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Further studies on microbiological ring-expansion of penicillin N.

The rate of microbiological ring-expansion of penicillin N to deacetoxycephalosporin C using protoplast lysates of the antibiotic-negative mutant Cephalosporium acremonium M-0198 has been increased some 70-fold over that of our earlier system. We confirmed the stimulatory effects of FeSO4 and ascorbate described by Hook et al. (Biochem. Biophys. Res. Commun. 87: 258, 1979); the optimum concentrations found were 0.04 mM FeSO4 and 0.67 mM ascorbate. Adenosine triphosphate concentration was lowered to 0.83 mM; phosphoenolpyruvate and pyruvate kinase were eliminated. The optimum pH and temperature for the reaction were 7.2 and 25 degrees C, respectively. Alpha-ketoglutarate and MnCl2 showed no marked effect on the reactions, MgSO4 and KCl were mildly stimulatory, and CuSO4 and ZnSO4 were very inhibitory. Penicillin N was optimal at a concentration of 0.07 mM. Specific ring-expansion activity reached its peak 13 hours after growth ceased and then disappeared rapidly.

Acremonium↗

Tetronic acid derivatives from Aspergillus panamensis. Production, isolation, characterization and biological activity.

When Aspergillus panamensis, CBS 120.45, was grown on a medium with high carbon and low nitrogen content, it was found to produce six antibiotically active metabolites. All compounds were identified as tetronic acid derivatives; three of them were found to be identical with gregatins A, B, and D, previously reported as metabolites of Cephalosporium gregatum. The other three compounds are new. All six compounds show antibacterial activity towards Gram-negative and Gram-positive bacteria. Five of the antibiotics exhibit inhibitory effects on the macromolecular syntheses in cells of the ascitic form of EHRLICH carcinoma (ECA) of mice.

Animals↗

Microbiological ring expansion of penicillin N.

A mutant of Cephalosporium acremonium producing high amounts of penicillin N was isolated. This antibiotic was purified and characterized. It was possible to convert this penicillin N to deacetoxycephalosporin C enzymatically. The reaction could be carried out with enzyme systems prepared from C. acremonium mutants producing either no beta-lactam antibiotics or excreting only penicillin N. It was surprising that a high level of transformation capacity was just found in a cephem negative mutant which overproduces penicillin N. For that reason the inability of the latter mutant to produce cephem compounds cannot be explained by a functional block of the ring expanding enzyme complex. The enzyme preparations used to carry out this reaction were made by ether-treatment or sonication of C. acraemonium cells, or by submitting them to osmotic shock. The ring expanding enzyme system is strongly dependent on ATP and behaves as a 2-oxoglutarate dependent dioxygenase.

Acremonium↗

High performance liquid chromatography (HPLC) of natural products. IV. The use of HPLC in biosynthetic studies of cephalosporin C in the cell-free system.

A new cepham metabolite has been isolated from the filtered broth of Cephalosporium acremonium by high performance liquid chromatography (HPLC) and identified as 7 beta-(5-D-amino-adipamido)-3 beta-hydroxy-3 alpha-methyl-cepham-4 alpha-carboxylic acid (I). Pure penicillin N was prepared using HPLC in the analytical mode. When I was added in place of penicillin N as substrate for the cell-free biosynthetic of cephalosporin, no formation of deacetoxycephalosporin C (II) was observed. A synthetic cepham derivative, 7 beta-(5-D-aminoadipamido)-3-exomethylene-cepham-4 alpha-carboxylic acid (III) was also tested in the cell-free system as a possible intermediate. The compound III was shown to be an inhibitor of the ring expansion enzyme that converts penicillin N to deacetoxycephalosporin C.

Acremonium↗

Enhancement of cerulenin production by a natural zeolite, an ammonium ion-trapping agent.

Addition of a natural zeolite, a known ammonium ion-trapping agent, to a complex medium resulted in a several fold increase in cerulenin production by Cephalosporium caerulens. In the presence of zeolite, ammonium ion in the medium decreased, while mycelial growth increased to a small extent, and pH values remained constant. Small amounts of ammonium bicarbonate inhibited cerulenin production without affecting mycelial growth and pH values. It is thus assumed that zeolite stimulated cerulenin production by releasing the biosynthesis from the suppression by ammonium ions in C. caerulens.

Acremonium↗

Carbon catabolite regulation of the conversion of penicillin N into cephalosporin C.

