrolase I (CBHI) from the fungus Trichoderma reesei were successfully expressed in A. gossypii from plasmids containing the two micron sequences from S. cerevisiae, under the S. cerevisiae PGK1 promoter. The native signal sequences of EGI and CBHI were able to direct the secretion of EGI and CBHI into the culture medium in A. gossypii. Although CBHI activity was not detected using 4-methylumbelliferyl-β-d-lactoside as substrate, the protein was detected by Western blot using monoclonal antibodies. EGI activity was detectable, the specific activity being comparable to that produced by a similar EGI producing S. cerevisiae construct. More EGI was secreted than CBHI, or more active protein was produced. Partial characterization of CBHI and EGI expressed in A. gossypii revealed overglycosylation when compared with the native T. reesei proteins, but the glycosylation was less extensive than on cellulases expressed in S. cerevisiae.     

Treatment of recycled kraft pulps with Trichoderma reesei hemicellulases and cellulases

Effects of recycling ECF-bleached softwood kraft pulp on pulp properties were evaluated in the laboratory. The tensile strength, fiber flexibility and WRV lost during drying of the pulp were recovered by refining between the cycles which, however, resulted in deteriorated drainage properties. The recycled pulps were treated with purified Trichoderma reesei cellulases and hemicellulases and the changes in fiber properties due to enzymatic treatments were characterized. The endoglucanases (EG I and EG II) significantly improved pulp drainage already at low dosage levels, and EG II was found to be more effective at a given level of carbohydrate solubilization. Combining hemicellulases with the endoglucanase treatments increased the positive effects of the endoglucanases on pulp drainage. However, as a result of the endoglucanase treatments a slight loss in strength was observed. Combining mannanase with endoglucanase treatments appeared to increase this negative effect, whereas the impact of xylanase was not significant. Although the drainage properties of the pulps could be improved by selected enzymes, the water retention capacity of the dried hornified fibers could not be recovered by any of the enzymes tested.     

Action of purified Trichoderma reesei cellulases on cotton fibers and yarn

In this work the possibility and potential of treating cotton fibers and yarns instead of fabrics with monocomponent cellulases was investigated. Different pretreatments on fibers were performed and tested in order to improve the accessibility of cotton to enzymatic modification. The enzymatic treatments were evaluated microscopically and by analysing the effects of treated fibers on spinnability, yarn evenness, tenacity and pilling. The accessibility of the cotton fibers for cellulases could be increased by different pretreatments. Steaming of fibers prior to enzymatic treatment was found to be an efficient way to increase hydrolysis levels. Cellulase treatments of carded yarns resulted in modification of yarn properties. Decrease in yarn hairiness was observed and the knitted fabric made of the treated yarn showed a lowered tendency towards pilling. In all cases endoglucanase activity rather than cellobiohydrolase activity was responsible for these modifications.     

Fragmentation of cellulose by the major Thermomonospora fusca cellulases, Trichoderma reesei CBHI, and their mixtures

In this study, the fragmentation activities of Thermomonospora fusca cellulases E(2), E(3), E(5), Trichoderma reesei CBHI, and their mixtures were measured to study synergism in fragmentation. Fragmentation studies revealed that only two pure cellulases, T. fusca E(2) and E(5) had significant fragmentation activity. T. fusca E(3) shows strong synergism in fragmentation both in the production of reducing sugars and in fragmentation with both T. fusca endoglucananses and with T. reesei CBHI. Most mixtures containing CBHI produced higher rates of fragmentation than comparable mixtures containing E(3). The highest rate and extent of reducing sugar formation and the highest fragmentation activity were obtained with a mixture of E(2), E(3), and CBHI. (c) 1992 John Wiley & Sons, Inc.     

Comparative study of the growth of Trichoderma reesei and Penicillium occitanie

During the last years, a great deal of research on the production of energetic substances was directed to the use of cellulosic by-products. A technique of special interest is the production of fermentissible sugars by the enzymatic hydrolysis of cellulose. Mandels and Reese (1960) showed that a fungi strain of Trichoderma reesei is the best performant microorganism in the production of cellulolytic enzymes. Nevertheless, recent investigation indicated that the rate and the yield of conversion of cellulose to glucose of this strain are limited by its poor beta-glucosidase activity. In order to increase the efficiency of the hydrolytic power of the cellulasic complex two approaches can be considered. Beta-glucosidase enrichment of Trichoderma reesei enzymes. The selection and use of strains with a better performance. In our laboratory, we chose the second approach using Penicillium occitanie comparing it to Trichoderma reesei.     

