Filter paper degrading ability of a Trichoderma strain with multinucleate conidia

The multinucleate conidia were produced from the green mature conidia of Trichoderma reesei Rut C-30 strain by colchicine treatment. The strain with higher Filter paper degrading ability was selected among those conidia using a double layer selection medium. The selected strain, JS-2 was able to collapse the filter paper within 15 min but the original strain took 25 min to collapse it completely. Moreover, the amount of reducing sugar in the L-type glass tube of the strain, JS-2 was greater than that of the original strain. The Avicel, CMC-Na, and Salicin hydrolyzing activity of the strain, JS-2, increased 2.1 times, 1.2 times, and 3.6 times higher than that of the original strain.     

Cellulase production by Trichoderma reesei using sawdust hydrolysate

Sawdust hydrolysates were investigated for their ability to support cell growth and cellulase production, and for potential inhibition of Trichoderma reesei Rut C30. Simultaneous fermentations were conducted to compare the hydrolysate-based media with the controls having equivalent concentrations of glucose and Avicel cellulose. Six hydrolysates differing in the boiling durations in the hydrolysis procedure were evaluated. The hydrolysates were found to support cell growth and induce active cellulase synthesis. The maximum specific cellulase production rate was 0.046 filter paper units (FPU)/(g of cells · h) in the hydrolysate-based systems, much higher than that (0.017 FPU/[g of cells · h]) in the controls.     

Rapid selection system of strains with higher avicel degrading ability in a cellulolytic fungus,Trichoderma

We have attempted to develop an active selection system for strains that have a higher potential for Avicel degradation using haploidized conidia from colchicine-treated Trichoderma reesei Rut C-30 as a model strain. Avicel, absorbent cotton, and wood powder were used as substrates for selection. It appeared that the strains that degrade Avicel actively could be effectively selected when the solid medium containing the selection substrate and the liquid medium containing Avicel were used.     

Polykaryon formation using a swollen conidium of Trichoderma reesei

The cellulolytic fungus, Trichoderma has oval and mononucleate conidia. When these conidia are incubated in a liquid medium, they begin to swell and their shape becomes spherical followed by an increase in inner space. In such swollen conidia, it is possible to produce a larger autopolyploid nucleus using a mitotic arrester compared with the case of the original conidia. In this study, polykaryon formation was attempted using these swollen conidia. Dried mature green conidia of Trichoderma reesei QM6a (IFO 31326) were incubated in Mandel's medium in order to swell. The swollen conidia were treated with a mitotic arrester, colchicine, for autopolyploidization. After autopolyploidization, polykary on formation was carried out using the swollen conidia. After the treatment, multiple smaller nuclei whose diameter was almost the same as that of the original strain were generated from an autopolyploid nucleus in a swollen conidium. A cellulase hyperproducer without decrease in growth rate could be selected using such swollen conidia.     

Production of cellulase/β-glucosidase by the mixed fungi culture of Trichoderma reesei and Aspergillus phoenicis on dairy manure

A cellulase production process was developed by growing the fungi Trichoderma reesei and Aspergillus phoenicis on dairy manure. T. reesei produced a high total cellulase titer (1.7 filter paper units [FPU]/mL, filter paper activity) in medium containing 10 g/L of manure (dry basis [w/w]), 2 g/L KH₂PO₄, 2 mL/L of Tween-80, and 2mg/L of CoCl₂. However, β-glucosidase activity in the T. reesei-enzyme system was very low. T. reesei was then cocultured with A. phoenicis to enhance the β-glucosidase level. The mixed culture resulted in a relatively high level of total cellulase (1.54 FPU/mL) and β-glucosidase (0.64 IU/mL). The ratio of β-glucosidase activity to filter paper activity was 0.41, suitable for hydrolyzing manure cellulose. The crude enzyme broth from the mixed culture was used for hydrolyzing the manure cellulose, and the produced glucose was significantly (p<0.01) higher than levels obtained by using the commercial enzyme or the enzyme broth of the pure culture T. reesei.     

Successive construction of cellulase hyperproducers of Trichoderma using hyperpolyploids

When the swollen conidia of Trichoderma reesei QM 6a are treated with 0.1% (w/v) colchicine solution, huge autopolyploid nuclei can be formed in those swollen conidia. When a mycelial mat derived from such a conidum is treated with a haploidizing reagent, benomyl, many fan-shaped sectors are produced from the colony, and cellulase hyperproducers are selected from conidia on the colony. When colchicine and benomyl treatments are repeated on cellulase hyperproducers, new hyperproducers can be constructed successively and systematically. Moreover, when conidia derived from autopolyploids are treated with ethylmethanesulfonate solution, another type of cellulase hyperproducers (polyploids) can be obtained.     

