Showing papers on "Trichoderma reesei published in 1986"

A highly active extracellular cellulase from the anaerobic rumen fungus Neocallimastix frontalis

Abstract: An extracellular cellulase which was highly active in solubilizing the highly hydrogen bond-ordered cellulose in cotton fibre was found in a culture filtrate of the anaerobic fungus, Neocallimastix frontalis, isolated from the rumen of a sheep. The cellulase was several-fold more active in solubilizing cotton fibre per unit of endo-1,4-β-glucanase than the cellulase of the aerobic fungus Trichoderma reesei mutant strain C-30, which is one of the most active cellulases isolated so far.

Kinetic, inhibition, and stability properties of a commercial β‐D‐glucosidase (cellobiase) preparation from aspergillus niger and its suitability in the hydrolysis of lignocellulose

Abstract: The rate and extent of cellulose conversion into glucose is dependent upon the amount of active ..beta..-glucosidase enzyme present in the cellulase preparation used for saccharification. This is because cellobiose produced during celluloysis is inhibitory to both exo- and endocellulases and, hence retards saccharification. The highly cellulytic fungus Trichoderma reesei and its mutant strains have been demonstrated to have potential for use in the practical saccharification of cellulosic materials. Although cellulase preparations derived from this fungus contain a very active cellulase complement of enzymes (i.e. both exo- and endo-cellulases) they are, nevertheless, deficient in ..beta..-glucosidase activity. Cellulase preparations of T. reesei containing higher levels of ..beta..-glucosidase activity can be obtained by supplementing with exogenous ..beta..-glucosidase preparations derived from another microorganism, which has been demonstrated to increase the rate and extent of saccharification of cellulose. Fungi belonging to the genus Aspergillus (e.g. A. niger and A. phoenicus) produce ..beta..-D-glucosidases in high yields which are compatible with T. reesei cellulases and promote saccharification of cellulose. Previous studies have shown that T. reesei cellulase preparations supplemented with Novozym 188 (Novo Industri A/S Denmark) were very effective in saccharifying pretreated sugarcane bagasse. The rather high cellulose conversions into glucose obtained in those studies promptedmore » an examination of the physicochemical and kinetic properties of the Novozym 188 ..beta..-glucosidase preparation which is described in this communication. Novo has provided some technical information on the pH and temperature properties of the enzyme. 15 references.« less

Specificity of cellulase and β-xylanase induction in Trichoderma reesei QM 9414

Abstract: Cellulose- and xylan-degrading enzymes of Trichoderma reesei QM 9414 induced by, sophorose, xylobiose, cellulose and xylan were analyzed by isoelectric focusing. The sophorose-induced enzyme system contained two types of endo-1,4-β-glucanases (EC 3.2.1.4), one specific for cellulose and the other non-specific, hydrolyzing both cellulose and xylan, and exo-1,4-β-glucanases (cellobiohydrolases I, EC 3.2.1.91), i.e. all types of glucanases that are produced during growth on cellulose. Specific endo-1,4-β-xylanases (EC 3.2.1.8) present in the cellulose-containing medium were less abundant in the sophorose-induced enzyme system. Xylobiose and xylan induced only specific endo-1,4-β-xylanases. It is concluded that syntheses of cellulases and β-xylanases in T. reesei QM 9414 are under separate control and that the non-specific endo-1,4-β-glucanases are constituents of the cellulose-degrading enzyme system.

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

Abstract: Cellulase production in Trichoderma reesei mutants was induced by l-sorbose, known to be an inhibitor of β-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 μg 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.

Enhanced cellulase production from Trichoderma reesei QM 9414 on physically treated wheat straw

Abstract: Trichoderma reesei QM 9414 was grown on wheat straw as the sole carbon source. The straw was pretreated by physical and chemical methods. The particle size of straw was less than 0.177 mm. Growth of T. reesei QM 9414 was maximal with alkali-pretreated straw whereas cellulase production was optimal when physically pretreated straw was used as substrate. Cellulase yields expressed as IU enzyme activity/g cellulose present in the cultures were considerably higher when alkali pretreatment of wheat straw was omitted. Cellulase yields of 666 IU/g cellulose for filter paper activity (FPA) are the highest described for cultures of T. reesei QM 9414 carried out in analogous conditions. Crystallinity index of the cellulose contained in wheat straw increased slightly after alkali pretreatment. This increase did not decrease cellulose accessibility to the fungus. Delignification of wheat straw was not necessary to achieve the best cellulase production.

