SEARCH · PubMed Health
Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
The synthesis of Cx-cellulase was de-repressed in Trichoderma lignorum growing on various easily metabolized carbon sources when their assimilation was limited. A reverse correlation has been established between the growth rate and the rate of the enzyme synthesis in the fungus.
The saccharification of agricultural woody wastes was studied using a commercial enzyme preparation, Cellulase onozuka, derived from Trichoderma viride or the solid culture extracts of the fungus. With the intention of producing sugar at low cost, a simple procedure of enzymatic saccharification of rice straw, bagasse, and sawdust was studied. Delignifying methods of these wastes were investigated using dilute sodium hydroxide solution and dilute peracetic acid. Rice straw and bagasse were effectively delignified by boiling in a 1% sodium hydroxide solution for 3 hr or by autoclaving at 120 degrees C in a 1% sodium hydroxide solution for 1 hr. The sawdust from a broad leaved tree (Machilus thunbergii) was delignified by autoclaving at 120 degrees C in a 1% sodium hydroxide solution for 1 hr and by subsequent boiling in diluted 1/5 peracetic acid for 1 hr. This type of sawdust was also delignified by boiling in 1/5 peracetic acid for 1 hr and by subsequent autoclaving at 120 degrees C in a 1% sodium hydroxide solution for 1 hr. The sawdust from a coniferous tree (Cryptomeria japonica) was delignified by boiling in 1/5 peracetic acid for 1 hr and by subsequent autoclaving at 120 degrees C in a 1% sodium hydroxide solution for 1 hr; however, the successive treatment by autoclaving with alkali solution and subsequent boiling with diluted peracetic acid failed to bring about the desired effect. The saccharification of delignified rice straw, bagasse, and sawdust was examined using Cellulase onozuka, wheat bran or rice straw solid culture at various substrate concentrations, resulting in the formation of 5 to 10% sugar solutions after incubation at pH 5.0, 45 degrees C for 48 hr. The optimum substrate concentration existed at around 10%. Reuse of cellulase solution and resaccharification of residual sawdust were considered to be inadequate.
The relative content of antioxidants in the mycelium of Trichoderma reesei 6/16 obtained by propagation of fungal protoplasts was shown to decrease (as compared to the initial culture taken for preparation of protoplasts) and restored only in the second generation of regenerated mycelium. In this respect, the effects of various antioxidants (beta-carotene, ascorbic acid, alpha-tocopherol, and ionol) on the frequency of regeneration of T. reesei 6/16 protoplasts were studied. beta-Carotene increased the viability of fungal protoplasts to the greatest extent. The effect of ascorbic acid depended on the presence of Fe ions. Ionol did not cause any measurable protective effect.
In the course of selection of Trichoderma viride, the producer of cellulolytic enzymes, the group of mutant strains characterized by a higher level of productivity are isolate. It is shown that the isolated mutants possess a number of common but differing them from original strains characters. These include: the small size of colonies ("dwarfs"), a lower capacity to carry out some biochemical reactions, and increased development rate and a higher resistance to lethal effect of nitrosoguanidine and nitrosoethyl urea. The data obtained indicate that in the series of populations of successively isolated mutants observed the stabilization of variability of the levels of C1 enzyme synthesis takes place. It is also shown that, unlike original cultures, the populations of mutant strains are characterized by a higher variability of Cx enzyme activity levels as compared with C1.
By using the developed display techniques of cellulase isozymes and the RAPD-PCR analysis guided by a deduced universal sequence of cellulase genes, the polymorphisms of genomic DNA fingerprints and cellulase isozymes were compared among three typical stable recombinants (3a, 3b, A7-1) and their two parents (Aspergillus niger AMS11, Trichoderma reesei QM9414) in order to provide the molecular evidence of gene recombination, to demonstrate the compatibility of heredity and expression of intergeneric genomes, and to assay on the molecular fundamentals of hybridization dominance. The results showed that in these recombinant strains the recombinantal fingerprints of genomic DNA could be stablly hereditary and the expression of recombinantal CMCase (carboxymethylcellulase) and beta GLase (beta-glucosidase) could be compatibly enhanced. The diversity of molecular fundamentals of cellulase hybridization dominance were (1) the compatible co-existence and enhanced expression of some hereditary beta GLase-coding genes from two parents in recombinant 3b; and (2) the compatibly enhancement of expression between the hereditary genes encoding beta GLase and CMCase from two parents, resulting in the dramatic increase of proteins of corresponding isozymes in recombinants 3a and A7-1. Based on these, a proposal model for the double synergism on cellulase activity in vitro and on its biosynthesis in vivo mediated by beta GLase was suggested. A practical method for assaying on the molecular fundamentals and the stability of hybridization dominance of recombinants was thereby established in this study.