Cephalosporin C biosynthesis by Cephalosporium acremonium was delayed until most glucose in the medium was used. Addition of increasing concentrations of glucose up to 55 g/liter decreased cephalosporin C biosynthesis but stimulated growth. Sequential formation of penicillin N (an intermediate in the cephalosporin C biosynthetic pathway) and cephalosporin C was found when the culture was developed synchronously. Little cephalosporin C formation was observed until most penicillin N had already been formed. The sequential formation of penicillin N and cephalosporin C was due to the sequential formation of the "penicillin N synthetase system" and the "cephalosporin C synthetase system". Cells grown in the presence of glucose showed an increased accumulation of penicillin N and clear reduction of the conversion of penicillin N to cephalosporin C. Resting cell studies indicated that the glucose effect was due to the repression of one or more of the enzymes converting penicillin N into cephalosporin C. Little inhibition by glucose of the activity of these enzymes, once formed, was observed. Glucose did not effect significantly the pool sizes of either precursor amino acids of cephalosporin (alpha-aminoadipic acid and valine) or methionine (an inducer of penicillin N and cephalosporin C biosynthesis). On the basis of these data it is suggested that glucose catabolism specifically represses the enzyme system converting penicillin N into cephalosporin C.

Cephalosporins↗

Enzymatic conversion of the unnatural tripeptide delta-(D-alpha-aminoadipyl)-L-cysteinyl-D-valine to beta-lactam antibiotics.

Incubation of the unnatural tripeptide delta-(D-alpha-aminoadipyl)-L-cysteinyl-D-valine (DLD-ACV) with a partially purified extract of Cephalosporium acremonium resulted in the production of deacetoxycephalosporin C. The extract contained isopenicillin N synthetase (cyclase) and deacetoxycephalosporin C synthetase (expandase) but no penicillin epimerase activity, and was incubated aerobically in the presence of the components of the cyclase and expandase reaction mixtures (Fe++, ascorbate, dithiothreitol, alpha-ketoglutarate and ATP). The reaction was sensitive to penicillinase, indicating penicillin N to be an intermediate. However, when ring expansion was prevented by omission of alpha-ketoglutarate and ATP, no penicillin N was detected.

Acremonium↗

Purification and characterization of cloned isopenicillin N synthetase.

Isopenicillin N synthetase (IPS) cloned from Cephalosporium acremonium has been isolated from transformed Escherichia coli and purified to homogeneity. The resulting, abundant, recombinant protein, whilst undergoing slightly different N-terminal processing to that observed for the fungally-derived protein, has identical kinetics for the conversion of LLD-aminoadipoyl-cysteinyl-valine to isopenicillin N. Recombinant IPS converts analogue substrates into unusual beta-lactam antibiotics in exactly the same way as the fungal protein.

Amino Acid Sequence↗

Synthesis and fungicidal activity of some 5-(2-aminoalkanoyl)-8-aryl-4(5H)-oxo-1,2,4-triazolo[4,3-b]-1,4,2, 6-dithiadiazine 6,6-dioxide derivatives.

Title compounds (5) were conveniently prepared from 5-aryl-3-mercapto-1,2,4-triazoles (1) and chloro-sulphonyl isocyanate (CSI) to give 8-aryl-4-(5H)-oxo-1,2,4-triazolo-[4,3-b]-1,4,2,6-dithiadiazine -6,6-dioxide (2). The Compound (2) and one suspension of 2-(benzyloxycarbonylamino) akanoic acid (3) in the presence of BOP reagent afforded Z-derivatives 8-aryl-5-(2-benzyloxycarbamoyl)alkanoyl-4(5H)-oxo- 1,2,4-triazolo[4,3-b]-1,4,2,6-dithiadiazine 6,6-dioxide (4). The Z-derivatives (4) gave aspected product (5) when treated with Pd-c 5% and formic acid in DMF. These compounds have been tested in vitro for their fungicidal activity against Aspergillus niger, Fusarium oxisporum and Cephalosporium saccharii and results were compared with their parent dithidiazines.

Antifungal Agents↗

Synthesis of some N1-phenyl-N3-[2-aryl/aryloxymethyl-1,3,4-oxa(thia)diazol-2-yl]sulphonyl ureas as potential pesticides.

N1-Phenyl-N3-aryl/aryloxymethyl[1,3,4-oxa(thia)diazol-2-yl]s ulphonyl ureas (4 and 7) were conveniently prepared from 2-aryl/aryloxymethyl- 1,3,4-oxa(thia)diazol-2-sulphonamide (3 and 6) and phenyl isothiocyanate. Sulphonamides were synthesised by the reaction of 2-aryl/aryloxymethyl-1,3,4-oxa(thia)diazol-2-sulphonyl chlorides (2 and 5) with ammonia. All the compounds have been tested for fungicidal activity against the fungal species Cephalosporium saccharii and Helminthosporium oryzae. The two species of the aquatic fungi viz. Saprolegnia parasitica and Achlya orion responsible for fish mycoses were tested and also found remarkably active. All the compounds were also tested for herbicidal activity against Digiteria ciliaris, Oryza sativa and Raphunus sativa.