Integrated process for the production of food, feed and fuel from biomass

Abstract: A feedstock containing a biomass such as lignocellulosic materials, e.g. forest biomass; agricultural residues; or manures, is pretreated and thereafter is fractionated into cellulose, lignin and hemicelluloses. New mutants are disclosed which include Chaetomium cellulolyticum IAF-101 (NRRL 18756), Aspergillus sp. IAF-201 (NRRL 18758), Penicillum sp. IAF-603 (NRRL 18759), and Trichoderma reesei QMY-1. With these new mutants and also known fungi including Pleurotus sajor-caju and other Pleurotus spp. unfractionated predetermined biomass is converted into feed. The same treatment can also be applied to hemicelluloses, and cellullose. Cellulose can also be hydrolyzed by means of a cellulase-system prepared from cellulose and Tricoderma reesei to prepare glucose which can be converted to alcohol with Saccharomyces cerevisiae, Kluyveromyces spp. and Zymomonas mobilis. The residual microbial biomass of these microorganisms from alcohol fermentation broth is also used as feed. The process is economical and non polluting.

Investigation of cellobiohydrolase from trichoderma reesei by small angle X-ray scattering

Abstract: Trichoderma reesei was grown on sulfite pulp and the major cellobiohydrolase of the culture filtrate was purified to homogeneity. The distance distribution function p(r) measured by the small angle X-ray scattering technique indicates that the enzyme molecule has a rather unusual tadpole like shape with an isotropic head and a long tail. The maximum length is 18 nm and the largest diameter is 4.4 nm.

A constitutive, plasma-membrane bound β-glucosidase in Trichoderma reesei

Abstract: Plasma membranes of Trichoderma reesei QM 9414, isolated from protoplasts by means of the concanavalin A procedure, contained β-glucosidase activity, which appeared constitutively upon growth on glucose. The enzyme had a pH optimum around 6, and was active on p-nitrophenyl-β-d-glucoside, cellobiose and sophorose (Km 0.7, 3.9 and 3.1 mM, respectively). Glucose was only weakly inhibitory (Ki 7 mM). Treatment of the plasma membranes with Triton X-100, Tween 80 or digitonin solubilized more than 60% of the membrane-bound β-glucosidase activity. The enzyme so solubilized exhibited an Mr of 70 000 ± 5000 and an isoelectric point at pH 8.2 ± 0.3.

Simultaneous saccharification/fermentation with Zymomonas mobilis.

Abstract: In recent years, an ethanol production process has been developed which utilizes Trichoderma reesei cellulase and Candida brassicae IFO 1664 in the simultaneous saccharification/fermentation (SSF) of cellulose to ethanol. The direct production of ethanol from cellulose in an SSF process alleviates the problem of end production inhibition. Glucose does not accumulate in this system, but rather is fermented to ethanol immediately following saccharification. The result is an increase in yield of 25% or greater as compared with separate processes of saccharification and fermentation. An alternative organisms which might be used in place of yeasts in ethanol production processes is Zymomonas mobilis. The optimum temperature for hydrolysis of cellulose by Trichoderma reesei cellulases is 50/sup 0/C. Since this hydrolysis is the rate limiting step in the SSF process, it is advantageous to utilize the most temperature tolerant ethanol producer available. Candida brassicae is currently the organism of choice due to its ability to produce ethanol efficiently at 40/sup 0/C. This investigation reports on the screening of Zymomonas strains and evaluating the feasibility of utilizing the most temperature tolerant strain in place of C. brassicae in SSF.

The hydrolysis of steamed birchwood hemicellulose by enzymes produced byTrichoderma reesei andAspergillus awamori

Abstract: The hemicellulase separated from birchwood by steaming and water extraction comprised mainly of acetyl- and 4-O-methyglucurono-substituted xylo-oligomers. The liberation of the acidic side groups affected the rate and yield of the enzymatic hydrolysis of the xylo-oligomers. The hemicellulase ofTrichoderma reesei was superior to that ofAspergillus awamori both with respect to side group cleavage and xylose yield in hydrolysis.