Endocellulase and exocellulase activities of two Streptomyces strains isolated from a forest soil

Two Streptomyces strains, M7a and M23, from a Brazilian forest soil were evaluated for the cellulase production of their superna tants after growth in a microcrystalline cellulose medium, using carboxy methylcellulose and filter paper as substrates at different temperatures and pH values. Endoglucanase and exoglucanase activities were compared to a commercial Trichoderma reesei cellulase using fluorogenic conjugated substrates Similar specific activities were observed for the enzyme preparations of strain M23 and T. reesei. For M7a the activities were about seven times higher than those obtained for T. reesei. Extracellular or cell-associated cellobiase activities were not detected in both strains.

     

Examining the Potential of Plasma-Assisted Pretreated Wheat Straw for Enzyme Production by <Emphasis Type="Italic">Trichoderma reesei</Emphasis>

Plasma-assisted pretreated wheat straw was investigated for cellulase and xylanase production by Trichoderma reesei fermentation. Fermentations were conducted with media containing washed and unwashed plasma-assisted pretreated wheat straw as carbon source which was sterilized by autoclavation. To account for any effects of autoclavation, a comparison was made with unsterilized media containing antibiotics. It was found that unsterilized washed plasma-assisted pretreated wheat straw (which contained antibiotics) was best suited for the production of xylanases (110 IU ml⁻¹) and cellulases (0.5 filter paper units (FPU) ml⁻¹). Addition of Avicel boosted enzyme titers with the highest cellulase titers (1.5 FPU ml⁻¹) found with addition of 50 % w/w Avicel and with the highest xylanase production (350 IU ml⁻¹) reached in the presence of 10 % w/w Avicel. Comparison with enzyme titers from other nonrefined feedstocks suggests that plasma pretreated wheat straw is a promising and suitable substrate for cellulase and hemicellulase production.     

Fingerprinting Trichoderma reesei hydrolases in a commercial cellulase preparation

Polysaccharide degrading enzymes from commercial T. reesei broth have been subjected to “fingerprint” analysis by high-resolution 2-D gelelectrophoresis. Forty-five spots from 11×25 cm Pharmacia gels have been analyzed by LC-MS/MS and the resulting peptide sequences were compared toexisting databases. Understanding the roles and relationships of component enzymes from the T. reesei cellulase system acting on complex substrates is key to the development of efficient artificial cellulase systems for the conversion of lignocellulosic biomass to sugars. These studies suggest follow-on work comparing induced and noninduced T. reesei cells at the proteome level, which may elucidate substrate-specific gene regulation and response.     

Synergism in binary mixtures of Thermobifida fusca cellulases Cel6B,Cel9A,and Cel5A on BMCC and Avicel

In an earlier binding study conducted in our laboratory using Thermobifida fusca cellulases Cel6B, Cel9A, and Cel5A (formally Thermomonospora fusca E₃, E₄, and E₅), it was observed that binding capacities for these three cellulases were 18–30 times higher on BMCC than on Avicel. These results stimulated an interest in how the difference in accessibility between the two cellulosic substrates would affect synergism observed with cellulase mixtures. To explore the impact of substrate, accessibility on the extent of conversion and synergism, three binary T. fusca cellulase mixtures were tested over a range of cellulase ratios and total molar cellulase concentrations on Avicel and BMCC. Higher extents of conversion were observed for BMCC due to the higher enzyme to substrate ratio resulting from the higher binding The processive endoglucanase, Cel9A, had four times the extent of conversion of the end endocellulase Cel5A, while the exocellulase Cel6B had three times the extent of conversion of Cel5A. Approximately 500 nmol/g of the cel9A+Cel6B mixture was needed to obtain 80% conversion, while the Cel6B+Cel5A and Cel9A+Cel5A mixtures required 1500 and 1250 nmol/g, respectively, to obtain 80% conversion. Thus, it appears that the more accessible structure of BMCC, as reflected by its binding capacity, results in relative higher processive activity.     