Several industrial yeast are developed as ideal expression hosts for the production of the commercially useful proteins. The expression levels in yeast cells of the heterologous proteins are affected by the regulation factors of the genes themselves. The full-length cDNA coding for EG I from Trichoderma reesei, the cellulose-degrading filamentous fungus, was expressed in Saccharomyces cerevisiae H158. EG I produced by the recombinant S. cerevisiae exhibits maximal activity at 50 degrees C-60 degrees C, pH 5.0. It was observed that removal of the 3'-untranslated region (3'-UTR) from EG I cDNA resulted in no active EG I produced by recombinant yeast. RT-PCR analysis indicated that unlike the yeast cells harboring full-length EG I cDNA, there was no detectable EG I mRNA in the yeast cells harboring EG I cDNA without 3'-UTR. The data suggested that 3'-UTR is important for the expression of EG I in Saccharomyces cerevisiae.
A mutant strain T199 producing about 8 times as much beta-glucanase as its parent strain trichoderma köningii T3 was obtained by treatment with ultraviolet light and N-methyl-N-nitro-N-nitrosoguanidine. Fermentation was conducted in 250 mL flask, each containing 30 mL of medium consisted of 5% corn cob powder, 3% wheat bran and 1.4% nitrogen source No. 10 ((NH4)2SO4 10%, Peptone 20%, Yeast extract 15%). The optima culture conditions were as below: initial pH 5.0, 30 degrees C, shaking speed 280 r/min, and cultivation time 5 d. Enzyme activity toward CMC-Na, lichenin, laminarin and barley beta-glucan at pH 5.0 and 60 degrees C for 10 min were 300, 1100, 12 and 1600 IU/mL, respectively. The optima pH and temperature for enzyme action toward barley beta-glucan were pH 5.0 and 70 degrees C, respectively. The enzyme was stable under below 50 degrees C and at pH 4.5-6.5.
Using Congo red-staining method, one positive clone with CMCase activity was isolated from the Trichoderma ressei cDNA gene bank constructed in Saccharomyces cerevasiae. Sequencing result showed that the 1.5 kb-length DNA fragment inserted in the recombinant plasmid encoded EG III gene from T. reesei. Enzymatic characterization of the EG III produced by recombinant S. cerevasiae was analyzed. The experimental results indicated that the optimum pH and temperature for EG III are 5.0 and 60 degrees C, respectively. The effects of secretory system components SSO 2 and SEB1 of S. cerevisiae on EG III secretion were examined. The results indicated that the amount of EG III secreted by the strain with SSO 2-overexpression was highest among the different recombinant S. cerevisiae strains, showed that SSO 2 is a rate-limiting component of the secretory machinery in the process of EG III secretion. Furthermore, the EG III expression level was increased 5.3 times by deletion. Furthermore, the EG III expression level was increased 5.3 times by deletion of the 98 bp in 5' untranslated region of eg3 mRNA sequence. This result suggested that the regulation region could exist in the 5' untranslated region of EG III mRNA, which is recognized by the gene expression related factors of S. cerevasiae.
The factors affecting Trichoderma sp. GXC for beta-glucanase (1,3-1,4-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.73) production have been investigated, the optimal conditions were that carbon and nitrogen sources were bran and ammonium sulfate, respectively, initial pH 4.0-5.0, shaken at 30 degrees C for 44 h. beta-glucanase was purified to electrophoretic homogeneity by using ammonium sulfate precipitation, Sephadex G-100 and DEAE-Sephadex A-50 chromatography. It's molecular weight was 35 kD by SDS-PAGE, and the enzyme functioned optimally at pH6.0 and 50 degrees C, respectively. In addition, the enzyme was stable at pH 4.0-5.0 and below 40 degrees C. The beta-glucanase activity was significantly inhibited by Fe3+ and stimulated by Co2+, respectively.
The expression of xylose reductase (XR), xylitol dehydrogenase (XDH) and transaldolase (TAL) genes from Trichoderma reesei, measured by Northern hybridization, were studied by adding different carbon sources (20 kinds, including single and mixed carbon sources) separately into the basal medium on which T. reesei QM9414 was grown. The experiment results indicated that the two disaccharides: sophrose and xylibiose act as a strong inducer for the expression of xr and xdh. The lactose and arabinose were identified as inducer also. The presence of glucose repressed the transcription of xr and xdh. When glucose depleted. xr and xdh were expressed at certain level, implying that expression of xr and xdh are controlled by the carbon catabolite repression mechanism and there existed a constitutive base level's expression of xr and xdh when the catabolite repression mechanism was derepressed. On the other hand, transaldolase gene strongly expressed on all the carbon sources used.
Numbers of strains of Trichoderma asperellum are known as biological control agents of certain root pathogens of tomato (Lycopersicon esculentum). The restricted use of fungicides is sometimes useful in combination with these biological control agents. Therefore some experiments were conducted to evaluate the growth of T. asperellum in the presence of fungicides as Previcur (active substance propamocarb) and Sumico (with the active substance carbendazim and diethofencarb). The influence of these fungicides was first examined in laboratory conditions. The fungus was brought on a potato dextrose agar where the fungicides Previcur, Sumico and carbendazim were added in a concentration of 0.1x, 1x and 10x the recommended dose. The growth of T. asperellum was totally inhibited by the three Sumico and carbendazim concentrations. T. asperellum knew a small but significant decrease of growth when the 10x dose of Previcur was added. Afterwards the influence of these fungicides on the fungus was tested in field conditions in the greenhouse. The fungus was applied to the roots of the tomato plant, which was grown on a rockwool medium. Previcur and Sumico were submitted to the plants using the normal procedure. The results of the tests showed that in field conditions there was no effect of the fungicide treatment on the presence of the fungus, although the laboratory tests showed the opposite for Sumico. To explain this contradiction two other experiments were conducted to follow the migration of the Sumico after treatment. A residue analysis showed that the highest concentration of Sumico was detected in the rockwool medium, and some residues were found in the drain water and the stems. Even with a 100x recommended dose of Sumico the fungus was still present the day after the treatment.
