Effect of different carbon sources on cellulase production by Hypocrea jecorina (Trichoderma reesei) strains

The ascomycete Hypocrea jecorina, an industrial (hemi)cellulase producer, can efficiently degrade plant polysaccharides. At present, the biology underlying cellulase hyperproduction of T. reesei, and the conditions for the enzyme induction, are not completely understood. In the current study, three different strains of T. reesei, including QM6a (wild-type), and mutants QM9414 and RUT-C30, were grown on 7 soluble and 7 insoluble carbon sources, with the later group including 4 pure polysaccharides and 3 lignocelluloses. Time course experiments showed that maximum cellulase activity of QM6a and QM9414 strains, for the majority of tested carbon sources, occurred at 120 hrs, while RUT-C30 had the greatest cellulase activity around 72 hrs. Maximum cellulase production was observed to be 0.035, 0.42 and 0.33 µmol glucose equivalents using microcrystalline celluloses for QM6a, QM9414, and RUTC-30, respectively. Increased cellulase production was positively correlated in QM9414 and negatively correlated in RUT-C30 with ability to grow on microcrystalline cellulose.     

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.     

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.     

Cellulase secretion from a hyper-cellulolytic mutant of Trichoderma reesei Rut-C30

Two strains of Trichoderma reesei, wild type QM6a and mutant Rut-C30, were grown in meida containing an inducer, insoluble crystalline cellulose (Avicel PH101), as carbon source for 11 days. The cell growth, expressed as myceliar protein content, of Rut-C30 was 4–5 times higher than QM6a. The lack of ultrastructural disorganization, and absence of intracellular enzyme release into the growth medium, indicated that none of these two strains had undergone any significant autolysis during the entire growth phase. Cellulase activities, mainly endoglucanase, cellobiase and filter paper degrading activity (disc) were enhanced in Rut-C30 cells. A major change was observed in the endoglucanase activity which was 30 times higher in Rut-C30 than QM6a, whereas, both -glucosidase and disc activities were 3 times enhanced in Rut-C30 compared to QM6a. In addition to synthesis, cellulase secretion was also enhanced in Rut-C30. Both the organisms contained same amounts of intracellular marker enzyme activities (e.g., inosine diphosphatase, thiamine pyrophosphatase, alkaline phosphatase). Finally, the enahncement of secretory activity of Rut-C30 was correlated with the proliferation of rough endoplasmic reticulum (RER) and increased phospholipid content. It appears that Rut-C30 is not only a hypercellulolytic but also a hypersecretor mutant.     

Reaction engineering analysis of cellulase production with Trichoderma reesei RUT-C30 with intermittent substrate supply

The effects of varying initial concentrations of microcrystalline cellulose on cellulase production with Trichoderma reesei RUT-C30 as well as the effects of varying lactose and ammonium sulfate concentrations in the feed medium were studied simultaneously in parallel-operated shake flasks and, alternatively, in parallel-operated stirred-tank bioreactors on a 10-mL scale. Fifteen experiments were performed as triplicates in shake flasks as well as in stirred-tank bioreactors in parallel to identify the parameters of second-order polynomials for the estimation of the final filter paper activity of T. reesei RUT-C30 after a process time of 96 h. Even though parameter estimation was not possible based on the results of the shake flasks due to final enzyme activities at or below the detection limit (with the exception of one shake flask), the identification of the second-order polynomial was successful with the results of the parallel-operated stirred-tank bioreactors on a 10-mL scale. Reaction conditions with 53.3 g L^?1 microcrystalline cellulose in the initial medium, no lactose feeding and 3.3 g L^?1 day^?1 intermittent ammonium sulfate addition were estimated to be optimal. The final experimental validation of the optimum substrate supply on a L-scale resulted in the production of 4.88 filter paper units (FPU) mL^?1 with T. reesei RUT-C30 after 96 h. This is an improvement by a factor of 3.6 compared to the reference batch process (1.35 FPU mL^?1).     

