Cellulase production and its localization inTrichoderma harzianum

The ability ofT. harzianum to produce the three components of cellulase,viz. filter paper activity, carboxymethylcellulase and β-glucosidase was investigated. The optimum pH and temperature for all the enzymes in the culture nitrate was 5–7 and 27°C, respectively. The shaken culture conditions gave low yields of the enzyme as compared to static cultures. Most of the FP-activity and CM-cellulase were located extracellularly but reasonable amount of β-glucosidase was retained in the cell debris fraction.     

Photoinduced conidiation inTrichoderma viride

Trichoderma viride is a deuteromycete in which conidiations is photo-inducible. Conidiation results when colonies grow in the day-night regime or when colonies grown in the dark are exposed to short pulses of near UV or blue light. Conidiation was induced by light pulses at intervals of 8, 16, 24, 48 or 72 hours. Several membrane-damaging agents, DNA-intercalating drugs and inhibitors of RNA or protein synthesis prevent photo-conidiation. A hypothetical scheme of photo-induced conidiation, based on the results with metabolic inhibitors, is presented. A sudden increase of intracellular ATP was observed as an immediate photo-response. The ATP level is dose-dependent, with a maximum at 1.2 klx. Drugs interfering with various signalling pathways were tested in an attempt to analyze the signal pathways whereby light pulses induce conidiation. Nonconidiating and color mutants have been obtained and used in complementation studied by means of heterokaryosis and protoplast fusion. In a color mutant with brown conidia, conidiation is accompanied by high production and excretion of anthraquinone metabolites. Modified version of a lecture given at the7th International Congress of IUMS Mycology Division, Prague, July 3rd–8th, 1994.     

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.     

Dose-Dependency of radiation on enzyme production inTrichoderma reesei

Effect of irradiation dose on the production of cellulase and amylase related enzymes inTrichoderma reesei was studied, in which post-irradiation time responce pattern was measured. The damage of the cells irradiated with certain irradiation doses (1.40±0.20x10⁵, 2.20±0.10x10⁵, 3.00±0.50 x 10 and 3.50±0.20 x 10 rad) was rapidly recovered. The increased enzyme production in the culture of the irradiated cells resulted from the recovery of radiation damage after irradiation. The function of cell growth was not affected by irradiation below dose of 5 x 10⁵ rad, though the function of enzyme synthesis was drastically affected.     

Partial Characterization of Cell-bound Lipase of Candida paralipolytica

In accordance with the regulation by aspartate of phosphoenolpyrubate (PEP*) carboxylase, glutamate formation in Brevibacterium flavum, a glutamate-producing bacterium, was inhibited by the addition of aspartate. Furthermore, an increase in aspartate formation caused by a mutational decrease in citrate synthase specific activity was accompanied by a decrease in the total amount of glutamate and aspartate formed. However, a mutational decrease in glutamate dehydrogenase activity caused a decrease in the total amount without increasing the asparate formation but with accumulation of 2-oxoglutarate, suggesting that the feedback inhibition by the aspartate of PEP carboxylase was enhanced by 2-oxoglutarate. In fact, partially purified PEP carboxylase from this organism was found to be synergistically inhibited by aspartate and 2-oxoglutarate, citrate, cis-aconitase, or isocitrate. Among them, the effects of tricarboxylic acids were attributed to their non-specific chelating action with Mn²⁺, an activator of the enzyme. The synergistic action of 2-oxoglutarate was accompanied by a decrease in Hill coefficient for the aspartate of the enzyme.     

Suppression of photo-induced sporulation inTrichoderma viride by inhibitors

The mycelium ofTrichoderma viride grown in the dark under submerged conditions and transferred to membrane filters sporulated only after photoinduction. The optimum photoinduction of sporulation was reached when applying daylight for 3 min and near ultraviolet radiation (366 nm) for 10 to 30 sec. After the photoinduction pronounced synthesis of DNA, RNA and protein was observed. The photoinduced sporulation was partially or fully inhibited in the presence of phenethyl alcohol, actinomycin D, 5-fluorouracil, cycloheximide and ethidium bromide. The same inhibitors blocked also the photoinduced sporulation of surface growing colonies ofTrichoderma viride. Various inhibitors of synthesis of nucleic acids and protein, inhibitors impairing the function of membranes and certain other compounds were also effective. A part of the results presented here was included in the dissertation of J.S. and defended at the Slovak Polytechnical University in Bratislava.     