Fusion of cellulose binding domain from Trichoderma reesei CBHI to Cryptococcus sp. S-2 cellulase enhances its binding affinity and its cellulolytic activity to insoluble cellulosic substrates

Cryptococcus sp. S-2 carboxymethyl cellulase (CSCMCase) is active in the acidic pH and lacks a binding domain. The absence of the binding domain makes the enzyme inefficient against insoluble cellulosic substrates. To enhance its binding affinity and its cellulolytic activity to insoluble cellulosic substrates, cellulose binding domain (CBD) of cellobiohydrolase I (CBHI) from Trichoderma reesei belonging to carbohydrate binding module (CBM) family 1 was fused at the C-terminus of CSCMCase. The constructed fusion enzymes (CSCMCase-CBD and CSCMCase-2CBD) were expressed in a newly recombinant expression system of Cryptococcus sp. S-2, purified to homogeneity, and then subject to detailed characterization. The recombinant fusion enzymes displayed optimal pH similar to those of the native enzyme. Compared with rCSCMCase, the recombinant fusion enzymes had acquired an increased binding affinity to insoluble cellulose and the cellulolytic activity toward insoluble cellulosic substrates (SIGMACELL^® and Avicel) was higher than that of native enzyme, confirming the presence of CBDs improve the binding and the cellulolytic activity of CSCMCase on insoluble substrates. This attribute should make CSCMCase an attractive applicant for various application.     

Comparative study of cellulases and hemicellulases from four fungi: mesophiles Trichoderma reesei and Penicillium sp. and thermophiles Thielavia terrestris and Sporotrichum cellulophilum

The enzymes produced by two thermophilic fungi claimed to produce heat-stable cellulases [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] have been compared to those of two mesophilic fungi on the basis of the following criteria: polysaccharolytic spectrum, heat and pH effects on stability and on activity of the different enzymes, and the ability to hydrolyse raw natural substrates. The cellulases produced by one of the thermophiles, Sporotrichum cellulophilum, appeared to be as heat-labile as those from the mesophile Trichoderma reesei; moreover, the former enzyme preparation is the least efficient of the four tested. Thielavia terrestris enzymes are the most thermostable; on the basis of the other properties tested, T. terrestris enzymes are comparable to, or in some cases better than, those from mesophilic strains. However, the differences are not so great as to compensate for the much lower productivity of T. terrestris compared to the improved T. reesei and Penicillium sp. strains.     

Improvement of cellulase activity in Trichoderma reesei by heterologous expression of a beta-glucosidase gene from Penicillium decumbens

Trichoderma reesei is a well-known cellulase producer and widely applied in enzyme industry. To increase its ability to efficiently decompose cellulose, the beta-glucosidase activity of its enzyme cocktail needs to be enhanced. In this study, a beta-glucosidase I coding sequence from Penicillium decumbens was ligated with the cellobiohydrolase I (cbh1) promoter of T. reesei and introduced into the genome of T. reesei strain Rut-C30 by Agrobacterium-mediated transformation. In comparison to that from the parent strain, the beta-glucosidase activity of the enzyme complexes from two selected transformants increased 6- to 8-fold and their filter paper activity (FPAs) was enhanced by 30% on average. The transformant's saccharifying ability towards pretreated cornstalk was also significantly enhanced. To further confirm the effect of heterologous beta-glucosidase on the cellulase activity of T. reesei, the heterologously expressed pBGL1 was purified and added to the enzyme complex produced by T. reesei Rut-C30. Supplementation of the Rut-C30 enzyme complex with pBGL1 brought about 80% increase of glucose yield during the saccharification of pretreated cornstalk. Our results indicated that the heterologous expression of a beta-glucosidase gene in T. reesei might produce balanced cellulase preparation.     

Production of cellulases and hemicellulases by Penicillium echinulatum grown on pretreated sugar cane bagasse and wheat bran in solid-state fermentation

AIM: To evaluate the solid-state fermentation (SSF) production of cellulase and hemicellulases (xylanases), by Penicillium echinulatum 9A02S1, in experiments carried out with different concentrations of the pretreated sugar cane bagasse (PSCB) and wheat bran (WB). METHODS AND RESULTS: This study reports the production of xylanolytic and cellulolytic enzymes by P. echinulatum 9A02S1 using a cheap medium containing PSCB and WB under SSF. The highest amounts of filter paper activity (FPA) could be measured on mixtures of PSCB and WB (32.89 +/- 1.90 U gdm(-1)). The highest beta-glucosidase activity was 58.95 +/- 2.58 U gdm(-1) on the fourth day. The highest activity for endoglucanases was 282.36 +/- 1.23 U gdm(-1) on the fourth day, and for xylanases the activity was around 10 U gdm(-1) from the second to the fourth day. CONCLUSIONS: The present work has established the potential of P. echinulatum for FPA, endoglucanase, beta-glucosidase and xylanase productions in SSF, indicating that WB may be partially substituted by PSCB. SIGNIFICANCE AND IMPACT OF THE STUDY: The incorporation of cheap sources, such as sugar cane bagasse, into media for the production of lignocellulose enzymes should help decrease the production costs of enzymatic complexes that can hydrolyse lignocellulose residues for the formation of fermented syrups, thus contributing to the economic production of bioethanol.     

