Isolation and metabolite production by Penicillium roqueforti,P. paneum and P. crustosum isolated in Canada

Penicillium roqueforti, P. crustosum and P. paneum grow on ensiled grain and recycled feed unless properly treated. The former two species occur also on cut lumber in Canada. These are known to produce a number of secondary metabolites including roquefortine. In cooler dairy production areas, including Scandinavia and North America, cattle toxicosis has been associated with silage contaminated by these fungi. We collected strains associated with cow or cattle toxicoses. The principal metabolites were determined making use of a new extraction method and analysis combining HPLC, LC/MS/MS, and LC/NMR. Penicillium roqueforti and P. crustosum required amino acid nitrogen for metabolite formation and their toxins were formed under conditions of low oxygen (20–30% saturation). Production of roquefortine C occurred on depletion of the available nitrogen and penitrem A on depletion of carbon source. Yield was reduced by excess carbon. Medium osmotic tension (a_w) affected metabolite production by the two species differently. Penicillium paneum was associated with ill-thrift of dairy cows and P. roqueforti was associated with more serious symptoms. Our data suggest a physiological basis for the common occurrence of roquefortine C in silage without serious consequences and the alternative, the presence of roquefortine C and toxicoses. The strain isolated from lumber was the best producer of the toxins studied. This is the first report of the toxigenic potential of P. roqueforti and P. paneum from Canada.     

Cellulases from two Penicillium sp. strains isolated from subtropical forest soil: production and characterization

AIMS: To isolate new fungal strains from subtropical soils and to identify those that produce high cellulase activity. To select microbial strains producing thermostable cellulases with potential application in industry. METHODS AND RESULTS: The new strains Penicillium sp. CR-316 and Penicillium sp. CR-313 have been identified and selected because they secreted a high level of cellulase in media supplemented with rice straw. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis, isoelectric focussing and zymography showed that the studied strains secreted multiple enzymes that hydrolyse cellulose. Cellulase activity of Penicillium sp. CR-316, the strain showing higher production, was analysed. Optimum temperature and pH of carboxymethyl cellulase activity were 65 degrees C and pH 4.5, respectively. Activity remained stable after incubation at 60 degrees C and pH 4.5 for 3 h. CONCLUSIONS: Fungal strains that secrete high levels of cellulase activity have been characterized and selected from soil. The isolated strains have complex sets of enzymes for cellulose degradation. Crude cellulase produced by Penicillium sp. CR-316 showed activity and stability at high temperature. SIGNIFICANCE AND IMPACT OF THE STUDY: Two fungal strains with biotechnological potential have been isolated. The strains secrete high levels of cellulase, and one of them, Penicillium sp. CR-316, produces a thermostable cellulase, that makes it a good candidate for industrial applications.     

Cyclopiazonic acid production by submerged cultures of Penicillium and Aspergillus strains

Abstract 62 isolates of Penicillium and Aspergillus were screened for cyclopiazonic acid (CPA) production by surface and submerged culture on different media. The production of this mycotoxin was restricted to Penicillium camembertii group II (and its domesticated form P. camembertii ), P. griseofulvum , and Aspergillus flavus (and its domesticated form A. oryzae ). The best yield of CPA was obtained by a strain of P. griseofulvum , but several strains of P. camembertii group II were also good producers. Propionic acid (500 and 1000 mg/l medium) did not enhance the production of CPA. The best yields of CPA were obtained in submerged culture, but in some cases growth and CPA production only occured in surface culture. A simplified procedure for isolation of CPA is described.     

<Emphasis Type="Italic">Penicillium svalbardense</Emphasis>, a new species from Arctic glacial ice

During investigations of mycobiota in the coastal Arctic polythermal glaciers, different species of the ubiquitous genus Penicillium were isolated from the extreme subglacial environment. A group of Penicillium strains was obtained that did not belong to any known Penicillium species. This species was isolated in high numbers from the Kongsvegen subglacial ice and was not detected in the surrounding environment. A detailed analysis of secondary metabolite profiles, physiological and morphological characteristics, and partial β-tubulin gene sequences showed that the proposed new species Penicillium svalbardense is closely related but not identical to Penicillium piscarium and Penicillium simplicissimum. It differs in the production of secondary metabolites and in the morphological features of conidia and penicilli, and it is therefore described as a new species.     

Toxins from strains of <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis> isolated from buildings and other sources

In 2004, Scott et al. (Mycologia 2004; 96: 1095–1105) determined that there are four molecular species within P. chrysogenum, one of which (clade 4) was dominant in isolates in house dust in ~100 homes in southern Ontario, Canada. We collected additional strains from buildings across Canada and obtained some from DAOM. The large majority of our strains were in clade 4, with a modest number of strains in Clade 1. Because these strains came from across Canada, the dominance of clade 4 in buildings is apparently widespread. Most strains tested produced penicillin G, roquefortine C and unexpectedly, meleagrin in high yield. Additionally, there appeared to be strains differentiated by their ability to accumulate xanthocillin X. These studies allowed focused toxicity studies in vivo and with primary lung cell cultures to be undertaken on the basis of reliable information of the toxins that should be studied.     