Extracellular hydrolase profiles of fungi isolated from koala faeces invite biotechnological interest

The extracellular enzymes of seven fungal strains isolated from koala faeces have been comprehensively characterised for the first time, revealing potential for biotechnological applications. The fungal isolates were grown in a hydrolase-inducing liquid medium and the supernatants were analysed using enzyme assays and zymogram gels. Temperature and pH profiles were established for xylanase (EC 3.2.1.8 endo-1,4-β-xylanase), mannanase (EC 3.2.1.78 mannan endo-1,4-β-mannosidase), endoglucanase (EC 3.2.1.4 cellulase), β-glucosidase (EC 3.2.1.21 β-glucosidase), amylase (EC 3.2.1.1 α-amylase), lipase (EC 3.1.1.3 triacylglycerol lipase) and protease (EC 3.4 peptidase) activities. Comparisons were made to the high-secreting hypercellulolytic mutant strain Trichoderma reesei RUT-C30 and the wild-type T. reesei QM6a. The isolates from koala faeces Gelasinospora cratophora A10 and Trichoderma atroviride A2 were good secretors of total protein and heat-tolerant enzymes. Doratomyces stemonitis C8 secreted hemicellulase(s), endoglucanase(s) and β-glucosidase(s) with neutral to alkaline pH optimums. A cold-tolerant lipase was secreted by Mariannaea camptospora A11. The characteristics displayed by the enzymes are highly sought after for industrial processes such as the manufacture of paper, detergents and food products. Furthermore, the enzymes were produced at good starting levels that could be increased further by strain improvement programs.     

Mutants of the white-rot fungus Sporotrichum pulverulentum with increased cellulase and β-d-glucosidase production

This paper reports the isolation of mutants of the white-rot fungus Sporotrichum pulverulentum and the results of a survey of enzymic activity among these mutants. The strains were screened for extracellular cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] and β-d-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) production in shake flask experiments. Apart from strain 63-2, strains 6, 63, 9, L5, E-1 and UV-18 showed equal or higher endo-1,4-β-d-glucanase (cellulase), filter paper-degrading and β-d-glucosidase activities than S. pulverulentum. The cellulase activity obtained, measured as filter paper activity, was comparable to that reported for Trichoderma reesei QM9414. However, the β-d-glucosidase activity was about six times higher than for the QM9414 strain. The pH and temperature-activity profiles of crude β-d-glucosidase preparations from the various strains were determined and were found to be identical. The thermal stability at pH 4.5 and 40°C was 5 days for all these preparations.     

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.     

Characterization of Cellulase Secretion and Cre1-Mediated Carbon Source Repression in the Potential Lignocellulose-Degrading Strain Trichoderma asperellum T-1

Trichoderma asperellum, a traditional bio-control species, was demonstrated to be an excellent candidate for lignocellulose degradation in this work. Comparing to the representatively industrial strain of Trichoderma reeseiQM6a, T. asperellum T-1 showed more robust growth, stronger spore production, faster secretion of lignocellulose-decomposing enzymes and better pH tolerance. The reducing sugar released by strain T-1 on the second day of fermentation was 87% higher than that of strain QM6a, although the maximum reducing sugar yield and the cellulase production persistence of the strain T-1 were lower. Our experiment found that the cellulase secretion was strongly inhibited by glucose, suggesting the existence of carbon source repression pathway in T. asperellum T-1. The inhibiting effect was enhanced with an increase in glucose concentration and was closely related to mycelium growth. SDS-PAGE and secondary mass-spectrum identification confirmed that the expression of endo-1,4-β-xylanase I in T. asperellum T-1 was down-regulated when glucose was added. The factor Cre1, which plays an important role in the down-regulation of the endo-1,4-β-xylanase I gene, was investigated by bioinformatics methods. The protein structure of Cre1, analyzed using multiple protein sequence alignment, indicates the existence of the Zn-fingers domain. Then, the binding sites of Cre1 on the endo-1,4-β-xylanase I gene promoter were further elucidated. This study is the first report about Cre1-mediated carbon repression in the bio-control strain T. asperellum T-1. All of the above results provided good references for better understanding T. asperellum T-1 and improving its application for lignocellulose degradation.     

