Penicillium griseofulvum F1959, high-production strain of pyripyropene a, specific inhibitor of acyl-CoA: cholesterol acyltransferase 2

Acyl-coenzyme A: cholesterol acyltransferase (ACAT) catalyzes cholesterol esterification and plays an important role in the intestinal absorption of cholesterol, hepatic production of lipoproteins, and accumulation of cholesteryl ester within cells. During the course of screening to find ACAT inhibitors from microbial sources, the present authors isolated pyripyropene A from Penicillium griseofulvum F1959. Pyripyropene A, an ACAT2-specific inhibitor, has already been produced from Aspergillus fumigatus. Yet, Aspergillus fumigatus is a pathogen and only produces a limited amount of pyripyropene A, making the isolation of pyripyropene A troublesome. In contrast, Penicillium griseofulvum F1959 was found to produce approximately 28 times more pyripyropene A than Aspergillus fumigatus, plus this report also describes the ideal conditions for the production of pyripyropene A by Penicillium griseofulvum F1959 and its subsequent purification.     

Production of extracellular lipases by Penicillium cyclopium purification and characterization of a partial acylglycerol lipase

Penicillium cyclopium, grown in stationary culture, produces a type I lipase specific for triacylglycerols while, in shaken culture, it produces a type II lipase only active on partial acylglycerols. Lipase II has been purified by ammonium sulfate precipitation and chromatographies on Sephadex G-75 and DEAE-Sephadex. The enzyme exists in several glycosylated forms of 40-43 kDa, which can be converted to a single protein of 37 kDa by enzymatic deglycosylation. Activity of lipase II is maximal at pH 7.0 and 40 degrees C. The enzyme is stable from pH 4.5 to 7.0. Activity is rapidly lost at temperatures above 50 degrees C. The enzyme specifically hydrolyzes monoacylglycerols and diacylglycerols, especially of medium chain fatty acids. The sequence of the 20 first amino acid residues is similar to the N-terminal region of P. camembertii lipase and partially similar to lipases from Humicola lanuginosa and Aspergillus oryzae, but is different from Penicillium cyclopium lipase I. However, it can be observed that residues of valine and serine at positions 2 and 5 in Penicillium cyclopium lipase II are conserved in Penicillium expansum lipase, of which 16 out of the 20 first amino acid residues are similar to Penicillium cyclopium lipase I.     

Cross-point determination of Penicillium conidia—Characterization of closely related fungi

The cross-points of conidia of Penicillium piceum were determined by partition in aqueous two-phase systems. No differences in cross-points or partition behaviour were registered for conidia subjected to u.v. radiation, 95°C dry heat or 120°C wet heat, compared with untreated conidia. The cultivation of fungi on various types of substrates affected the surface characteristics and thereby the cross-points of the conidia. When the method was applied to three closely related and morphologically similar isolates in the P. viridicatum group, significant differences were observed between the range of cross-points for conidia of P. crustosum, P. camembertii group II and P. camembertii group III grown on various media.     

Enumeration and identification of Aspergillus group and Penicillium species in poultry feeds from Argentina

A total of 180 samples of poultry feeds were collected during 1996 and 1997 from different factories in the south of the province of Córdoba-Argentina. They were examined for the occurrence of Penicillium spp. and Aspergillus group species. Likewise, the capacity to produce aflatoxins by the Aspergillus section flavi group was determined. The predominant species of Aspergillus were A. flavus and A. parasiticus. For Penicillium spp., P. brevicompactum, P. purpurogenum and P. oxalicum were identified. Less frequently isolated were A. candidus, A. fumigatus, A. niger, A. orizae, A. parvulus, A. tamarii, A. terreus, and P. expansum, P. funiculosum, P. minioluteum, P. pinophylum, P. restrictum, P. variabile and others. The mean value counts ranged from 1 × 103 to 9.5 × 104 CFU/g for the Aspergillus spp. and from 1.2 × 103 to 2.5 × 105 CFU/g for the Penicillium spp. When cultured on autoclaved rice kernels for 1 week in the dark at 25°C, mycotoxin production by strains of A. flavus was as follows: 21 of the 45 assayed strains (47%) produced aflatoxins. From them, 24% of the isolates produced AFB1 and AFB2 with levels from 181 to 14 545 and 6 to 3640 μg/kg respectively. Only 10 strains produced AFB1 with levels from 10 to 920 μg/kg. Fifty percent of the A. parasiticus strain was toxicogenic; six aflatoxicogenic profiles were identified. Only 10% of the strains produced all of the aflatoxins. These results showed that a potential exists for the production of mycotoxins by the Aspergillus section flavi and the Penicillium spp. They also suggested an association of mycotoxicosis with poultry feeds in Argentina. This revised version was published online in June 2006 with corrections to the Cover Date.     

