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L-lysine-alpha-oxidase, isolated from a strain of Trichoderma sp. exhibited enzymological properties suitable for a chemotherapeutic drug used in experimental oncology.
The present study describes the production of cellulases by a native strain of Trichoderma pseudokoningii in the presence and absence of a catabolic repressor. We report a plate assay for selecting fungal mutants capable of synthesizing the cellulase enzymes under conditions of catabolite repression. This method was used for the selection of catabolite repression-resistant mutants. One mutant, B-1, secreted 3.3 times the extracellular protein and 3 times the endoglucanase and filter paper activity in submerged cultures when compared with the wild type strain. The cellobiase activity remained at similar levels in both strains. Although this mutant was isolated as i cellulase producer in the presence of 500 mM glycerol on agar plates, in liquid medium containing the repressor B-1 it exhibits only partial derepression of the cellulase complex.
Cellulosic material was inoculated with Trichoderma album by solid state fermentation and the change of protein, total nitrogen, mineral nitrogen, cellulose and total fiber contents of raw material were measured. It was found that the water holding capacity and bulk density of substrate increased as the fermentation progressed. The optimum C/N ratio for the conidia production was between 4 and 7, and the optimum pH was around 5. The protein enrichment of sugar beet residue were the best at the initial moisture content 76%, initial pH 4.5 and supplement with 1% nitrogen. After 4 days fermentation, the final product contained 22% of protein. From the present state of technological development, the protein enrichment of cellulosic materials by solid state fermentation proved to be valuable.
Combined cultivation of the following microorganisms was studied: the fungus Trichoderma longibrachiatum producing cellulases and the yeast Endomycopsis fibuligera producing glucoamylase. A growth medium was found to maintain the activity of these enzymes at a high level in the both microbial monocultures. The effect of the yeast inoculation time on the enzyme activity was studied during combined cultivation of the two organisms. When the yeast was inoculated during the first two days of the fungal growth, the enzyme activities were 40-70% of those during the growth of the monocultures. The yeast did not grow when it was inoculated by the 4th day of the fungal growth. When the yeast was added to the fungus earlier, the activity of cellulases fell down. Possible reasons for these phenomena are discussed.
An improved and relatively rapid procedure is developed for isolation and purification of a new antitumor enzyme L-lysyl-alpha-oxidase from Trichoderma sp. The method involves four steps, instead of six steps described previously, with a yield of 22.4%. The purified enzyme preparation was homogeneous as shown by polyacrylamide gel disc electrophoresis and ultracentrifugation. Physico-chemical and antitumor properties of the enzyme are under study.
A potent cellulase producing mold, Tichoderma sp. W-10 suitable for enzyme production with solid state culture was isolated from infected mushroom spawn, its characteristics and taxonomic studies were carried out. As a result, this mold was identified as Trichoderma koningii (Oudemans) according to the keys of Gilman and Rifai's classification system and the morphological features described by Komatsu. Meanwhile, a detail survey of physiological, cultural and biochemical characteristics were also described.
L-Lysine alpha-oxidase from Trichoderma viride Y244-2 has been purified to homogeneity. The enzyme shows absorption maxima at 277, 388, and 466 nm and a shoulder around 490 nm and contains 2 mol of FAD/mol of enzyme. The enzyme has a molecular weight of approximately 116,000 and consists of two subunits identical in molecular weight (about 56,000). In addition to L-lysine, L-ornithine, L-phenylalanine, L-tyrosine, L-arginine, and L-histidine are oxidized by the enzyme to a lesser extent. Several lysine analogs such as delta-hydroxylysine are oxidized efficiently. Balance studies showed that 1 mol of L-lysine is converted to an equimolar amount of alpha-keto-epsilon-aminocaproate, ammonia, and hydrogen peroxide with the consumption of 1 mol of oxygen. alpha-Keto-epsilon-aminocaproate spontaneously is dehydrated intramolecularly into delta 1-piperideine-2-carboxylate in the presence of catalase, and is oxidatively decarboxylated into delta-aminovalerate in the absence of catalase. The Michaelis constants are as follows: 0.04 mM for L-lysine, 0.44 mM for L-ornithine, 14 mM for L-phenylalanine, and 1.6 mM for oxygen with L-lysine.