Localization of Cell-bound Penicillinase in Bacillus licheniformis

When protoplasts are prepared from Bacillus licheniformis (strain 749/C, constitutive for penicillinase), approximately 60% of the cell-bound penicillinase is released. The remainder is retained by the protoplast and cannot be removed by washing. This release is specific, in that less than 7% of the cellular reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase and alpha-glucosidase is liberated by the treatment. The freed penicillinase is excluded from G-200 Sephadex, and it is partially sedimented with a force of 65,000 x g for 20 hr. It is probably attached to characteristic tubular and vesicular structures with single-layered membranes that are comparable to structures previously described in intact penicillinase-forming cells. The specific activity of the organelle is more than six times that of twice washed peripheral membrane; furthermore, about 8% of the protein of the structure is penicillinase. At substrate concentrations (benzylpenicillin) of about one-fifth the K(m) value, whole cells show a slight permeability restriction, although this does not occur in isolated particles and protoplasts.     

Cell-bound and secreted proteases of Serratia marcescens.

Exoprotease of Serratia marcescens ATCC 25419 is exceptional among members of the family Enterobacteriaceae in that it is secreted in large amounts by viable cells into the culture medium. Labeling of cells with radioactive amino acids revealed no intracellular protein that could be precipitated with antibodies raised against purified exoproteases. With substances known to interfere with the excretion of some proteins--tosyl-L-lysine chloromethyl ketone, phenethyl alcohol, procaine, and sodium azide--and with rifampin, an intracellular form (apparent molecular weight, 52,000) larger than the major exoform (molecular weight, 51,000) was identified. Moreover, the 52,000-molecular-weight form was the main protein in immunoprecipitates of a cysteine-auxotrophic mutant starved for cysteine. Beside the major exoform, protease I, two additional exoproteases, termed II and III, appeared in the medium of stationary cultures. They were precipitated by antibodies against protease I, were identical in the Ouchterlony double-diffusion assay, and exhibited only a small difference, if any at all, in the peptide pattern after partial hydrolysis with protease V8 of Staphylococcus aureus. The amino- and carboxy-terminal amino acid sequences of protease I and II were determined and found to be identical, NH2-Ala-Ala-Thr-Gly-Gly-Tyr-Asp-Ala-Val-Asp and Phe-Ile-Val-COOH, respectively. The microheterogeneity of the isolated exoforms revealed by anion-exchange chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was also observed in samples pulse-labeled with radioactive amino acids. It remains to be determined whether the different protease forms are the result of processing (modification) reactions or whether they constitute isoenzymes encoded by very similar genes.     

Photoinduced conidiation inTrichoderma viride: A study with inhibitors

The influence of a number of inhibitors affecting respiration, oxidative phosphorylation, cAMP-phosphodiesterase and of the antioxidant 1,4-dithiothreitol on growth and photoinduced conidiation ofTrichoderma viride were investigated. In all cases, growth and conidiation were influenced to a different extent. Among the first group of compounds, antimycin A was the most potent inhibitor of conidiation while it influenced growth much less. A similar effect was obtained with 2,4-dinitrophenol and 1,4-dithiothreitol. On the other hand, 3-isobutyl-1-methylxanthine (inhibitor of phosphodiesterase) greatly stimulated the conidiation induced by light without affecting growth. It is concluded that the redox reactions represent a vital component of the differentiation pathway and that cAMP may play a regulatory role in this process.     

Stimulation of conidiation by derivatives of cAMP inTrichoderma viride

Derivatives of cyclic-3′,5′-adenosine monophosphate, N⁶,2′-O-dibutyryl-cAMP, 8-(chlorophenylthio)-cAMP and 8-bromoadenosine-cAMP added at 0.1–10 μmol/L concentrations into the growth medium have markedly stimulated conidiation inTrichoderma viride. Their stimulatory effect on conidiation was best observed in colonies that were constantly kept in the dark but was also marked in colonies illuminated by sub-saturating doses of light. The relative stimulation of conidiation depended not only on the concentration of exogenous cyclic nucleotides but also on the concentration of glucose in the medium. It was more pronounced in glucose-rich media than in media where the concentration of the sugar was low. Concentrations of cAMP analogues equal to 50 μmol/L and higher inhibited the conidiation.     