Production and Some Properties of a New Type of Acid Carboxypeptidase of Penicillium Molds

Among some 38 strains of the genus Penicillium we investigated seven wild-type strains (P. daleae IFO-6087, P. frequentans AHU-8328, P. funiculosum IAM-7013, P. janthinellum IFO-8070, IAM-7026, P. lividum IAM-7200, and P. oxalicum AHU-8336) that were found to be excellent strains for a new type of acid carboxypeptidase production in a surface koji culture at 25 C. The production of acid carboxypeptidase was determined in various culture conditions in a koji culture. The maximum yields of acid carboxypeptidase were obtained by P. janthinellum IFO-8070. Partial purification and isolation of the acid carboxypeptidase from strains of Penicillium were performed with gel filtration on Bio-Gel P-100. Characterization studies indicate that the acid carboxypeptidases from P. daleae IFO-6087, P. funiculosum IAM-7013, P. janthinellum IFO-8070, and P. oxalicum AHU-8336 have some properties similar to those of the enzyme of Aspergillus saitoi with regard to the hydrolysis of several peptides at acidic pH range but have other slightly different properties with regard to stability, pH optima, inhibitors, and molecular weights.     

A monoclonal antibody specific for conidia and mycelium wall layer of Penicillium and Aspergillus.

A monoclonal antibody was obtained from BALB/c mice immunized with Penicillium frequentans mycelium. The specificity of the antibody was evaluated by enzyme-linked immunosorbent and indirect immunofluorescence assays against the same mycelium. This IgM antibody cross-reacted with various strains of the Penicillium and Aspergillus genera. By indirect immunofluorescence assays, the antibody was able to stain about 10% of Penicillium and Aspergillus conidia, but major part of conidia did not absorb the fluorescence-labeled antibody before swelling. During germination of P. frequentans conidia, the germ tube wall which constitutes a continuation of an inner wall layer was also stained. During germination of P. griseofulvum, the protrusion of the germ tube wall was not always recognized by the antibody because the germ tube wall was constituted by a continuation of an outer spore wall layer. The study of the staining patterns of the spores and the protrusions suggests that the antibody specifically recognizes an antigen of the inner spore wall layer. The monoclonal antibody reacts with extracellular galactomannans produced by genera Aspergillus and Penicillium but is not directed against beta-(1,5)-linked galactofuranose units.     

Penicillium notatum 1 a new source of dextranase

Summary Two hundred and fifteen fungal strains were screened for extracellular dextranase production with a diffusion plate method. The best enzymatic activity (12–19 DU ml^–1) was achieved byPenicillium notatum 1, a species for which the dextranase productivity has not yet been published. Some of the parameters affecting enzyme production have been standardized. The enzyme in crude state was relatively stable, its maximal activity was at 50°C and at pH 5.0. Conidia of the selected strain were mutagenized, and isolated mutants were tested for production of dextranase in submerged culture. The most active mutant,P. notatum 1-I-77, showed over two-times higher dextranase activity than the parentP. notatum 1     

Assimilation of peptides and amino acids and dissimilation of lactate during submerged pure cultures of Penicillium camembertii and Geotrichum candidum

The behavior of Penicillium camembertii and Geotrichum candidum growing in submerged pure cultures on simple (glutamate) or complex (peptones) substrates as nitrogen and carbon sources and an lactate as a second carbon source was examined. Similar to the behavior previously recorded on a simple substrate (glutamate), a clear differentiation between the carbon source and the energy source was also shown on peptones and lactate during P. camembertii growth, since throughout growth, lactate was only dissimilated, viz., used for energy supply by oxidation into CO2, whereas peptides and amino acids from peptones were used for carbon (and nitrogen) assimilation. Because of its deaminating activity, G. candidum preferred peptides and amino acids to lactate as energy sources, in addition to being assimilated as carbon and nitrogen sources. From this, on peptones and lactate, G. candidum grew faster than P. camembertii (0.19 and 0.08 g/l/h, respectively) by assimilating the most readily utilizable peptides and amino acids; however, owing to its lower proteolytic activity, the maximum biomass was lower than that of P. camembertii (3.7 and 5.5 g/l, respectively), for which continuous proteolysis and assimilation of peptides were shown.     