The production of extracellular 1,3-, 1,6-beta-glucanases and chitinase was studied during submerged cultivation of a Trichoderma viride strain 3/78 on various carbon sources: glycerol, glucose, lactose, sucrose, laminaran, starch, pustulan, chitin, and Agaricus bisporus fruit bodies. The synthesis of these enzymes and cellulase was studied also under the conditions of depression at low concentrations (10(-2) and 10(-3)M) of the first five aforementioned carbon sources as well as cellobiose, gentiobiose, N-acetyl-beta-D-glucosamine and 0.1% chitooligosaccharides and A. bisporus cell walls. The experiments were conducted with the washed mycelium of this strain grown for 2 days in a medium with glycerol as a carbon source. The results indicated that 1,3- and 1,6-beta-glucanases of the strain were of the constitutive nature and were repressed by such carbon sources as glycerol and glucose. Chitinase and cellulase were shown to be inducible enzymes. Chitinase was induced by N-acetyl-beta-D-glucosamine, chitooligosaccharides and A. bisporus cell walls as well as by lactose when the fungus was grown on this carbon source. Cellulase biosynthesis was induced by lactose, cellobiose and gentiobiose.
Two cellobiohydrolases and two endoglucanases were purified from a culture filtrate of the fungus Trichoderma reesei QM 9414 by simple and straightforward purification techniques. Molecular weights, isoelectric points, amino acid compositions, and carbohydrate contents are reported.
Trichoderma cultures were tested for their ability to produce L-lysine-alpha-oxidase. The highest enzyme activity was manifested by T. harzianum (MGU), T. longibrachiatum Rifai VKM F-2025 and T. aureoviride Rifai VKM F-2026. The biosynthesis of the enzyme did not depend on the growth of the cultures and did not vary among the species.
The effect of various nitrogen sources on cellulase biosynthesis by the mutant strain Trichoderma viride 44 was examined. This strain may utilized nitrogen in the nitrate, ammonium of organic form. When cultivating this strain, it appears advantageous to add to the nutrient medium yeast and yeast lyzates as well as their mixture with ammonium sulfate. Cellulase reached its maximum activity of 20.2, 21.5 and 23.2 mu/ml when grown on the medium containing ammonium phosphate, peptone and brewing yeast plus ammonium sulfate, respectively. It is useful to apply nitrogen in its organic forms in small quantities and in combination with mineral forms. The nitrogen presence in the medium is necessary only at the exponential stage of fungal growth. The lack of nitrogen in the stationary stage characterized by the maximum cellulase formation does not inhibit an increase in the enzyme activity.
The mode of action of two purified endoglucanase (Mr 42 000) from Trichoderma longibrachiatum on soluble CM-cellulose as well as on amorphous and crystalline celluloses was studied. Both enzymes had the same Km values for CM cellulose (1.4 g/l), similar V values (51 and 47 mumole . min . mg of protein) and synthesized glucose and cellobiose from CM-cellulose in comparable amounts. However, the enzymes differed essentially in their ability to be adsorbed on soluble cellulose, i. e. their partition coefficients (cellulose surface/bulk solution) differed by one order of magnitude. When the enzyme concentrations on the cellulose surface were equal (in two independent experiments), the rate of degradation of amorphous (but not crystalline) cellulose was also almost identical. However, when crystalline cellulose was subjected to hydrolysis, the more tightly adsorbed enzyme revealed the initial solubilizing activity which was 20 times greater than that of the less tightly adsorbed enzyme (at the same endoglucanase activity on the surface). In the presence of high cellobiase concentrations sufficient to convert all intermediate cellobiose into glucose the effectively adsorbed endoglucanase was capable to convert at least 50% of crystalline cellulose to glucose.
Microcristalline cellulose (cellulose Avicel, Merck) supported growth of Trichoderma harzianum and induced production of cellulases in liquid cultures. After 50 h growth, the total cellulasic activities present in both the supernatant and the mycelium were 3,000 IU/l of carboxymethyl cellulose, 400 IU/l of filter paper activity, and 4 IU/l of cotton activity corresponding to 1.7 g/l of proteins. Cellulase production could be increased by a preliminary treatment of cellulose, and pH regulation during growth. The influence of inoculum concentration was studied and an optimum of 3 x 10(7) conidia/g dry weight of substrate was demonstrated. Using a synthetic culture medium, a soluble factor of germination was demonstrated which could be leached out by 3 successive washings of conidia.