A Thermophilic Cellulase Complex from <Emphasis Type="Italic">Phialophora</Emphasis> sp. G5 Showing High Capacity in Cellulose Hydrolysis

A cellulase-producing mesophilic fungal strain, named G5, was isolated from the acidic wastewater and mud of a tin mine and identified as Phialophora sp. based on the internal transcribed spacer sequence. The volumetric activities and specific activities of cellulase induced by different carbon sources (Avicel, corn cob, wheat bran and corn stover) were compared. The cellulase complex of Phialophora sp. G5 exhibited the optimal activities at 60–65 °C and pH 4.0–5.0, and had good long-term thermostability at 50 °C. Compared with the commercial cellulase (Accellerase 1500, Genencor), the enzyme under study showed 60% and 80% of the capacity to hydrolyze pure cellulose and natural cellulose, respectively. This is the first study to report that a cellulytic enzymes complex from Phialophora genus, and the superior properties of this enzyme complex make strain G5 a potential microbial source to produce cellulase for industrial applications, and the production ability could be improved by mutagenesis.     

Active nuclear shuffling system using a swollen conidium of Trichoderma reesei

Cellulase hyperproducers of Trichoderma reesei can be constructed using autopolyploidization and haploidization techniques. To increase the efficiency of this method, the active nuclear shuffling system in a swollen conidium was effective. A dried mature green conidium of a model strain, T. reesei QM6a (IFO 31326), was swollen to make room for a larger autopolyploid nucleus. After colchicine treatment, a larger autopolyploid nucleus was produced in such a swollen conidium. Benomyl treatment of swollen conidia generated multiple smaller nuclei from one larger autopolyploid nucleus. Those smaller nuclei were transported through conidia to mycelia after germination. This system could contribute to increasing the efficiency of genetic shuffling.     

Expression and high-level secretion of Trichoderma reesei endoglucanase I in Yarrowia lipolytica

The endoglucanase I (EGI) from fungus Trichoderma reesei was cloned, expressed, and secreted from Yarrowia lipolytica using the XPR2 promoter. The signal sequence of EGI transferred from T. reesei was efficiently processed in the Y. lipolytica secretory pathway and directed the secretion of active EGI into the culture medium. However, the recombinant EGI produced from YLCSIn strain was hyperglycosylated and significantly larger than the native enzyme produced by the parent strain. The expression of EGI using XPR2 preproregion has caused secretion of modified proteins that still retained cellulase activity. This resulted from imprecise processing of the N-terminus of recombinant protein. While the batch culture produced 5 mg EGI/L from YLCSIn strain, the EGI yield was increased approx 20-fold when the fed-batch fermentation process strategy in combination with the high-cell density cultivation technique was employed. These results showed that the Y. lipolytica is a useful host organism for production of a large amount of large size heterologous proteins, especially when used in combination with high-cell density and fed-batch culture techniques.     

The effect of additional autopolyploidization in a slow growing cellulase hyperproducer of Trichoderma

M14-2 is a cellulase hyperproducer derived from Trichderma recesei QM 6a, but with a growth rate lower than that of the original strain. When M14-2 was autopolyploidized followed by haploidization and selection, the strain with both a higher cellulase productivity per mycelia and a higher growth rate could be obtained as M14-2B. This strain seemed to be constructed using gene sources amplified by additional autopolyploidization.     

Enzyme production, growth, and adaptation of T. reesei strains QM9414, L-27, RL-P37, and rut C-30 to conditioned yellow poplar sawdust hydrolysate

National Renewable Energy Laboratory (NREL) has developed a conditioning process that decreases acetic acid levels in pretreated yellow poplar hydrolysate. Trichoderma reesei is sensitive to acetic acid and this conditioning method has enabled applied cellulase production with hardwoods. T. reesei strains QM9414, L-27, RL-P37, and Rut C-30 were screened for growth on conditioned hydrolysate liquor. Tolerance to hydrolysate was found to be strain-dependent. Strain QM9414 was adapted to grow in 80% (v/v) conditioned hydrolysate (40 g/L of soluble sugars and 1.6 g/L acetic acid from pretreated poplar). However, enzyme production was highest at 20% (v/v) hydrolysateusing strain L-27. Cellulasetiters of 2–3 International Filter Paper Units (IFPU)/mL were achieved using pretreated yellow poplar liquors and solids as the sole carbon sources.     