Method for purifying xylanase

Abstract: The separation of xylanases from mixtures thereof with other hemicellulases, particulary cellulase produced by the culturing of hemicellulolytic microorganisms, particularly the fungus Trichoderma harzianum E58 and Trichoderma reesei by ultrafiltration through an ultrafiltration membrane having a low molecular weight cut-off point in the range of about 1,000 to 20,000 daltons to obtain a cellulase rich retentate and xylanase rich ultrafiltrate. The dilute xylanase rich filtrate from the ultrafiltration is concentrated and purified by adsorption and elution from an insoluble matrix, e.g. a cationic exchange resin. The xylanase obtained is suitable for use in the hydrolysis of hemicellulose for which it is selective, particularly in the presence of cellulose and the cellulase rich retentate is suitable for the hydrolysis of cellulose.

Production of cellulase enzymes and hydrolysis of steam-exploded wood

Abstract: Steam-exploded aspen has been examined as a candidate feedstock for both cellulose production and enzymatic hydrolysis of wood. Batch and fed-batch cultivation methods were evaluated and compared with previous experiments using ball-milled, crystalline cellulose (Solka Floe). Batch cultivation of Trichoderma reesei Rut C-30 on 9 wt% water-washed aspen yielded enzyme productivities and activities comparable to those obtained on Solka Floe (40 FP IU/L-h; 7. 5 FP IU/mL). Fed-batch cultivation of Rut C-30 resulted in higher enzyme productivities and tilers than batch cultivation (50 FP IU/L-h; 15 FP IU/mL). However, the overall enzyme production performance was lower than on Solka Floe at comparable cellulose feeding rates and concentrations. This may be due to the accumulation of steam explosion by-products and lignin in the fermentor.The hydrolysis of water-washed steam-exploded aspen was performed at different enzyme loadings and wood concentrations. Glucose production, using 10 and 15 wt% suspension, showed that while glucose concentration increased with wood load, the yield of glucose derived from cellulose decreased. With 10 wt% suspensions, it was possible to obtain a cellulose conversion to glucose above 95%. Low cellulose levels in the hydrolyzates indicated that the filter paper activity ratios (approximately 1.5), a significant result since the fungus was grown exclusively on wood. mIt also suggested that the observed yield decrease is more likely to be caused by glucose than cellobiose inhibition of the enzymes.

Production and Characteristics of Avicel-Disintegrating Endoglucanase from a Protease-Negative Humicola grisea var. thermoidea Mutant.

Abstract: Mutational experiments were performed to decrease the protease productivity of Humicola grisea var. thermoidea YH-78 using UV light and N-methyl-N'-nitro-N-nitrosoguanidine. A protease-negative mutant, no. 140, exhibited higher endoglucanase activity than the parent strain in mold bran culture at 50 degrees C for 4 days. The culture extract rapidly disintegrated filter paper but produced a small amount of reducing sugar. About 30% of total endoglucanase activity in the extract was adsorbed onto Avicel. The electrophoretically homogeneous preparation of Avicel-adsorbable endoglucanase (molecular weight, 128,000) showed intensive filter-paper-disintegrating activity but did not release reducing sugar. The preparation also exhibited a highly synergistic effect with the cellulase preparation from Trichoderma reesei in the hydrolysis of microcrystalline cellulose. This endoglucanase was observed via scanning electron microscopy to disintegrate Avicel fibrils layer by layer from the surface, yielding thin sections with exposed chain ends. A mutant, no. 191, producing higher protease activity and an Avicel-unadsorbable, Avicel-nondisintegrating endoglucanase was isolated. The purified enzyme (molecular weight, 63,000) showed no disintegrating activity on filter paper and Avicel and a less synergistic effect with the T. reesei cellulase in hydrolyzing microcrystalline cellulose than did the former enzyme. Endoglucanase was therefore divided into two types, Avicel disintegrating and Avicel nondisintegrating.