A carboxylic proteinase has been isolated from a commercial preparation of Trichoderma lignorum used as a source of cellulolytic enzymes. The purification procedure included precipitation by (NH4)2SO4 (65% saturation), gel-filtration through Acrylex P-10, affinity chromatography on gramicidin S bound to an inorganic matrix, gel-filtration through Acrylex P-10, affinity chromatography on bacitracin-Sepharose and separation on Ultrogel AcA 54 followed by gel-filtration through Sephadex G-50. A 400-fold purification of enzyme was achieved, the enzyme yield being 7,2%. The molecular weight of carboxylic proteinase as determined by gel-filtration is 33 000; its amino acid composition is found to be similar to that of carboxylic proteinases isolated from other fungal species. The enzyme is stable within the pH range of 3,0-6,0. The enzyme was fully inhibited by the specific inhibitors of carboxyliec proteinases-N-diazoacetyl-N'-2,4-dinitrophenylethylenediamine and pepstatin.
Conidiation mutants were isolated from the fungus Trichoderma viride, and tested for complementation by both anastomosis and protoplast fusion. They could be grouped into three classes: (1) colour mutants; (2) mutants with reduced conidiation; and (3) nonconidiating mutants. Several mutants of classes (2) and (3) were incapable of anastomosis, but via protoplast fusion they produced heterokaryons suitable for complementation tests.
The major cellulase components produced by Trichoderma reesei are composed of distinct catalytic and cellulose-binding domains. A simple two-step procedure is described for the purification of the catalytic domains, also termed core proteins (cp), of the major components, cellobiohydrolase (CBH) I and II. The novel aspect of this procedure is that native CBH I and II do not have to be purified initially. Papain digestion of a commercial T. reesei cellulase preparation followed by gel filtration on a Superdex 75 column resulted in the separation of fractions containing CBH I cp and CBH II cp; chromatofocusing purified the latter to homogeneity. N-terminal protein sequencing of CBH II cp provided good evidence for its identity. A comparison of the catalytic activity and cellulose-binding ability of these cp was made. A major difference between them was that CBH II cp bound to microcrystalline cellulose, unlike CBH I cp. CBH I cp readily hydrolysed the bond between the aglycone and cellobiose in p-nitrophenyl cellobioside unlike the CBH II cp preparation. Neither CBH I cp nor CBH II cp had activity toward carboxymethylcellulose, but both were able to hydrolyse barley beta-glucan. It was also shown that incubation of cellulose fibres with native CBH I, CBH I cp or CBH II cp resulted in a smoothing of the fibre surface.
Three forms of cellobiohydrolase I (CBH I) (65, 58 and 54 kDa) were isolated to apparent homogeneity from culture filtrates of Trichoderma reesei. The N-terminal sequence of amino acid residues is the same for all of them. The 65 kDa CBH I (pI 4.1) is the intact protein which is fully active against small, soluble substrates and an insoluble substrate Avicel. The 58 kDa CBH I (pI 3.8) and 54 kDa CBH I (pI 3.6) are two truncated forms of the intact CBH I, which are fully active against small, soluble substrates, but have decreased adsorption on and activity against Avicel. Limited proteolysis of the 65 kDa and 58 kDa CBH I by papain yields the same core protein (pI 3.6, 54 kDa). It appears that there are mainly two different specific proteolytic cleavage points in the intact CBH I, one the site for a papain-like protease action cutting at the hinge area (54 kDa CBH I) and the other in the B block (58 kDa CBH I).
The effect of cadmium ion on growth and differentiation of the filamentous fungus Trichoderma viride was studied. Cd++ at a concentration of 10 mM strongly retarded growth and caused morphological changes. From cultures treated with cadmium, two white mutants and one yellow mutant were isolated. The white mutants formed no conidiophores and one of them produced colourless crystals in the cultivation medium. The yellow mutant differed from the parental strain mainly by the production of differently coloured conidia and by a slower growth rate.