Cellulase screening by iodine staining: An artefact

Hydrolysis zones visualized by iodine (KI/I₂) staining of cellulose-agar media after growth of Trichoderma reesei QM6a, RUT C30, QM9136 (cellulase negative) or Thielavia terrestris cultures, or incubation of crude endoglucanases and amylases, were due primarily to degradation of a small amount of starch contaminant in commercial agar and not to cellulolysis as recently suggested. No zones were evident when amylase-digested agar or Gelrite was used as the gelling agent or when purified cellobiohydrolase and endoglucanase were used. Cellulase screening free from artefacts is best obtained by growing cultures on acid-swollen or crystalline cellulose with Gelrite as optimal gelling agent, followed by incubation at elevated temperature to enhance visualization of hydrolysis zones while restricting fungal growth, but without additional staining.     

Novel Penicillium cellulases for total hydrolysis of lignocellulosics

The (hemi)cellulolytic systems of two novel lignocellulolytic Penicillium strains (Penicillium pulvillorum TUB F-2220 and P. cf. simplicissimum TUB F-2378) have been studied. The cultures of the Penicillium strains were characterized by high cellulase and β-glucosidase as well moderate xylanase activities compared to the Trichoderma reesei reference strains QM 6a and RUTC30 (volumetric or per secreted protein, respectively). Comparison of the novel Penicillium and T. reesei secreted enzyme mixtures in the hydrolysis of (ligno)cellulose substrates showed that the F-2220 enzyme mixture gave higher yields in the hydrolysis of crystalline cellulose (Avicel) and similar yields in hydrolysis of pre-treated spruce and wheat straw than enzyme mixture secreted by the T. reesei reference strain. The sensitivity of the Penicillium cellulase complexes to softwood (spruce) and grass (wheat straw) lignins was lignin and temperature dependent: inhibition of cellulose hydrolysis in the presence of wheat straw lignin was minor at 35 °C while at 45 °C by spruce lignin a clear inhibition was observed. The two main proteins in the F-2220 (hemi)cellulase complex were partially purified and identified by peptide sequence similarity as glycosyl hydrolases (cellobiohydrolases) of families 7 and 6. Adsorption of the GH7 enzyme PpCBH1 on cellulose and lignins was studied showing that the lignin adsorption of the enzyme is temperature and pH dependent. The ppcbh1 coding sequence was obtained using PCR cloning and the translated amino acid sequence of PpCBH1 showed up to 82% amino acid sequence identity to known Penicillium cellobiohydrolases.     

The effects of disruption of phosphoglucose isomerase gene on carbon utilisation and cellulase production in Trichoderma reesei Rut-C30

Background

Cellulase and hemicellulase genes in the fungus Trichoderma reesei are repressed by glucose and induced by lactose. Regulation of the cellulase genes is mediated by the repressor CRE1 and the activator XYR1. T. reesei strain Rut-C30 is a hypercellulolytic mutant, obtained from the natural strain QM6a, that has a truncated version of the catabolite repressor gene, cre1. It has been previously shown that bacterial mutants lacking phosphoglucose isomerase (PGI) produce more nucleotide precursors and amino acids. PGI catalyzes the second step of glycolysis, the formation of fructose-6-P from glucose-6-P.

