Electrophoretic karyotype ofTrichoderma reesei

Summary Pulsed field electrophoresis was used to separate the intact chromosomes of the fungi imperfectiTrichoderma reesei for which no previous karyotype has been available.T. reesei was shown to have six chromosomes with molecular weights ranging from 3.3 to 11.9 megabasepairs (Mbp). The genome size was estimated to be 38 Mbp, indicating thatTrichoderma has a genome larger thanAspergillus nidulans (31 Mbp) but smaller thanNeurospora crassa (47 Mbp).     

Product inhibition ofTrichoderma reesei andCellulomonas glycosidase activities

Summary A comparison has been made of the effects of glucose, xylose and arabinose on the ability ofTrichoderma reesei andCellulomonas preparations to hydrolyse the glycosides of these three sugars. The stronger resistance of theCellulomonas preparations to product inhibition meant that these were comparable to theT.reesei preparations in the presence of 500mM sugar product.     

Adsorption ofTrichoderma reesei cellulases on protein-extracted lucerne fibers

Summary Protein-extracted lucerne fibers (PELF) had a higher adsorptive capacity forTrichoderma reesei cellulases than a variety of other cellulosic substrates compared on an equal carbohydrate basis. Adsorption at room temperature reached a maximum at about 5 min; desorption was directly proportional to the extent of carbohydrate solubilization. Cellulase binding conformed to a Langmuir isotherm; the maximum cellulasebinding capacity of PELF was 111 filter paper units per g dry weight. About 85% of the cellulase was recovered in the soluble fraction after PELF hydrolysis. Soluble carbohydrates in the hydrolysate inhibited cellulase adsorption to fresh substrate (50% inhibition at a hydrolysate concentration of 7% glucose equivalents). The effect of these carbohydrates on cellulase adsorption was a complex one composed of both enhancing and inhibitory influences. Artificial hydrolysates (known sugars in proportions identical to actual hydrolysates) inhibited adsorption, but glucose, cellobiose and xylose resulted in adsorption enhancement. Acid treatment of the hydrolysate to convert oligosaccharides to monomers increased reducing sugar concentrations and eliminated its capacity for adsorption inhibition.     

Isolation and characterization of a new antifungal metabolite ofTrichoderma reesei

A mycelial extract ofTrichoderma reesei (P-12) was separated into four fractions by high performance liquid chromatography (HPLC). Out of these, two were found to exhibit antifungal activity when tested against plant pathogenic fungi. The level of antifungal compounds was higher in media containing glucose as the carbon source as compared to one containing cellulose. The synthesis of these antifungal compounds started after 3 days of inoculation at 30°C and continued upto 8 days. No further increase was recorded beyond this period.     

Sequential release of cellulose enzymes during germination ofTrichoderma reesei spores

The pattern of release of extracellular cellulase during the germination ofTrichoderma reesei spores has been examined. The four enzymes namely, filter paper degrading enzyme, Β-1,4 endoglucanase, Β-glucosidase and xylanase appear sequentially in the culture broth during germination of the spores. The order of enzyme appearance is not altered by the type of carbon source in the germination medium. Measureable quantities of filter paper degrading enzyme is detected only after the outgrowth has occurred. A possible mechanism of spore germination and induction of the enzymes by insoluble cellulose is suggested. An erratum to this article is available at .     

Reactions of reversion occuring with theTrichoderma reesei CL-847 cellulase system

Summary Poplar wood chips were pretreated by steam explosion and the cellulosic residue was hydrolysed with the cellulase complex ofTrichoderma reesei CL 847. The hydrolysate contains the (1–6) disaccharide gentiobiose. Incubation of cellobiose with the same cellulase system led to the synthesis of Glc-Glc dimers which were characterized and quantified as gentiobiose, cellobiose, laminarabiose, sophorose and trehalose, whose origin was clarified by using carbon-13 glucose selectively labelled on C1. We propose a mechanism to explain these reversion reactions.     