Comparison of extracellular proteases produced by Aeromonas salmonicida strains, isolated from various fish species

Extracellular products (ECPs) of five typical and 25 atypical Aeromonas salmonicida isolates from various fish species and geographical locations were analysed by substrate specificity, inhibition of proteolytic activity and substrate SDS-PAGE. The type strains of Aer. salmonicida subsp. salmonicida and Aer. salmonicida subsp. achromogenes were included for comparison. The results indicated that the strains formed six protease groups. The proteases produced by the two type strains were of a different nature. All the typical strains belonged to one group and showed proteolytic activities comparable to P1 and P2 proteases. Three atypical (oxidase-negative) strains secreted a protease comparable to P1. With the exception of these three, all strains produced metallo-gelatinases. A metallo-caseinase (AsaP1) was detected in the ECP of subsp. achromogenes type strain and 10 of the atypical strains. A number of proteolytic components with different apparent molecular weights (AMWs) were identified. These include caseinases with AMWs of > 100, 80, 60 and 30 kDa and gelatinolytic components with different AMWs, including some with AMW higher than P1 and lower than P2. The protease production of the isolates was not found to be host specific.     

Optimization of fermentation conditions for production of xylanase by a newly isolated strain,Penicillium thiersii ZH-19

The objective of this study was to maximize production of xylanase by a newly isolated strain Penicillium thiersii ZH-19. Response surface methodology was employed to study the effects of significant factors such as pH, temperature, xylan concentration, and cultivation time, on the production of xylanase by Penicillium thiersii ZH-19. The optimal fermentation parameters for enhanced xylanase production were found to be pH 7.72, temperature 24.8°C, xylan 13.2 g l⁻¹ and the fermentation time 125.8 h. The model predicted a xylanase activity of 75.24 U ml⁻¹. Verification of the optimization showed that the maximum xylanase production reached 73.50 U mL⁻¹ in the flask experiments and 80.23 U mL⁻¹ in the scale of 15-L fermenter under the optimal condition.     

Comparison of pepsins isolated from porcine, bovine and Penicillium jathiuellum

1. The reactivities of pepsins, isolated from three different sources (porcine, bovine, and Penicillium jathiuelum), toward ester and peptide substrates were compared. 2. Porcine pepsin showed the highest activity followed by penicillopepsin with bovine pepsin being the least active. 3. The esterase activity of penicillopepsin was greater than that of porcine pepsin with bovine pepsin again showing the least activity. 4. The CD spectra indicate that porcine and bovine pepsin have similar conformations, even though bovine pepsin shows less ellipticity at 220 nm. 5. Penicillopepsin showed a completely opposite sign in the near-u.v. region of the CD spectrum. 6. The far-u.v. region of the CD spectrum of penicillopepsin strongly suggests a beta-sheet structure. 7. Previously reported X-ray crystallographic data suggest that porcine pepsin has a compact three-dimensional structure, while the structures of bovine and penicillopepsins are partially unfolded.     

Production and partial characterization of lipases from a newly isolated Penicillium sp. using experimental design

Aims:  The objective of this work was to investigate the lipase production by a newly isolated Penicillium sp . , using experimental design technique, in submerged fermentation using a medium based on peptone, yeast extract, NaCl and olive oil, as well as to characterize the crude enzymatic extracts obtained.
Methods and Results:  Lipase activity values of 9·5 U ml⁻¹ in 96 h of fermentation was obtained at the maximized operational conditions of peptone, yeast extract, NaCl and olive oil concentrations (g l⁻¹) of 20·0, 5·0, 5·0 and of 10·0 respectively. The partial characterization of crude enzymatic extract obtained by submerged fermentation showed optimum activity at pH range from 4·9 to 5·5 and temperature from 37°C to 42°C. The crude extract maintained its initial activity at freezing temperatures up to 100 days.
Conclusions:  A newly isolated strain of Penicillium sp . used in this work yielded good lipase activities compared to the literature.
Significance and Impact of the Study:  The growing interest in lipase production is related to the potential biotechnological applications that these enzymes present. New lipase producers are relevant to finding enzymes with different catalytic properties of commercial interest could be obtained, without using genetically modified organisms (GMO).     