Reducing end-specific fluorescence labeled celluloses for cellulase mode of action

Cellobiohydrolases (CBH-s) attack primarily from the cellulose chain ends. It is known that CBH-s can differ in their chain end preference. Here the cellulose reducing end-specific fluorescence labeling was studied with the aim to find the most suitable labeled cellulose for easy determination of CBH-s chain end preference. Anthranilic acid (AA) was used as fluorescence label. Bacterial cellulose, amorphous Avicel and filter paper were subjected to the labeling. Suitability of modified bicincoic acid method for determination of reducing groups on cellulose was also confirmed. AA labeled celluloses were hydrolysed with family 7 and 6 CBH-s from Trichoderma reesei and Phanerochaete chrysosporium. Hydrolysis data were plotted as released label (%) versus total degradation (%) (progress curves). Most characteristic progress curves were obtained with AA labeled regenerated amorphous Avicel where the family 6 CBH-s produced clearly upward curved progress curves confirming their preference for non-reducing ends.     

Improvement of cellulase activity inTrichoderma

The conditions for the production and regeneration of protoplasts fromTrichoderma reesei QM 9414 were optimized. Mutants obtained fromTrichoderma reesei QM 9414 were crossed, using protoplast fusion. In the crosses attempted, the diploids showed enhanced yields of exoglucanase, whereas the yield of endoglucanase and β-glucosidase was intermediate as compared to their respective parents. In some of the segregants, the yields were further enhanced, indicating the use of protoplast fusion techniques in developing breeding strategies for strain improvement. The study of cellulase mutants as such and the segregants revealed that the three cellulase components appear to be regulated independently of each other.

     

Dynamics of cellulase production by glucose grown cultures of Trichoderma reesei Rut-C30 as a response to addition of cellulose

An economic process for the enzymatic hydrolysis of cellulose would allow utilization of cellulosic biomass for the production of easily fermentable low-cost sugars. New and more efficient fermentation processes are emerging to convert this biologic currency to a variety of commodity products with a special emphasis on fuel ethanol production. Since the cost of cellulase production currently accounts for a large fraction of the estimated total production costs of bioethanol, a significantly less expensive process for cellulase enzyme production is needed. It will most likely be desirable to obtain cellulase production on different carbon sources—including both polymeric carbohydrates and monosaccharides. The relation between enzyme production and growth profile of the microorganism is key for designing such processes. We conducted a careful characterization of growth and cellulase production by the soft-rot fungus Trichoderma reesei. Glucosegrown cultures of T. reesei Rut-C30 were subjected to pulse additions of Solka-floc (delignified pine pulp), and the response was monitored in terms of CO₂ evolution and increased enzyme activity. There was an immediate and unexpectedly strong CO₂ evolution at the point of Solka-floc addition. The time profiles of induction of cellulase activity, cellulose degradation, and CO₂ evolution are analyzed and discussed herein.     

Cloning of Thermostable Cellulase Genes of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> and Their Secretive Expression in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Screening for the powerful cellulase genes with improved activities remains a challenge for the biorefinery research. In this study, five cellobiohydrolase genes and one endoglucanase gene sourced from Clostridium thermocellum DSM 1237, cbhA, celK, celO, cel48Y, cel48S, and celA were cloned into a newly established tool vector pP43JM2 and expressed in two Bacillus subtilis strains, B. subtilis WB600 and B. subtilis WB800, respectively. Most of the cellulases produced in the B. subtilis recombinants were efficiently secreted into the culture medium. These secreted soluble proteins showed distinct cellulase activities using phosphoric acid swollen cellulose (PASC) as the substrate and they also demonstrated strong synergistic effects for PASC, Avicel cellulose, and the dilute acid pretreated corn stover. The current work provided a quick secretive cloning method for screening cellulase genes and may provide a host strain for constructing a consolidated bioprocessing platform with the capacity of secretive expression of multiple cellulases.     

Effect of pH on cellulase production of Trichoderma ressei RUT C30

Currently, the high market price of cellulases prohibits commercialization of the lignocellulosics-to-fuel ethanol process, which utilizes enzymes for saccharification of cellulose. For this reason research aimed at understanding and improving cellulase production is still a hot topic in cellulase research. Trichoderma reesei RUT C30 is known to be one of the best hyper producing cellulolytic fungi, which makes it an ideal test organism for research. New findings could be adopted for industrial strains in the hope of improving enzyme yields, which in turn may result in lower market price of cellulases, thus making fuel ethanol more cost competitive with fossil fuels. Being one of the factors affecting the growth and cellulase production of T. reesei, the pH of cultivation is of major interest. In the present work, numerous pH-controlling strategies were compared both in shake-flask cultures and in a fermentor. Application of various buffer systems in shake-flask experiments was also tested. Although application of buffers resulted in slightly lower cellulase activity than that obtained in non-buffered medium, β-glucosidase production was increased greatly.