Fungi↗

Microbial contamination of advanced life support (ALS) systems poses a moderate threat to the long-term stability of space-based bioregenerative systems.

Microbial contamination of terrestrial hydroponic plant-growing systems provides an effective analogue for studying microbial contamination issues for space-based advanced life support (ALS) systems. If aggressive root or foliar pathogens are introduced into ALS hydroponic systems, severe epidemics are probable. Greater than 80% of the reported outbreaks of root pathogens in terrestrial hydroponic systems describe epidemics caused by fungal species of Fusarium, Phytophthora, and Pythium. However, it is likely that a comprehensive sanitation and quarantine program for space-based ALS modules will prevent contamination by Phytophthora and Pythium spp. because these pathogens are general soilborne. However, Fusarium spp. are typically airborne, can grow saprophytically on diverse substrates, and have been common contaminants of American spacecraft. If comprehensive sanitation and quarantine programs are established for space-based ALS modules, then the threat of pathogen introductions into these systems will be significantly mitigated. Microbial contamination studies in spacecraft over the last 30 years indicate that a high diversity of bacteria, fungi, and actinomycetes are commonly carried on board probably via clothing, equipment, air currents during spacecraft handling and loading, food, and the astronauts themselves. Species of Alternaria, Aspergillus, Botrytis, Candida, Cephalosporium, Cladosporium, Fusarium, Mucor, Penicillium, Phoma, and Trichoderma were the most prevalent fungi recovered, and species of Bacillus, Escherichia, Klebsiella, Micrococcus, Pseudomonas, Staphylococcus, and Streptococcus were the most prevalent bacteria recovered from spacecraft. Most of these genera contain species that have been reported as plant pathogens. Strict quarantine procedures were not effective in preventing contamination of spacecraft during these missions. Research must be initiated to better understand how microorganisms interact with plants and animals in microgravity environments because microbial contamination of spacecraft and ALS modules cannot be avoided. An integrated pest management (IPM) program likely will be developed for managing plant disease outbreaks in space-based ALS systems.

Ecological Systems, Closed↗

Impact of industrial waste water effluents on mycoflora of the shore sediments of the 3rd oxidation pond, with reference to biosorption of heavy metals.

The third oxidation pond at 10th of Ramadan desert receives a number of industrial waste water effluents contaminated with the heavy metal ions Zn, Cd, Cu and Ni. The species diversity and fungal community structure of seven different sites at the onshore sediments and offshore were studied. Mycological analysis resulted in isolation of 3912 fungal colonies, 11.7% of this count were recovered from the onshore sediment sites (4 sites) whereas 88.3% were from the offshore sites (3 sites), in the desert. Fungal counts and species diversity at the onshore sites tend to increase with increasing distance far from the waste water input. A complete accordance was observed among the total fungal counts and species variabilities with organic matter content at each sampling site. This relationship was reversed in case of heavy metal contents with both counts and diversity. Seventeen fungal species belonging to seven genera were isolated from all sites under study. Aspergillus spp. constituted the majority of the isolates (51.7% of the total isolates), followed by Curvularia, Cephalosporium, and Humicola. Of nine isolated Aspergillus spp., A. humicola was the most dominant (37.4% of the total catch) and appeared at all polluted sites. Therefore, A. humicola was chosen to investigate its potential for heavy metals sorption from the contaminated waste water effluent. Four days old biomass pellets could sorb a large amount of heavy metals according to the following sequence: Zn>Cd>Cu>Ni ions. Agitation significantly increased Zn and Cd sorption, but not Cu and Ni. Heavy metals sorption took place at a wide pH range and particularly increased at alkaline pH (8-9).

Aspergillus↗

[The origin of cephalosporins].

The origin of cephalosporins is investigated. In 1945, Giuseppe Brotzu, who was the rector of the University of Cagliari in Sardina, Italy, isolated a cephalosporin-producing strain, Cephalosporium acremonium. Although as many as 48 cephalosporin derivatives have been developed in Japan, how a cephalosporin-producing organism was discovered is not widely known here. This article contains the first Japanese translation of Brotzu's Italian publication entitled "Ricerche su di un nuovo antibiotico (Research on a new Antibiotic)" and reveals how cephalosporin was developed, together with a cross reference to the first report of penicillin, a similar antibiotic compound, which was discovered by A. Fleming in 1928. Brotzu's brief academic and social background is also presented.