Preparation of mutants ofTrichoderma viride with increased production of cellulase

Four mutant strains exhibiting increased production of cullulases were prepared by UV irradiation of conidia ofTrichoderma viride QM 9414. Selected mutants were tested for production of cellulases in submerged cultivations in shake flasks and in a 30-L fermentor in a synthetic medium containing 1 % microcrystaline cellulose as the carbon source. Some mutants showed considerable morphological differences when compared to the parent strain, the most noticeable being a higher degree of branching of the mutant hyphae. The branched mutants produced 2 to 3 times higher levels of β-glucosidase than the parent strain QM 9414.     

Shear inactivation of cellulase of Trichoderma reesei

Inactivation of the cellulase of Trichoderma reesei (EC 3.2.1.4) by shear, is of sufficient magnitude to merit consideration in the design of equipment for the enzymatic hydrolysis of cellulose. The inac inactivation constant, k_d, is a function of the flow rate of the enzyme solution through a fine capillary tube. k_d increased slowly at low shear stress, and much more rapidly when the shear stress was greater than 15 dynes cm^?2.     

Different temperature profiles of enzyme secretion by two common strains ofTrichoderma reesei

Summary We have investigated the effects of high and low temperature on the synthesis and secretion of cellulases and other enzymes by two common and readily available strains ofTrichoderma reesei. While some effects were similar in both strains QM9414 and RUT-C30 (a reduction in cellulase production but stimulation of xylanase production at high temperature, and alterations in expression of the cellulase complex at low temperature), some specific differences between the strains were determined, most significantly an enhanced specific secretion rate (secretion/growth) at low growth temperature for QM9414.     

Transglycosylation of cellobiose by partially purified Trichoderma viride cellulase.

A commercial cellulase from Trichoderma viride was fractionated into three fractions, F1, F2, and F3, in order to investigate transglycosylation activities. Among these fractions, F3, which demonstrated highly hydrolytic activity toward p-nitrophenyl beta-D-glucopyranoside and Avicel, most effectively catalyzed the transglycosylation of cellobiose and converted cellobiose into beta-Glc-(1-->6)-beta-glc-(1-->4)-Glc and beta-Glc-(1-->6)-beta-Glc-(1-->6)-beta-Glc(1-->4)-Glc. The F3 fraction contained the enzyme to catalyze beta-glucosyl transfer toward only the C-6 position of the sugar acceptor, and thus it is expected to be of use for syntheses of functional oligosaccharides.     

Formation of cellulases by co-culturing ofTrichoderma reesei andAspergillus niger on cellulosic waste

A cellulase system possessing high hydrolytic and -glucosidase activity was obtained by co-culturingTrichoderma reesei andAspergillus niger by a new approach using semi-solid fermentation of lignocellulosic materials. Various types of pretreatments were used for making the cellulose easily accessible to enzymatic attack. The optimal water content for maximum activity of the mixed fermentation was investigated. A more concentrated enzyme preparation could be obtained by semi-solid state fermentation than by conventional submerged fermentation.     

Enzymatic studies on a cellulase system of Trichoderma viride. III. Transglycosylation properties of two cellulase components of random type.

Two highly purified cellulases [EC 3.2.1.4], II-A, and II-B, were obtained from the cellulase system of Trichoderma viride. Both cellulases split cellopentaose retaining the beta-configuration of the anomeric carbon atoms in the hydrolysis products at both pH 3.5 and 5.0. The Km values of cellulases II-A and II-B for cellotetraose were different, but their Vmax values were similar and those for cellooligosaccharides increased in parallel with chain length. Both cellulases produced predominantly cellobiose and glucose from various cellulosic substrates as well as from higher cellooligosaccharides. Cellulase II-A preferentially attacked the holoside linkage of rho-nitrophenyl beta-D-cellobioside, whereas cellulase II-B attacked mainly the aglycone linkage of this cellobioside. Both cellulases were found to catalyze the synthesis of cellotriose from rho-nitrophenyl beta-D-cellobioside by transfer of a glucosyl residue, possibly to cellobiose produced in the reaction mixture. They were also found to catalyze the rapid synthesis of cellotetraose from cellobiose, with accompanying formation of cellotriose and glucose, which seemed to be produced by secondary random hydrolysis of the cellotetraose produced. The capacity to synthesize cellotetraose from cellobiose appeared to be greater with cellulase II-B than with cellulase II-A.     