Mycotoxin-Producing Potential of Mold Flora of Dried Beans

To evaluate the potential for mycotoxin production by molds in dried beans, the mold flora of 114 samples was determined both before and after surface disinfection of the beans with 5% NaOCl. Surface disinfection substantially reduced mold incidence, indicating that contamination was mainly on the surface. The flora, both before and after disinfection, was dominated by species of the Aspergillus glaucus group, the toxicogenic species A ochracues, Penicillium cyclopium, and P. viridicatum, and species of Alternaria, Cladosporium, and Fusarium. The toxicogenic species Aspergillus flavis, A. versicolor, Penicillium Citrinum, P. expansum, P. islandicum, and P. urticae were encountered less frequently. Of 209 species of Aspergillus and Penicillium screened for mycotoxin production on sterile rice substrate, 114 produced one or more of the following mycotoxins: A. flavus, aflatoxins; A. ochraceus, ochratoxins; A. nidulans, A. unguis, and A. versicolor, sterigmatocystin; P. cyclopium, penicillic acid; P. citrinum and P. viridicatum, citrinin; P. urticae, patulin and griseofulvin. Sterigmatocystin production by A. unguis is reported for the first time.     

Mites and fungi in heavily infested stores in the Czech Republic

Toxigenic and allergen-producing fungi represent a serious hazard to human food and animal feed safety. Ninety-four fungal species were isolated from mite-infested samples of seeds taken from Czech seed stores. Fungi were isolated from the surface of four kinds of seeds (wheat, poppy, lettuce, and mustard) and from the gut and external surface of five species of mites (i.e., Acarus siro L., 1758, Caloglyphus rhizoglyphoides (Zachvatkin, 1973), Lepidoglyphus destructor (Schrank, 1781), Tyrophagus putrescentnae (Schrank, 1781) and Cheyletus malaccensis Oudemans 1903) separately. Multivariate analysis of fungi complex composition showed that the frequency of fungal was species significantly influenced by the kind of seed. Fungal frequencies differed between mites gut and exoskeleton surface and between the surfaces of mites and seeds. Three groups of fungal species were recognized: 1) mite surface-associated fungi: Penicillium brevicompactum, Alternaria alternata, and Aspergillus versicolor; 2) mite surface- and seed-associated fungi: Aspergillus niger, Penicillium crustosum, Penicillium aurantiogriseum, Penicillium chrysogenum, and Aspergillus flavus; and 3) seed-associated fungi: Cladosporium herbarum, Mucor dimorphosporus f. dimorphosporus, Botrytis cinerea, Penicillium griseofulvum, and Eurotium repens. Mite-carried species of microfungi are known to produce serious mycotoxins (e.g., aflatoxin B1, cyclopiazonic acid, sterigmatocystin, ochratoxin A, and nephrotoxic glycopeptides) as well as allergen producers (e.g., A. alternata and P. brevicompactum). Storage mites may play an important role in the spread of some medically hazardous micromycetes. In addition, these mite-fungi associations may heighten the risk of occurrence of mycotoxins in food and feed stuffs and cause mixed contamination by fungal and mite allergens.     

Mycoflora and ochratoxin-producing strains of Aspergillus section Nigri in wine grapes in Argentina

AIMS: The aims of this work were to evaluate the mycoflora and to identify the species of Aspergillus with the potential to produce ochratoxin A (OA) from different wine grape varieties from Mendoza, Argentina. Likewise, the capacity to produce OA by Aspergillus section Nigri was studied. METHODS AND RESULTS: Fifty samples of wine grapes were obtained from a winery of Mendoza province, Argentina. The surface-disinfection method was used for mycoflora determination using the medium dichloran 18% glycerol agar (DG18). Alternaria, Aspergillus and Penicillium were identified at species level. OA production was tested in 63 strains belonging to section Nigri. Alternaria genus was the most frequent (80% of the samples) followed by Aspergillus (70%). Alternaria alternata was the only specie identified from the Alternaria genus, followed by A. niger var. niger, A. flavus among others. From Penicillium genus, P. crysogenum was the most frequent specie. From 63 strains of Aspergillus section Nigri, 41.3% were OA producers. The levels of produced toxin ranged from 2 to 24.5 ng ml-1 of culture medium. CONCLUSIONS: The presence of ochratoxigenic strains of Nigri section in this substrate suggests that they may be an important source of OA in grapes from tropical and subtropical zones. Therefore, the industry should work further to diminish the growth of these fungi and mycotoxins formation in grapes, with the aim to reduce OA content in wine products. SIGNIFICANCE AND IMPACT OF THE STUDY: The wine grape contamination with A. alternata and Aspergillus section Nigri was significant.     