Penicillium ochrochloron MTCC 517 chitinase: An effective tool in commercial enzyme cocktail for production and regeneration of protoplasts from various fungi

Penicillium ochrochloron MTCC 517 is a potent producer of chitinolytic enzymes. Novozyme 234, traditional enzyme cocktail for protoplast generation is not available in the market. So, new enzyme cocktail is prepared for protoplast formation from various filamentous fungi which consists of 5 mg ml⁻¹ lysing enzymes from Trichoderma harzianum, 0.06 mg ml⁻¹ β-glucuronidase from Helix pomatia and 1 mg ml⁻¹P. ochrochloron chitinase. The greatest number of protoplasts could be produced from most of the fungi in 0.8 M sorbitol and by incubation for about 2 h at 37 °C, but the number was decreased by incubation for more than 3 h. About twice as many protoplasts were produced from different species of fungi by involvement of P. ochrochloron chitinase than with combined commercial enzymes.     

Thiabendazole resistance and mutations in the β-tubulin gene of Penicillium expansum strains isolated from apples and pears with blue mold decay

Penicillium expansum is the causal agent of blue mold rot, a postharvest decay of stored fruits. This fungus also produces the mycotoxins patulin and citrinin. Control of P. expansum still relies mainly on the use of fungicides such as thiabendazole. Since its introduction, resistant strains have been reported. The aim of this work was to investigate the thiabendazole resistance and mutations in the β-tubulin gene of P. expansum strains isolated from apples and pears with blue mold decay from Spain. A total of 71 strains of P. expansum were scored for resistance to thiabendazole and the β-tubulin gene was sequenced. Out of 71 strains, 37 were sensitive and 34 were resistant to thiabendazole. Regarding the β-tubulin gene sequence, 10 different genetic types were determined, with a 99.7–100% similarity. When the amino acid sequence was deduced, five different amino acid sequences were found. All except one of the sensitive strains lacked mutations in the region sequenced. Of the 34 resistant strains, only eight had mutations that involved the residues 198 and 240. All the strains with mutations at position 198 always corresponded to resistant isolates. However, a high percentage of resistant strains had no mutations in the region of the β-tubulin gene sequenced, and so other mechanisms may be involved in thiabendazole resistance.     

Production and fungitoxic activity of Sch 642305, a secondary metabolite of <Emphasis Type="Italic">Penicillium canescens</Emphasis>

Production of fungitoxic extrolites was evaluated in culture filtrates of several isolates belonging to Penicillium canescens and P. janczewskii that showed some extent of inhibitory activity against the plant pathogenic fungus Rhizoctonia solani. In addition to griseofulvin and dechlorogriseofulvin that are already known in these species, curvulinic acid, previously unreported in Penicillium, was produced by all isolates assayed. Another extrolite recently characterized from a P. verrucosum strain by the name of Sch 642305 was detected in 5 isolates of P. canescens only. The purified compound completely inhibited mycelial growth of isolates of Rhizoctonia solani and other plant pathogenic fungi in␣vitro. The role of this extrolite as a possible biochemical determinant of antagonism toward plant pathogenic fungi, and implications concerning chemotaxonomy are discussed.     

Production of culmorin compounds and other secondary metabolites by Fusarium culmorum and F. graminearum strains isolated from Norwegian cereals

Twenty-three Fusarium culmorum and 21 F. graminearumisolates were studied for their ability to produce mycotoxins and other secondary metabolites. The strains were cultivated on rice, and the extracts analysed by gas chromatography mass spectrometry (GC-MS) after derivatization with pentafluoropropionic (PFP) reagent. Two F. culmorum strains formed nivalenol and its acetylated derivatives (chemotype II), while all F. graminearum and the otherF. culmorum isolates produced deoxynivalenol (DON) via 3-acetyldeoxynivalenol (3-acetyl-DON) (chemotype IA). 15-hydroxy-culmorin, followed by 5-hydroxy-culmorin were the main other metabolites produced F. culmorum, while 5-, 12- and an unidentified hydroxy-culmorin, suggested to be 14-hydroxy-culmorin, were the main metabolites of F. graminearum. The hydroxy-culmorin profile was found to be significantly different for the two Fusarium species. Minor amounts of about ten other hydroxy-culmorins, four hydroxy-culmorones and 3,13-dihydroxy-epiapotrichothecene were also detected in most cultures. Traces of sambucinol seemed to be present in some of the isolates, but were not detected in any significant amounts. The precursors in the biosynthetic sequence to 3-acetyldeoxynivalenol,7,8-dihydroxycalonectrin and 15-deacetyl-7,8-dihydroxycalonectrin,were detected in most cultures. We also report the assignment of both the ¹H and¹³C NMR data of 15-deacetyl-7,8-dihydroxycalonectrin, which has only been reported incorrectly before. This revised version was published online in August 2006 with corrections to the Cover Date.     