Anti-Bacterial Agents↗

[Fungal detection of American ginseng seeds from Beijing and northeast area in China].

OBJECTIVE: The quantity and genus of fungi on 13 samples of American ginseng seed from Beijing and Northeast area in China were detected. METHOD: Washing and surface sterilization were used to isolate the fungi existed on seed surface and inside of the seed, respectively. RESULT: The genera of the fungi in embryo and shell of the seeds mainly belonged to Fusarium spp., Alternaria spp., Penicilium spp., Cephalosporium sp., Rhizopus spp., and a small quantity of Mucor, Aspergillus, Tritroderma, Doratomyces and so on. The isolated fungal frequencies on the shell and in the embryo were 36%-100% and 40%-100% respectively. CONCLUSION: The fungal frequency of American ginseng seed was higher. There were significant differences among different seed samples, the main fungal genera and isolation frequency in the seed embryo were identical with the results from the seed shell.

Alternaria↗

Production and optimization studies of cephalosporin C by solid state fermentation.

Production of cephalosporin C employing a strain, Cephalosporium sp. NCIM 1039 under solid state fermentation was optimized. Different substrates like wheat bran, wheat grains, rice grains, barley and rice bran were studied to optimize the best substrate. Wheat bran showed the highest antibiotic yield. The physical and chemical parameters were optimized. The maximum productivity of cephalosporin C (750 U/g) was achieved by employing wheat bran and with optimized nutritional and process parameters such as potato starch as additive 1% w/w, urea as additive 1% w/w, incubation period of 7 days, incubation temperature at 30 degrees C, inoculum level 10% w/v, moisture content of solid substrate 80% and pH 7.0.

Acremonium↗

[Decolorization conditions for reactive brilliant blue KN-R by three fungi].

Seventeen strains with wideranging decolorization ability were screened from contaminated soil and were applied on the decoloring of dye wastewater. Three strains were selected due to their high decolorization capacity on azo dyes, anthraquinone dyes and triphenylmethane dyes. The three strains were named as Strain I, Strain II, Strain III. This three superior strains were identified as penicillium link (Strain I & strain II) and Cephalosporium corda (strain III). Using aqueous samples, the influences of several factors on decolorization were reported, such as pH, carbon source, temperature and so on. And then applying of these strains for treatment of actual wastewater have also been done. The results showed that the optimal pH, temperature and carbon source were 5-9, 18-37 degrees C and 0.5% respectively, under those conditions the decolorization removal rate was 70%. And these fungi have a good prospect for the treatment of dye wastewater.

Acremonium↗

Increased systemic efficacy of aphidicolin encapsulated in liposomes.

Aphidicolin, a tetracyclic diterpene antibiotic produced by Cephalosporium aphidicola, is under investigation as anti-cancer drug. Because of its poor solubility in water, it cannot be administered directly in vivo. Systemic application of aphidicolin glycinate or aphidicolin gamma-cyclodextrin complexes resulted in tumour growth inhibition but not in cures. To improve the pharmacokinetics, a liposomal preparation of aphidicolin was developed and tested in neuroblastoma-bearing (UKF-NB-3) mice. The loading capacity of these liposomes was limited. Therefore, 4.5 mg aphidicolin/kg body weight was the maximum aphidicolin dose that could be applied as liposomal preparation in this approach. Comparison of effects on tumour growth exhibited by aphidicolin liposomes (4.5 mg aphidicolin/kg) given for 15 consecutive days to those of gamma-cyclodextrin inclusion complexes (15 mg aphidicolin/kg) revealed comparable tumour growth inhibition, although aphidicolin concentrations were approximately 3-fold lower. This shows that liposomal encapsulation is a promising strategy for the improvement of systemic anti-cancer activity of aphidicolin.

Animals↗

Antifungal activity and mode of action of saponins from Madhuca butyracea Macb.

The antifungal activity of saponins isolated as a byproduct from the defatted cake of M. butyracea oil seed is reported. The inhibitory concentrations against plant pathogenic fungi ranged from 500 to 2000 ppm. Maximum sensitivity to saponins was shown by Penicillium expansum. Cephalosporium acrimonium, Helminthosporium oryzae and Trichoderma viride. The saponins caused leakage of cell components and underwent degradation by fungus, Trichoderma viride. The usefulness of saponins as antimycotic agents is discussed.

Antifungal Agents↗