Characterization of the secreted cellulases of Trichoderma reesei wild type and mutants during controlled fermentations

Summary The cellulases produced under pH controlled fermentation conditions with 5% Solka Floc and cornsteep liquor as substrates by Trichoderma reesei wild type QM6a and two mutants, Rut-C30 and RL-P37, have been separated by isoelectric focusing in polyacrylamide gels. The total complement of secreted proteins of the two mutants was distinct from the parent. However, the number and isoelectric points of the various enzymes in the cellulase complex were unchanged in the mutants. All secreted proteins stained with Schiff's reagent which indicated they were glycoproteins. One mutant, Rut-C30, exhibited a dramatic shift in the CBH I proteins during the course of the fermentation. RL-P37 showed a two-fold increase in the specific activity of both the total cellulase complex and endoglucanase. In addition a productivity on the order of 100 IU/l/h was achieved. Co-produced with the cellulases were at least two acid proteases with differential activity towards azocoll and azocasein.     

The effect of oxidative pretreatment on cellulose degradation by Poria placenta and Trichoderma reesei cellulases

The possible role of hydrogen peroxide in brown-rot decay was investigated by studying the effects of pretreatment of spruce wood and microcrystalline Avicel cellulose with H₂O₂ and Fe²⁺ (Fenton's reagent) on the subsequent enzymatic hydrolysis of the substrates. A crude endoglucanase preparation from the brown-rot fungus Poria placenta, a purified endoglucanase from Trichoderma reesei and a commercial Trichoderma cellulase were used as enzymes. Avicel cellulose and spruce dust were depolymerized in the H₂O₂/Fe²⁺ treatment. Mainly hemicelluloses were lost in the treatment of spruce dust. The effect of the pretreatment on subsequent enzymatic hydrolysis was found to depend on the nature of the substrate and the enzyme preparation used. Pretreatment with H₂O₂/Fe²⁺ clearly increased the amount of enzymatic hydrolysis of spruce dust with both the endoglucanases and the commercial cellulase. In all cases the amount of hydrolysis was increased about threefold. The hydrolysis of Avicel with the endoglucanases was also enhanced, whereas the hydrolysis with the commercial cellulase was decreased. Received: 23 December 1996 / Received revision: 17 April 1997 / Accepted: 19 April 1997     

Production of cellulases by Trichoderma reesei QM 9414 in batch and fed-batch cultures on wheat straw

Trichoderma reesei QM 9414 was grown in batch fermentation on wheat straw pretreated by different methods as the sole carbon source. Cellulase production was maximal with NaOH treated wheat straw at a concentration of 10 g/l and an initial pH of 5.5. The addition of fresh straw produced an elongation of the exponential phase or the beginning of a new exponential phase when the additions were carried out at 50 and 120 h, respectively. Filter paper and carboxymethylcellulase activities decreased as an answer to the addition of wheat straw and the levels were regained at the end of fermentation. The decreases of activities were accompanied by the increases of soluble sugar levels, which decreased at the end of fermentation. β-glucosidase activity was stimulated by wheat straw addition at 50 h while not by addition at 120 h; however, at the end of the fermentation the levels of activities were both similar to control. The studies of pH stabilities of these enzymes allow assurance that the effect of the addition of wheat straw on the enzyme activities is not produced by the changes of the pH during the fermentation.     

The relation between solution association and surface activity of the hydrophobin HFBI from Trichoderma reesei

Hydrophobins are small fungal surface active proteins that self-assemble at interfaces into films with nanoscale structures. The hydrophobin HFBI from Trichoderma reesei has been shown to associate in solution into tetramers but the role of this association on the function of HFBI has remained unclear. We produced two HFBI variants that showed a significant shift in solution association equilibrium towards the tetramer state. However, this enhanced solution association did not alter the surface properties of the variant HFBIs. The results show that there is not a strong relationship between HFBI solution association state and surface properties such as surface activity.     

Fungal cellulase systems. Comparison of the specificities of the cellobiohydrolases isolated from Penicillium pinophilum and Trichoderma reesei.

Reaction patterns for the hydrolysis of chromophoric glycosides from cello-oligosaccharides and lactose by the cellobiohydrolases (CBH I and CBH II) purified from Trichoderma reesei and Penicillium pinophilum were determined. They coincide with those found for the parent unsubstituted sugars. CBH I enzyme from both organisms attacks these substrates in a random manner. Turnover numbers are, however, low and do not increase appreciably as a function of the degree of polymerization of the substrates. The active-site topology of the CBH I from T. reesei was further probed by equilibrium binding experiments with cellobiose, cellotriose, lactose and some of their derivatives. These point to a single interaction site (ABC), spatially restricted as deduced from the apparent independency of the thermodynamic parameters. It appears that the putative subsite A can accommodate a galactopyranosyl or glucopyranosyl group, and subsite B a glucopyranosyl group, whereas in subsite C either a glucopyranosyl or a chromophoric group can be bound, scission occurring between subsites B and C. The apparent kinetic parameters (turnover numbers) for the hydrolysis of cello-oligosaccharides (and their derivatives) by the CBH II type enzyme increase as a function of chain length, indicative of an extended binding site (A-F). Its architecture allows for specific binding of beta-(1----4)-glucopyranosyl groups in subsites A, B and C. Binding of a chromophore in subsite C produces a non-hydrolysable complex. The thermodynamic interaction parameters of some ligands common to both type of enzyme were compared: these substantiate the conclusions reached above.