Results

We deleted the gene pgi1, encoding PGI, in the T. reesei strain Rut-C30 and we introduced the cre1 gene in a Δpgi1 mutant. Both Δpgi1 and cre1 ⁺ Δpgi1 mutants showed a pellet-like and growth as well as morphological alterations compared with Rut-C30. None of the mutants grew in media with fructose, galactose, xylose, glycerol or lactose but they grew in media with glucose, with fructose and glucose, with galactose and fructose or with lactose and fructose. No growth was observed in media with xylose and glucose. On glucose, Δpgi1 and cre1 ⁺ Δpgi1 mutants showed higher cellulase activity than Rut-C30 and QM6a, respectively. But in media with lactose, none of the mutants improved the production of the reference strains. The increase in the activity did not correlate with the expression of mRNA of the xylanase regulator gene, xyr1. Δpgi1 mutants were also affected in the extracellular β-galactosidase activity. Levels of mRNA of the glucose 6-phosphate dehydrogenase did not increase in Δpgi1 during growth on glucose.

Conclusions

The ability to grow in media with glucose as the sole carbon source indicated that Trichoderma Δpgi1 mutants were able to use the pentose phosphate pathway. But, they did not increase the expression of gpdh. Morphological characteristics were the result of the pgi1 deletion. Deletion of pgi1 in Rut-C30 increased cellulase production, but only under repressing conditions. This increase resulted partly from the deletion itself and partly from a genetic interaction with the cre1-1 mutation. The lower cellulase activity of these mutants in media with lactose could be attributed to a reduced ability to hydrolyse this sugar but not to an effect on the expression of xyr1.     

A high cellulase-producing mutant strain of Trichoderma reesei

A mutant strain with increased production of cellulolytic enzymes was induced from the good cellulase producer Trichoderma reesei QM 9414. Cellulase activities of the mutant in fermenter cultivations were increased two- to three-fold and β-glucosidase activity up to six-fold when compared to the corresponding activities produced by QM 9414.     

Formation of enzymes required for the hydrolysis of plant cell wall polysaccharides byTrichoderma reesei

Summary Enzyme preparations fromTrichoderma reesei RUT-C30, in addition to cellulase, contained various glucanase and glucosidase, acetylxylan esterase, glucuronidase and xylanase activities. These preparations were able to hydrolyse endosperm cell walls of corn and wheat and commercially-available xylans and plant gums having stright chains, but lacked the ability to hydrolyse branched or substituted hemicelluloses.

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Formation d'enzymes requises pour l'hydrolyse des polysaccharides de la paroi de plantes chez Trichoderma reesei

Résumé Les préparations enzymatiques deTrichoderma reesei RUT-C30, contiennent, outre la cellulase, diverses glucanases et glucosidases, acétyl-xylane esterases, glucuronidases et xylanases. Ces préparations demeurent stables pour l'hydrolyse de parois cellulaires de l'endosperme de maïs et de froment ainsi que des xylanes et gommes de plantes à chaînes linéaires, disponibles dans le commerce, mais ne présentent pas le pouvoir d'hydrolyser les hémicelluloses branchées ou substituées.

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Issued as NRCC No. 29855     

The glucose repressor genecre1 ofTrichoderma: Isolation and expression of a full-length and a truncated mutant form

Thecre1 genes of the filamentous fungiTrichoderma reesei andT. harzianum were isolated and characterized. The deduced CREI proteins are 46% identical to the product of the glucose repressor genecreA ofAspergillus nidulans, encoding a DNA-binding protein with zinc fingers of the C₂H₂ type. Thecre1 promoters contain several sequence elements that are identical to the previously identified binding sites forA. nidulans CREA. Steady-state mRNA levels forcre1 of theT. reesei strain QM9414 varied depending on the carbon source, being low on glucose-containing media. These observations suggest thatcre1 expression may be autoregulated. TheT. reesei strain Rut-C30, a hyperproducer of cellulolytic enzymes, was found to express a truncated form of thecre1 gene (cre1-1) with an ORF corresponding to a protein of 95 amino acids with only one zinc finger. Unlike QM9414 the strain Rut-C30 produced cellulase mRNAs on glucose-containing medium and transformation of the full-lengthcre1 gene into this strain caused glucose repression ofcbh1 expression, demonstrating thatcre1 regulates cellulase expression.     