Sorbose mediated enhancement of cellulase biosynthesis inTrichoderma reesei

Addition of L-sorbose, a non-metabolizable non-inducing ketohexose, toTrichoderma reesei cultures growing on cellobiose or Avicel-cellulose lead to increased cellulase activities. Addition of sorbose resulted in a 6-fold increase in cellodextrins (cellotriose, cellotetraose, cellopentaose) concentration on day 3 in cellobiose cultures and 1.3-fold increase in cellodextrins concentrations on day 4 in Avicel cellulose cultures. This increase in intracellular cellodextrins concentration matched closely with the increase in endoglucanase activity at these time points. Treatment of the cell-free extracts with cellulase preparation led to disappearance of the cellodextrins and increase of glucose. These observations suggested a more direct involvement of cellodextrins in cellulase induction process. The cellulases produced in sorbose-supplemented cellobiose medium hydrolyzed microcrystalline cellulose as effectively as the ones produced on Avicel cellulose medium.     

里氏木霉纤维素酶的分离纯化及酶学性质

通过(NH4)2SO4分级沉淀、HiPrep 26/10 Desalting凝胶色谱脱盐、Source 15 Q阴离子交换色谱技术,里氏木霉(Rut C-30)纤维素酶主要组分得以初步分开,再经过Source 15 S阳离子交换色谱、HiPrep Sephacryl S-100 HR凝胶过滤色谱、Superdex 75 PrepGrade凝胶过滤色谱进一步分离纯化,得到2个纯化的内切葡聚糖酶组分EGⅡ、EGⅠ和一个外切葡聚糖酶组分CBHⅠ;经过SDS-PAGE电泳鉴定为电泳纯,测得相对分子质量分别为5.22×104,5.62×104和6.90×104。EGⅡ的最适反应pH是5.6,最适反应温度为65℃;EGⅠ的最适反应pH是4.4,最适反应温度为55℃;以羧甲基纤维素(CMC)为底物时,EGⅠ、EGⅡ的米氏常数(Km)分别为2.20 mg/mL、3.38 mg/mL。CBHⅠ的最适反应pH是5.8,最适反应温度为60℃,以对硝基苯基-β-D-纤维二糖苷(PNPC)为底物时,米氏常数(Km)为0.12 mg/mL。     

Production of cellulase by Trichoderma reesei from dairy manure

Cellulase production by the fungi Trichoderma reesei was studied using dairy manure as a substrate. Data showed that T. reesei RUT-C30 had higher cellulase production than T. reesei QM 9414 and that a homogenized manure, treated by a blender to reduce fiber size, led to higher cellulase production. The cellulase production was further optimized by growing T. reesei RUT-C30 on homogenized manure. The effects of manure concentration, pH, and temperature on cellulase production were investigated with optimal parameter values determined to be 10 g/l manure (dry basis), 25.5 degrees C, and pH 5.7, respectively. Elimination of CaCl2, MgSO4, nitrogen sources (NH4+ and urea) and trace elements (Fe2+, Zn2+, Co2+ and Mn2+) from the original salt solution had no negative influence on the cellulase production, while phosphate elimination did reduce cellulase production. Based on above results, the final medium composition was simplified with manure additives being KH2PO4, tween-80 and CoCl2 only. Using this medium composition and a reaction time of 6-8 days, a maximum cellulase production activity of 1.74 IU/ml of filter paper activity, 12.22 IU/ml of CMCase activity, and 0.0978 IU/ml of beta-glucosidase was obtained. This filter paper activity is the highest ever reported in cellulase production from agricultural wastes.     