AFP (antifungal protein) secreted by the filamentous fungi Aspergillus giganteus MDH18894 is a strong basic protein (pI>10.65). Its mRNA has an 430 bp open reading frame encoding the 94 amino acids' precursor of AFP. The precursor of AFP is processed to form the mature AFP of 51 amino acids by a putative two-step processing. A 285 bp AFP-like gene (i.e. AFP cDNA) lacking introns was reported to exist in the genome of Trichoderma viride, another species of filamentous fungi, but the gene was silent. In order to investigate whether the genomic AFP gene from A. giganteus could be expressed in T. viride, an expression plasmid with the open reading frame of AFP gene fused between the promoter and terminator of trpC gene was constructed and was transformed into T. viride. SDS-PAGE and Western blot analysis showed that AFP was expressed and secreted into the culture supernatant of the transformant of T. viride. Our study provides a basis for further secretory expression of heterologous eukaryotic genes of important value in the system of T. viride.
The variation of kinetic parameters of beta-glucosidase from Trichoderma reesei QM 9414 with pH was used to gain information about the chemical mechanism of the reaction catalysed by this enzyme. The pH-dependence of Vmax. and Vmax./Km for p-nitrophenyl beta-D-glucopyranoside showed that a group with a pK value of 4.3 must be unprotonated and a group with a pK value of 5.9 must be protonated for activity. Temperature and solvent-perturbation studies indicated that these groups are a histidine residue and a carboxy group respectively. Profiles of pKi for maltose as competitive inhibitor showed that binding is prevented when a group on the enzyme with a pK value of 4.5 becomes protonated.
In this work the influence of 12 carbon and 9 nitrogen sources on pigment production of brown mutant Trichoderma viride M-108 was tested. There were two concentrations of nutrient sources used and the tests were carried out in three light regimes. The pigmentation was good only on media containing sodium nitrate, sodium nitrite, sucrose, starch and glycerol and there was no pigment production on media with adenine, ammonium chloride and ammonium sulphate. In mixtures of pigments, individual compounds were determined by using thin-layer chromatography and UV spectra of colour fractions.
By using UV light 13 years ago from Trichoderma viride 8-7 Pers. ex S. F. Gray 82 colour and morphological mutants were prepared. The reversion of these mutants was high and in 1985 only 26 stable mutants (i.e. 32%) could be used for genetic studies. In 1991, from these 26 mutants we had only 6 stable types (i.e. 7.3% from the original state). These mutants were permanently transferred on fresh CzDA medium every 3-4 months and checked for morphology, growth, conidiation, heterokaryons and diploids production. The group of mentioned mutants was enlarged with mutants which were after 3-4 transfers on fresh medium, preserved dried to the present time. The largest differences were observed in microscopic appearance of colonies. Differences between growth rates, intensity of conidiation were comparable with those made 13 years ago.
An efficient heterologous protein production system was developed in Trichoderma viride, a very efficient cellulase producer. An expression vector containing the Taka-amylase A gene from Aspergillus oryzae, which was fused to the strong promoter and signal peptide sequence of the cellobiohydrolase 1 gene (cbh1) of T. viride, and the hygromycin B resistance gene was used to transform protoplasts of T. viride. Using hygromycin B resistance, a frequency of 3 transformants per microgram DNA on average was obtained. One transformant showed highly elevated alpha-amylase production, 1.0 g/l, which was shown to be under the control of the cbh1 gene promoter. Analysis of the chromosomal DNA of the transformant showed the integration of more than one copy of the vector.
beta-N-Acetylhexosaminidase was produced by Trichoderma harzianum cultivated with chitin as the growth substrate. The enzyme was purified 13.2-fold to homogeneity by ultrafiltration and sequential chromatography on SP-Toyopearl and Sephacryl S-200. The molecular weight of the enzyme was estimated to be about 150,000 by gel filtration. The pH and temperature optima were 4.0-5.5 and 50 degrees C, respectively. The enzyme hydrolyzed N-acetylchitooligosaccharides at the non-reducing ends to release GlcNAc monomer. The enzyme showed a strict substrate specificity to the sugar chains in complex carbohydrates, hydrolyzing only the linkage of GlcNAc beta 1-3Gal, but not hydrolyzing the other linkages such as GalNAc beta 1-3Gal and GlcNAc beta 1-2Man.
The amount of germinated conidia of microcytes belonging to the genus Trichoderma considerably decreased with an increase in the population density. Strains exhibited different ecological strategies. The maximum number of germinated conidia (30-70%) was recorded when the average distance between conidia was 50 microns.