Preparation and Application of Trichoderma reesei Mutants with Enhanced β-Glucosidase

Abstract: Mutants with enhanced β-glucosidase production were isolated by the plate method with 4-methylumbelliferyl-β-d-glucoside as a substrate of β-glucosidase in Trichoderma reesei. Among them, the mutant strain CDU-11 had the highest production of β-glucosidase. In 5-liter jar fermentor culture of CDU-11 with 6% Avicel as a carbon source, β-glucosidase production was significantly increased by keeping the pH at 4.0 during the first 3 days, then keeping the pH at 5.5 for 4 days. CMCase reached 320U/ml, Avicelase 29 U/ml, and β-glucosidase 21 U/ml. The ratio of β-glucosidase to CMCase in the CDU-11 culture was about three times that in KY 746, the parent strain. The saccharification of Avicel and alkali-treated bagasse by the cellulolytic system of CDU-11 became much more effective than with other strains.

Production of cellulases from alkali-treated bagasse in Trichoderma reesei

Abstract: Most of the mutants of Trichoderma reesei had good cellulase productivity on Avicel but this was low on alkali-treated bagasse, which could be a most promising cellulosic biomass to use as an inexpensive carbon source for cellulase production. Two T. reesei mutants, PC-3-7 and X-31, in which strong cellulase activity is inducible by l-sorbose, were, however, found to produce cellulase on alkali-treated bagasse. They produced about 100 units of CMCase per ml in 5-1 jar fermentor culture with 4% alkali-treated bagasse as carbon source. They also showed higher cellulase productivity than other mutants on other easily saccharified substrates, such as alkali-treated rice straw and Walseth's cellulose.

Comparison of different cellulolytic fungi for bioconversion of apple distillery waste

Abstract: The suitability of three ascomycetous fungi, Aspergillus niger, A. awamori and Trichoderma reesei, as well as two basidiomycetes, Pleurotus ostreatus and Phanerochaete chrysosporium, for bioconversion of apple distillery slop was compared. Trichoderma and Phanerochaete degraded raw fiberes by 20%, producing filter cakes with 17% to 22% raw protein contents. Aspergillus spp. were superior in filtration time and COD reduction and were of the same efficiency in protein synthesis as Trichoderma and Phanerochaete, but did not degrade fibres. Pleurotus ostreatus did not degrade lignin under fermentation conditions used and could not compete with other fungi due to its slower growth.

Localisation of β‐glucosidase in Trichoderma reesei cell walls with immunoelectron microscopy

Abstract: Using ferritin-conjugated antibodies as an electron microscopic marker, β-glucosidase was localized within the cell walls of the imperfect fungus Trichoderma reesei QM9414 With different states of cell wall degradation obtained with a cell wall-lysing culture filtrate of Micromonospora chalcea, β-glucosidase was mainly detected within the outer, fibrous exopolysaccharide layer and the outer face of the plasma membrane

Effect of l(–)Sorbose on the Release of β-Glucosidase by Trichoderma reesei QM9414

Abstract: Summary: l(–)Sorbose, a sugar known to cause paramorphogenesis in fungi, was tested for its effect on morphology and release of cell-wall bound β-glucosidase (EC 3.2.1.21) in the cellulolytic fungus Trichoderma reesei QM9414. Sorbose caused an increase in branching and septation in the growing mycelium. Extracellular β-glucosidase activity was enhanced when cellobiose or cellulose growth medium was supplemented with sorbose. In sorbose-supplemented cultures, the β-glucosidase activity associated with the wall fraction was less than half that in unsupplemented cultures. The intracellular activity was also lower in the sorbose-supplemented cultures than in unsupplemented controls. The glucosamine/glucose ratio of wall hydrolysates from sorbose-supplemented cultures was about twice that of control hydrolysates. Since β-glucosidase is closely associated with 1,3-β-glucan in the walls of T. reesei, a decrease in wall glucan content, and the resulting weakened association of the enzyme with the walls, is probably responsible for its increased release into the culture medium in the presence of sorbose.