青霉菌聚半乳糖醛酸酶基因在毕赤酵母中的表达及酶学性质研究

筛选得到一株能分解果胶的青霉菌(Penicillium sp.),使用简并引物PCR和TAIL-PCR方法从该菌中克隆了一个聚半乳糖醛酸酶基因pgp1.pgp1基因全长1 225 bp,包含2个内含子,其cDNA全长1 104 bp,编码367个氨基酸和一个终止密码子,前18个氨基酸为信号肽序列.将pgp1基因连接pPIC9载体,在巴斯德毕赤酵母表达系统中进行了异源表达.在3L发酵罐水平,培养基中聚半乳糖醛酸酶活力达到700 U/mL.酶学性质测定表明,重组酶蛋白PGP1的最适pH为5.0,在pH4.0 -6.0下处理1h后,剩余酶活力超过90％;最适温度为38℃,以聚半乳糖醛酸为底物,PGP1的Km=(1.172±0.169)mg/mL,Vmax=(0.061±0.002) mg/min/mL.     

The biosynthesis of low-molecular-weight nitrogen-containing secondary metabolite-alkaloids by the resident strains of Penicillium chrysogenum and Penicillium expansum isolated on the board of the Mir space station

The analysis of the absorption spectra of the low-molecular-weight nitrogen-containing secondary metabolites--alkaloids--of 4 Penicillium chrysogenum strains and 6 Penicillium expansum strains isolated on board the Mir space station showed that all these strains synthesize metabolites of alkaloid origin (roquefortine, 3,12-dihydroroquefortine, meleagrin, viridicatin, viridicatol, isorugulosuvin, rugulosuvin B, N-acetyl-tryptamine, and a "yellow metabolite" containing the benzoquinone chromophore).     

Comparison of growth characteristics and roquefortin C production of<Emphasis Type="Italic">Penicillium roqueforti</Emphasis> from blue-veined cheese

The properties of 21 isolates ofPenicillium roqueforti from just as many commercial blue-veined cheeses, purchased from the Argentinean market (domestic and imported products) were comparatively examined. Isolates were investigated for their ability to grow at different temperatures, pH values and concentration of NaCl, as well as for their proteolytic and lipolytic activities, respectively. The potential of these strains to produce roquefortin in vitro, and the actual levels of roquefortin in 10 of these cheeses were analysed by TLC. All strains showed similar growth properties in aspects of salt concentration and pH-value of the medium, and all grew well at 10 °C. Only four strains showed proteolytic activity on casein agar, while all strains were lipolytic on trybutirin agar. After incubation at 25 °C for 16 days, all strains produced roquefortin in Yeast Extract Sucrose (25.6–426.7 μg/g) and in reconstituted (10%) sterile skim milk (26.9–488 μg/g). Roquefortin at >0.1 μg/g was also found in 9 out of 10 analysed samples of blue-veined cheeses (8 from Argentine, 1 from Spain), with a maximum value 3.6 μg/g. During the ripening process of blueveined cheese, production of roquefortin seems to be unavoidable. Care should be taken to select strains with low toxin production characteristics, to minimize potential health risks. Roquefortin C production byP. roqueforti in vitro was not correlated with roquefortin C levels found in cheese. Financial support: Research grants from the National University of Quilmes, Argentina     

Polymorphism of hyaluronidase in serum from man, various mouse strains and other vertebrate species revealed by electrophoresis

Polymorphism of hyaluronidase (EC 3.2.1.35) was detected in the serum from 6 out of 20 vertebrate species by electrophoretic analysis. Electrophoresis was performed on a hyaluronan-containing polyacrylamide gel, that visualizes hyaluronidase activity upon incubation at acid pH. No hyaluronidase activity was detected in the sera of horse, swine, cattle, goat, sheep, rabbit, chicken, Triton alpestris, Triton palmatus, Triton vulgaris, pleurodeles, axolotl, eel and dog-fish. The 6 positive sera were from man, mouse, rat, Syrian hamster, dog and Triton cristatus. In each of these species, the electrophoretic banding pattern of hyaluronidase was different, as was the activity per unit volume of serum. Furthermore, in mice, the 12 strains analyzed could be divided into 3 groups, containing the following numbers of hyaluronidase bands; 8 (BALB/c/J, BALB/c/By, ICFW, SW, XVIInc/Z), 5 (DBA/2 Mrc Ico, DBA/2 Mrc Ico nu/nu, B10.D2/nSn), and 1 (C57B/Rho Ico, C57BL/6/By, C57BL/6/J Ico, C57BL/6/J Ico nu/nu). Human serum generally displayed only 1 band, although there was a 2nd faint band in a few cases and a 3rd in 1 case. Rat serum displayed 4 bands, Syrian hamster serum, 3, and dog and Triton cristatus serum, 1.