Cell growth and cellulase production inTrichoderma viride on microcrystalline cellulose

The production of CM and FP cellulases was studied during the growth of a wild strain ofTrichoderma viride on microcrystalline cellulose. Part of the enzymes was found to be released into the medium while another part remained bound to the cell. Bound cellulases are released into the medium at the stage of cell lysis which takes place in the post-stationary phase. In this period extracellular CM and FP cellulases attain maximum activities. When the hyphae are subjected to a cold shock, maximum cellulase activity is detected already at the beginning of the stationary phase. An indirect method of dry cell mass determination showed that during exponential growth of cells on microcrystalline cellulose the μmax was 0.23 and the yield coefficient was 41 %.     

Effect of spore concentration on germination and autotropism inTrichoderma hamatum

Summary The effect of spore concentration on spore germination and germtube growth ofTrichoderma hamatum on water agar and on potato dextrose agar (PDA) was studied. Increasing inoculum size up to 10⁹ spores/plate on PDA and up to 10⁷ spores/plate on water agar shortened the incubation period required for germtubes emergence and increased germination rate. However, on water agar germination was inhibited at 10⁸ and was completely arrested at 10⁹ spores/plate. Inhibition in germination of 10⁷ spores/plate was observed on water agar when the plates were preincubated with 10⁹ spores/plate for 5 h or more. Addition of glucose and ammonium nitrate to the water agar medium allowed only 25% of the spores to germinate at 10⁹ as compared to 78% at 10⁷ spores/plate after 8 h of incubation. Addition of polysaccharides to the C+N supplemented medium, significantly increased germination up to 84% as compared to 100% on PDA, after 8 h of incubation. Germlings ofTrichoderma hamatum phialospores exhibited positive autotropism and anastamosis on both media. The phenomenon was positively related to inoculum size, being most pronounced at 10⁷ spores/plate.     

Cell-bound cations of the moderately halophilic bacterium Vibrio costicola.

Over most of the range of salt concentrations in which the moderately halophilic bacterium Vibrio costicola could grow, the sum of the cell-associated Na+ + K+ ions was at least as high as in the external medium. This is in contrast to other moderate halophiles, which have substantially lower internal than external salt concentrations for most of their growth range. The relative amounts of Na+ and K+ in V. costicola varied with environmental conditions. The K+/Na+ ratio fell during anaerobic incubation or when cells were poisoned. As Na+ ions left the cells, K+ ions entered. However, movement of these ions was not tightly coupled, since K+ content of cells could increase without a corresponding decrease in Na+ content. The Mg2+ contents of cells varied little with environmental conditions.     

Nonparental progeny resulting from protoplast fusion inTrichoderma in the absence of parasexuality

The purpose of this study was to characterize a number of progeny from intra- and interstrain protoplast fusion within the genusTrichoderma. We wished to determine whether parasexuality or other genetic mechanisms occur in these fungi. When two different auxotrophs of the same strain were fused, rapidly growing prototrophic progeny were obtained in high frequencies. When single spore isolates of these strains were prepared, equal numbers of strains indistinguishable from the two parental auxotrophic strains were obtained, even though 10⁹–10¹⁰ conidia were tested per strain. Thus, progeny from intrastrain fusions all appeared to be balanced heterokaryons, and no evidence of recombination between the two parental strains was obtained. When 16 separate interstrain fusions were conducted, very different results were obtained, regardless of whether fusions were within or between species. Following interstrain fusions, presumptive somatic hybrids developed very slowly and in low numbers as compared with hybrids from intrastrain fusions. Most were weakly prototrophic. These slow-growing progeny were unstable and sectors developed from them. Such sectors themselves were unstable and gave rise to other progeny. Usually sectors were more strongly prototrophic and more rapid growing than the original progeny strain. Sectoring gave rise to a very wide range of morphotypes. Most of these morphotype variants were stable through conidiation; thus, these types did not occur as a consequence of heterokaryosis. Isozyme analysis was conducted on over 1000 progeny strains. Nearly all progeny were identical to one or the other parental isozyme phenotypes. A few progeny, when tested as soon as possible after fusion, exhibited the isozyme phenotypes of both parents, but such biparental banding patterns were rapidly lost upon subsequent reculturing. Isozyme banding patterns of multimeric enzymes never gave band patterns indicative of heterokaryosis or heterozygosis. Banding patterns indicative of heterozygous diploids or recombinants were never detected. Despite the extreme variation in morphotype and nutritional requirements among progeny, isozyme banding patterns of derived progeny from any fusion were invariably identical to one or the other parental strains. From these results, we conclude that protoplast fusion in the genusTrichoderma gives rise to great variability, but that the classical parasexual cycle is not required for variation to occur.     