Penicillium persicinum, a new griseofulvin, chrysogine and roquefortine C producing species from Qinghai province, China

A unique Penicillium isolate from Chinese soil with terverticillate penicilli and ellipsoidal to cylindrical smooth-walled conidia, produces, in addition to the common metabolite ergosterol, copious amounts of an unknown peach-red pigment and the following secondary metabolites: griseofulvin, dechlorogriseofulvin, lichexanthone, roquefortine C, roquefortine D, chrysogine, 2-pyrovoylaminobenzamide, 2-acetyl-quinazolin-4(3H)-one. This isolate, CBS 111235, is described as Penicillium persicinum sp. nov., which belongs to subgenus Penicillium section Chrysogena but is morphologically similar to P. italicum. On the basis of the production of secondary metabolites it resembles P. griseofulvum and P. coprophilum. Sequence data using part of the beta-tubulin gene showed that it is phylogenetically related to P. chrysogenum and P. aethiopicum in section Chrysogena with which it shares both secondary metabolites and ability to grow at 37 degrees C.     

近岸污染指示半知菌灰黄青霉(Penicillium griseo fulvum)的分子检测

【目的】通过分子方法检测近海污染环境优势种灰黄青霉,并为由此而推断污染程度做准备。【方法】根据GenBank中青霉属不同种和相近属种的ITS序列差异和灰黄青霉特有的IAO序列,设计了污染区优势种灰黄青霉的特异性引物AS1/RS4和IAO1/IAO2,建立相应的特异探针检测体系。通过PCR和套式PCR技术,分析比较两对特异序列检测灰黄青霉的差异。【结果】建立的分子检测体系可以排除其它近似或相关菌株干扰,从环境中扩增到目的基因片段。利用引物AS1/RS4作为核酸探针,通过套式PCR菌株DNA的检测灵敏度可达到10fg/μL,当仅有10个数量级分生孢子时即可检测出,从沉积物中检测灵敏度为102个数量级孢子/0.25g。特异酶基因IAO1/IAO2检测灵敏度较前者稍低。【结论】利用特异序列作为探针检测污染环境优势种灰黄青霉的方法可行,在一定范围内,灰黄青霉的出现频率及数量对污染程度有较好的指示作用。     

Identification of a novel polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) gene required for the biosynthesis of cyclopiazonic acid in Aspergillus oryzae

Cyclopiazonic acid (CPA) is a mycotoxin produced by several strains of Penicillium and Aspergillus species. Aspergillus oryzae strains used in fermented foods do not produce CPA; however, several wild-type A. oryzae strains produce CPA. Here, we identified a novel polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) gene involved in CPA production by comparing the telomere-adjacent region of a CPA-producing strain (A. oryzae NBRC 4177) with that of a nonproducing strain (A. oryzae RIB40). NBRC 4177 has an additional 17-18-kb sequence beyond the region corresponding to the telomere repeat in RIB40 and this additional regions contains 3' region of the PKS-NRPS gene, while RIB40 has only the 5' region of the PKS-NRPS gene. Gene disruption of the PKS-NRPS gene in NBRC 4177 resulted in elimination of CPA production. Thus, the PKS-NRPS gene is required for CPA biosynthesis, and the truncation of this gene is presumed as one of the determinants of CPA nonproductivity in A. oryzae RIB40.     

Isolation and screening of glucose oxidase producing microorganisms from natural sources.

Among 1486 mould strains isolated from natural sources (screened for extracellular glucose oxidase) only 119 (Aspergillus and Penicillium) showed this enzyme activity. As the best glucose oxidase producer, A. niger 0-1 was isolated from decaying tree. The dynamics of glucose oxidase synthesis in A. niger 0-1 during its culture by submerged method show that the intracellular activity of this enzyme is 10-times higher than its extracellular level. Some properties of the crude glucose oxidase preparation, isolated from the postculture liquids by lyophilization, were examined.     

Effect of Mycelial Forms on Citric Acid Fermentation in Submerged Mold Culture

A number of reports have been published on the production of citric acid by submerged mold culture. Most of them, however, have laid stresses on the effects of chemical factors, such as metal ions, nitrogen sources, potassium ferrocyanide and methanol, and very little has been reported on the effect of other factors.