Adsorption of Trichoderma reesei CBHI cellulase on silanized silica

Adsorption kinetics and surfactant-mediated elution of Trichoderma reesei CBHI cellulase were recorded in situ, at hydrophobic, silanized silica. Experiments were performed at different solution concentrations, ranging from 0.001 to 0.98 mg/mL. Adsorbed enzyme was partially elutable upon rinsing, with the amount of adsorbed mass remaining being highest at intermediate concentrations. In addition, the resistance to elution with buffer was generally lower at higher concentrations, and the resistance to elution with surfactant was generally lower at intermediate concentrations. These observations are tentatively explained with reference to a mechanism allowing for adsorption of associated monomers of CBHI as well as free monomers.     

Semicontinuous cellulase production in an aqueous two-phase system with Trichoderma reesei rutgers C30

Cellulases [see 1,4(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] from Trichoderma reesei, Rutgers C30, can be semicontinuously produced in an aqueous two-phase system composed of dextran and poly(ethylene glycol) using Solka Floc BW 200 as substrate. When substrate was intermittently added along with fresh top phase, which replaced the withdrawn top phase containing the produced enzymes, a yield of 1740 U endo-β-d-glucanase/g cellulose and 59.3 FPU/g cellulose was extracted with the top phase. Without fresh substrate added, a yield of 3920 U endo-β-d-glucanase/g cellulose and 127.7 FPU/g cellulose was extracted after five runs.     

Secretome characteristics of pelletized Trichoderma reesei and cellulase production

Trichoderma reesei is an important cellulase producer and its secondary mycelial phase is responsible for cellulase expression and secretion in submerged fermentation. Little is known regarding the effects of fungal morphology on cellulase production by Trichoderma sp. In this study we aimed to extend the understanding of cellulase production by T. reesei, especially correlating cellulase productivity with pellet morphology and with its secretome characteristics. We found that T. reesei was more likely to form pellets in malt extract broth than in potato dextrose broth. CaCO(3) helped in formation of fine pellets in malt extract broth. 10(9) spores/ml resulted in formation of pellets with the size of 0.13 ± 0.047 mm. LC/MS spectrometry analysis indicated that the secretomes from pellet was different from that of mycelial mat under the same fermentation conditions. Optimization tests showed that lactose, xylose and Pluronic F68 are important for efficient production of cellulases with FPU activity in the pellets fermentation. This is the first report on the artificial formation of pellets by Trichoderma sp. as well as correlation between physiological characteristic of the pellets and cellulase production by T. reesei. The findings from this study can be used for improvement of cellulase productivity.     

Optimization of cellulase production in batch fermentation byTrichoderma reesei

Maximum cellulase production was sought by comparing the activities of the cellulases produced by differentTrichoderma reesei strains andAspergillus niger. Trichoderma reesei Rut-C30 showed higher cellulase activity than otherTrichoderma reesei strains andAspergillus niger that was isolated from soil. By optimizing the cultivation condition during shake flask culture, higher cellulase production could be achieved. The FP (filter paper) activity of 3.7 U/ml and CMCase (Carboxymethylcellulase) activity of 60 U/ml were obtained from shake flask culture. When it was grown in 2.5L fermentor, where pH and DO levels are controlled, the Enzyme activities were 133.35 U/ml (CMCase) and 11.67 U./ml (FP), respectively. Ammonium sulfate precipitation method was used to recover enzymes from fermentation broth. The dried cellulase powder showed 3074.9 U/g of CMCase activity and 166.7 U/g of FP activity with 83.5% CMCase recovery.     

Inhibition of Trichoderma reesei cellulase by sugars and solvents

Inhibition of Trichoderma reesei cellulase by sugars (glucose, delta-gluconolactone, and cellobiose) and solvents (ethanol, butanol, and acetone) was studied using cellulose azure. Glucose, cellobiose, ethanol, and butanol were noncompetitive inhibitors, delta-gluconolactone was a mixed inhibitor, and acetone was a noncompetitive activator. Converting cellobiose to glucose reduces the effective inhibitor binding constant by 6 times and converting cellobiose to ethanol reduces it by 16 times.