Cell-bound and extracellular carboxylesterases from Streptomyces: hydrolytic and synthetic activities

AIM: The distribution of cell-bound and extracellular carboxylesterases was investigated among the genus Streptomyces using 420 strains. METHODS AND RESULTS: Primary screening was carried out on solid media using tributyrin, triolein and Tween 60 as current substrates. Eleven representative strains were selected and grown in submerged cultures for evaluating their cell-bound and extracellular hydrolytic activity independently on various naphthyl and aliphatic esters. The best lipolytic strain was lyophilized and used as dry mycelium for catalysing the synthesis of various aliphatic esters in heptane, with molar conversions ranging from 28 to 78% after 3 days. CONCLUSIONS: Carboxylesterase activities can easily be found among the Streptomyces, often being cell-bound and also employable for catalysing esterification in organic solvent. SIGNIFICANCE AND IMPACT OF THE STUDY: A wide screening among Streptomyces, a genus poorly studied for the production of carboxylesterases, has allowed the selection of several strains with interesting enzymatic activities to be used in commercially valuable biotransformation.     

Cell-bound antiglial immunity in patients with malignant tumors of the brain

Peripheral blood lymphocytes from patients with malignant brain tumors were found to have a cytotoxic effect against cultured autologous tumor cells as well as normal adult and fetal glial cells obtained from 18- to 20-week surgical abortions. In a blind series of 32 patients, five of nine patients with a malignant glioma could be detected by using ⁵¹Cr-labeled fetal glial cells as targets and appropriate controls. Lymphocytes from patients with “benign” gliomas, nonglial, or metastatic tumors were characterized by a low or absent cytotoxicity.The results are interpreted to show a development of cell-bound immunity against normal glial antigens in patients with destructive infiltrating glial tumors carrying the antigenic determinants through the immunological barrier normally isolating the central nervous tissues.     

Cell-bound peptidase activities of Treponema denticola ATCC 33520 in continuous culture.

The oral spirochaete Treponema denticola ATCC 33520 was grown at a mean generation time of 10 h in anaerobic continuous culture in a serum- and carbohydrate-free medium at pH 7.0. The extracellular proteolytic activities of this spirochaete were then investigated by incubating washed cells with 68 2-naphthylamide derivatives of the Extended API System. Chymotrypsin-like, trypsin-like, elastase-like and iminopeptidase activities were demonstrated. The phenylalanine peptidase or chymotrypsin-like activity of T. denticola ATCC 33520, estimated with N-succinyl-L-phenylalanyl-L-leucyl-L-phenylalanine-thiobenzyl ester (SPLP) had a pH optimum at pH 8.5, a specific activity of 36.6 nmol min-1 (mg dry wt)-1 and was inhibited only slightly by HgCl2. The trypsin-like activity, estimated with benzoyl-DL-arginine-7-amido-4-methylcoumarin (BAMC), had a pH optimum at pH9, and a specific activity of 0.3 nmol min-1 (mg dry wt)-1; inhibition by HgCl2 indicated the involvement of active thiol groups. The activity should preferably be termed arginine peptidase activity, according to the carboxy-terminal amino acid of the test substrate. The extracellular proline peptidase activity, estimated with L-proline-7-amido-4-methylcoumarin. HBr (PRAMC), had an activity of 1.5 nmol min-1 (mg dry wt)-1, an optimum at pH 8.5 and the properties of a thiol protease. The main cell-bound and extracellular active peptidase activities of fast-growing cells of T. denticola ATCC 33520 are phenylalanine peptidase, proline peptidase, arginine peptidase and an oligopeptide-dependent alanine peptidase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Raffinose-induced mutanase production from Trichoderma harzianum

Abstract The enzyme α (1 → 3),3-glucanohydrolase (referred to as mutanase) from the filamentous fungus Trichoderma harzianum OMZ 779 is capable of degrading the water-insoluble glucan in dental plaque. Previously, it was necessary to produce the glucan (referred to as mutan) in vitro for use as the sole carbon source and inducer of mutanase synthesis in fungal cultures. We report here that raffinose also induces the production of mutanase. The metabolism of raffinose differed from that of other sugars in metabolic end products and secreted protein profile. In addition to mutanase, we observed an approximately 15 000 M _r protein that was also regulated by carbon source and by illumination conditions.