The form of mycelia of mold changes depending on the physical characters cf broth, and it was found that Aspergillus niger 93A, which showed a constant activity of citric acid production, could increase its acid production activity when mycelial forms were controlled to filamentous by adding suitable non-ionic surface active agents to the broth.     

Surface hydrophobicity, viability and efficacy in biological control of Penicillium oxalicum spores produced in aerial and submerged culture

The surface hydrophobicity, viability and biocontrol ability of Penicillium oxalicum spores, produced either in aerial or submerged culture, were characterized. A phase distribution test showed that spores produced in both methods of culture were highly hydrophobic, but those produced in aerial culture were more hydrophobic. Spores stored fresh at either 4 or 25 degrees C retained a high viability (80%) after 27 weeks of storage, although aerial spores survived better. Freeze-drying severely affected viability, especially of submerged spores. Biocontrol ability against Fusarium oxysporum f. sp. lycopersici was studied in the growth chamber. Aerially- produced spores were more effective than submerged ones. Aerially-produced P. oxalicum spores appeared to have more advantages than those produced by submerged culture, in relation to both viability and efficacy. These results demonstrate that physiological changes occur depending on production conditions which significantly influences quality of the biocontrol agent.     

Bioremediation of aflatoxins by some reference fungal strains.

Aspergillus parasiticus RCMB 002001 (2) producing four types of aflatoxins B1, B2, G1, and G2 was used in this study as an aflatoxin-producer. Penicillium griseofulvum, P. urticae, Paecilomyces lilacinus, Trichoderma viride, Candida utilis, Saccharomyces cerevisiae as well as a non-toxigenic strain of Aspergillus flavus were found to be able to exhibit growth on aflatoxin B1-containing medium up to a concentration of 500 ppb. It was also found that several fungal strains exhibited the growth in co-culture with A. parasiticus, natural aflatoxins producer, and were able to decreased the total aflatoxin concentration, resulting in the highest inhibition percentage of 67.2% by T viride, followed by P. lilacinus, P. griseofulvum, S. cerevisiae, C. utilis, P. urticae, Rhizopus nigricans and Mucor rouxii with total aflatoxin inhibition percentage of 53.9, 52.4, 52, 51.7, 44, 38.2 and 35.4%, respectively. The separation of bioremediation products using GC/MS revealed that the toxins were degraded into furan moieties.     

A new Penicillium echinulatum strain with faster cellulase secretion obtained using hydrogen peroxide mutagenesis and screening with 2-deoxyglucose

Aims: The aim of this study is to improve cellulase production and secretion by Penicillium echinulatum using mutagenesis and selection in association with microfermentation and microanalysis methods. Methods and Results: A new genetic variant was isolated from strain 9A02S1 and named S1M29. It was obtained by mutagenesis with H₂O₂ and two screening steps, which involved selection in Petri dishes using the medium supplemented with 2‐deoxyglucose and microfermentations in submerged culture. The mutant showed higher cellulase productivity than 9A02S1 based on the Filter Paper Activity assay and endoglucanase; the peak activities for these enzymes were reached significantly faster than for the parent strain. Conclusions: The mutant obtained after mutagenesis and selection could produce and secrete cellulase faster than the parent strain. Significance and Impact of the Study: Mutagenesis followed by selection is a useful tool for rapidly generating new cellulase‐producing phenotypes in fungi. Faster production and higher titers of cellulases in mutant strains contribute to reduce the production costs for enzymatic complexes that hydrolyse lignocellulose residues and form fermented syrups, thus contributing to the economic production of bioethanol.     

Production of feruloyl/ρ-coumaroyl esterase activity by Penicillium expansum, Penicillium brevicompactum and Aspergillus niger

Extracellular esterase production by Penicillium expansum, Penicillium brevicompactum and Aspergillus niger was determined in both liquid and solid-state culture. Methyl ferulate was used as the main carbon source in liquid culture whereas wheat bran and sugar beet pulp were used in solid-state culture. Extracted enzyme for each fungus showed activity in the presence of ONP butyrate, methyl ferulate, methyl coumarate and two 'natural'feruloylated carbohydrate esters. Higher enzyme recoveries were obtained using wheat bran in solid-state culture. Higher levels of feruloyl esterase activity were recovered from P. expansum on all feruloylated substrates than from P. brevicompactum or A. niger. Using ONP butyrate as substrate the pH and temperature optima for the esterases of both Penicillium spp. were 6.0 and 25–30°C. Aspergillus niger esterase activity showed a broader temperature range with an optimum at 40°C.