Influence of L-Sorbose on Growth and Enzyme Synthesis of Trichoderma reesei C-5

Abstract: SUMMARY: The effects of L-sorbose on the growth and biosynthesis of cellulases and other polysaccharide-degrading enzymes of Trichoderma reesei C-5 were studied. The specific growth rate and yield of this strain in batch culture were reduced by 23% and 46% respectively on addition of 1% (w/v) sorbose to Vogel's medium containing 1% (w/v) glucose. The specific consumption rate of both sorbose and glucose decreased in the presence of the other sugar at 1% (w/v) concentration. The addition of sorbose (1-5%) to cultures grown in 1% glucose resulted in enhanced activities of all cellulase enzymes, and particularly endoglucanase activity, which increased sevenfold in the presence of 5% sorbose. There was no significant effect on the activities of β-glucosidase, acid phosphatase and amylase. While the increased enzyme activities seemed to be correlated with a decreased rate of glucose consumption, a direct effect on some extracellular enzymes could not be ruled out.

Fed-batch production of cellulases using Trichoderma reesei Rutgers C-30

Abstract: In the past ten years there has been considerable attention devoted to improving the microbial production of cellulases for their potential in saccharification of cellulosic materials. The Rut. C-30 strain of the fungus Trichoderma reesei has yielded some of the best enzyme activities and productivities reported to date. The use of fed-batch techniques has also been shown to improve enzyme productivities while maintaining or increasing enzyme activities and reducing problems associated with aeration, mixing and foaming. In this work on fed-batch production of cellulases using Rut. C-30 the effects of both the size of the substrate injection and the time between injections are examined. The results indicate an appropriate feeding policy and provide insight into the mechanism for cellulase production. Une grande attention a ete portee ces dix dernieres annees a l'amelioration de la production microbienne des cellulases en raison de leur potentiel dans la sacchariflcation des substances cellulosiques. La souche Rut. C-30 du champignon Trichoderma reesei a donne quelques-unes des meilleures activites et productivites enzymatiques obtenues jusqu'a ce jour. On a montre egalement que l'utilisation des techniques d'alimentation discontinue permet d'ameliorer les productivites enzymatiques tout en maintenant ou en augmentant les activites des enzymes et de reduire les problemes lies a l'aeration, a l'agitation et au moussage. Ce travail qui porte sur la production avec alimentation discontinue de cellulases a partir de Rut. C-30, a permis d'etudier l'effet de la taille de l'injection de substrats ainsi que l'effet de l'intervalle de temps entre les injections. Les resultats permettent d'etablir une procedure d'alimentation appropriee et de mieux comprendre le mecanisme de la production de cellulase.

The effect of different cellulosic growth substrates and pH on the production of cellulolytic enzymes by Trichoderma reesei

Abstract: The effect of different cellulosic growth substrates on the production of cellulolytic enzymes by Trichoderma reesei was investigated. It was observed that growth on Avicel, Solka Floc and wheat straw produced different pH/time profiles in cultures. Over a range of controlled pH it was demonstrated that the production of cellulolytic and xylanolytic activity by T. reesei is dependent on culture pH and the type of growth substrate. The effect of pH on enzyme production varies with the nature of the growth substrate. Furthermore, it was shown that the optimum culture pH and growth substrate for the production of enzyme preparations for the extensive saccharification of cellulosic materials depends on the type of material to be saccharified.

Substrate conditions that influence the assays used for determining the β‐glucosidase activity of cellulolytic microorganisms

Abstract: Culture filtrates from Trichoderma harzianum E58, T. reesei CL 847 and Penicillium sp. C 462 were assayed for beta-glucosidase activity using a range of substrates and sugar analysis methods. Although sugar analyses by the dinitrosalicylic acid (DNS) and Nelson-Somogyi methods gave a similar profile, when increasing concentrations of salicin were assayed, considerably higher values were obtained with the DNS assay. The salicin concentration used for the assay greatly influenced the final beta-glucosidase values with higher values obtained for T. harzianum E58 and T. reesei CL 847 at substrate concentrations of 1 mg/mL while optimum values for Penicillium sp. C 462 were obtained at substrate concentrations greater than 3 mg/mL. Low concentrations of salicin and p-nitro-phenyl-beta-D-glucopyranoside (PNPG) gave the same response as cellobiose. Cellobiose should be used at concentrations greater than 3.74 mg/mL to avoid substrate limitation of the beta-glucosidase assay.