A continuous spectrophotometric assay for the determination of cellulase solubilizing activity

A cellulase assay was developed for the continuous measurement of colored cellulose oligosaccharides (total carbohydrates) released during enzymatic hydrolysis of dyed crystal-line cellulose. Several cellulosic substrates were uniformly dyed by Remalzol brilliant blue R salt without altering their physical properties. Dyed Avicel (6.5%, w/w) was selected as the most representative substrate for the assay procedure. The assay was performed continuously in a simple, thermally controlled apparatus designed for filtration of the reaction mixture via a 5-μm-pore-size nylon filter to retain the crystalline dyed cellulose while spectrophotometrically monitoring the absorbance at 595 nm of the reaction filtrate. Crude supernatant cellulase of Trichoderma viride QM9414 was used to test the assay procedure. The activity of cellulase on dyed Avicel as measured by ΔA_595nm correlated directly with the total carbohydrates formed. The initial reaction rate of cellulase solubilizing activity was readily determined with high sensitivity. The continuous assay has utility for the study of cellulase kinetics and for the comparison of activities from different microorganisms.     

Folding, quality control, and secretion of pancreatic ribonuclease in live cells

Although bovine pancreatic RNase is one of the best characterized proteins in respect to structure and in vitro refolding, little is known about its synthesis and maturation in the endoplasmic reticulum (ER) of live cells. We expressed the RNase in live cells and analyzed its folding, quality control, and secretion using pulse-chase analysis and other cell biological techniques. In contrast to the slow in vitro refolding, the protein folded almost instantly after translation and translocation into the ER lumen (t_½ < 3 min). Despite high stability of the native protein, only about half of the RNase reached a secretion competent, monomeric form and was rapidly transported from the rough ER via the Golgi complex (t_½ = 16 min) to the extracellular space (t_½ = 35 min). The rest remained in the ER mainly in the form of dimers and was slowly degraded. The dimers were most likely formed by C-terminal domain swapping since mutation of Asn¹¹³, a residue that stabilizes such dimers, to Ser increased the efficiency of secretion from 59 to 75%. Consistent with stringent ER quality control in vivo, the secreted RNase in the bovine pancreas was mainly monomeric, whereas the enzyme present in the cells also contained 20% dimers. These results suggest that the efficiency of secretion is not only determined by the stability of the native protein but by multiple factors including the stability of secretion-incompetent side products of folding. The presence of N-glycans had little effect on the folding and secretion process.     

Characterization of cellobiohydrolase I (Cel7A) glycoforms from extracts of Trichoderma reesei using capillary isoelectric focusing and electrospray mass spectrometry

Capillary isoelectric focusing (CIEF) was used to profile the cellulase composition in complex fermentation samples of secreted proteins from Trichoderma reesei. The enzyme cellobiohydrolase I (CBH I, also referred to as Cel7A), a major component in these extracts, was purified from different strains and characterized using analytical methods such as CIEF, high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC–PAD), and capillary liquid chromatography–electrospray mass spectrometry (cLC–ESMS). ESMS was also used to monitor the extent of glycosylation in CBH I isolated from T. reesei strain RUT-C30 and two derivative mutant strains. Selective identification of tryptic N-linked glycopeptides was achieved using LC–ESMS on a quadrupole/time-of-flight instrument with a mixed scan function. The suspected glycopeptides were further analyzed by on-line tandem mass spectrometry to determine the nature of N-linked glycans and their attachment sites. This strategy enabled the identification of a high mannose glycan attached to Asn270 (predominantly Man₈GlcNAc₂) and single GlcNAc occupancy at Asn45 and Asn384 with some site heterogeneity depending on strains and fermentation conditions. The linker region of CBH I was shown to be extensively glycosylated with di-, and tri-saccharides at Thr and Ser residues as indicated by MALDI-TOF and HPAEC–PAD experiments. Additional heterogeneity was noted in the CBH I linker peptide of RUT-C30 strain with the presence of a phosphorylated di-saccharide.