Production of lipase free of citrinin by Penicillium citrinum

Lipase (Glycerol ester hydrolase E.G. 3.1.1.3) from a Brazilian strain of Penicillium citrinum free of the mycotoxin citrinin has been investigated. Citrinin production was inhibited by using culture medium containing olive oil, soybean oil and corn oil as carbon sources. Potassium concentration and pH play an important role in citrinin production. Potassium concentration lower than 30 mM and pH below 4.5 inhibited the mycotoxin production. P. citrinum produced lipase free of extraneous proteins and citrinin when cultured using, as nitrogen source, ammonium sulphate (lipase activity of 7.88 U/mg) and yeast extract (lipase activity of 4.95 U/mg) with olive oil as carbon source. This data is relevant to the larger scale production of lipases for food technology applications, from Penicillium citrinum.     

A chemically-defined medium for production of compactin by Penicillium citrinum

A mutant strain of Penicillium citrinum grown in a chemically-defined production medium, yielded 145 mg compactin l^–1. The medium also facilitated spectrophotometric analysis of compactin. Addition of KH₂PO₄in the production medium did not increase the compactin production, while addition of a surfactant, Tween 80, increased compactin to 175 mg l^–1. Inoculation with 10⁷ spores ml^–1 and initial pH of 6.5–7 were the most suitable for compactin production.     

Production of a novel syrup containing neofructo-oligosaccharides by the cells of Penicillium citrinum

A novel syrup containing neofructo-oligosaccharides was produced from sucrose (Brix 70) by whole cells of Penicillium citrinum. The efficiency of fructo-oligosaccharides production was more than 55% and those of the main carbohydrate components, 1-kestose (Fruf 21Fruf 21 Glc), nystose (Fruf 21Fruf 21 Fruf 21 Glc) and neokestose (Fruf 26 Glc12 Fruf), were 22, 14 and 11%, respectively.     

Production of patulin and citrinin by <Emphasis Type="Italic">Penicillium expansum</Emphasis> from British Columbia (Canada) apples

Twenty-four isolates of Penicillium expansum Link from British Columbia (Canada) apples were cultured in yeast-extract sucrose (YES) at 25°C for 28 days to investigate production of patulin and citrinin. These isolates proved to be potent producers of citrinin, patulin, or in most cases, both mycotoxins. In every isolate, citrinin, patulin, or both compounds were produced at levels as high as 565 μg/mL (mean 269 μg/mL) and 100 μg/mL (mean 31 μg/mL), respectively. Of the 24 isolates, 4 produced citrinin only, and 2 produced patulin only. Overall, 83% of the isolates formed patulin and 91% formed citrinin. YES broth proved to be an effective medium for patulin and citrinin production. Other workers have noted that production of these mycotoxins in culture often presages production in fruits, so these results might help Canadian fruit processors evaluate and minimize mycotoxin levels in their products.     

Continuous production of neo-fructooligosaccharides by immobilization of whole cells of <Emphasis Type="Italic">Penicillium citrinum</Emphasis>

To produce neo-fructooligosaccharides (neo-FOSs) in a 500ml continuous packed-bed reactor using whole cell immobilization of Penicillium citrinum KCCM 11663, the optimum reaction conditions were 50 °C, pH 6 with 600 g sucrose l^-1 being fed as substrate at 1.3 ml min^-1 . Under these conditions, the maximum neo-FOSs production was 49 g l^-1. In a packed-bed reactor, continuous production of neo-FOSs was possible for 50 d indicating a potential for industrial production. Revisions requested 6 September 2004/14 October 2004; Revisions received 7 October 2004/29 November 2004     

Penicillium marneffei: types and drug susceptibility

The PCR fingerprints of 30 Penicillium marneffei isolates from Chiang Rai in northern Thailand and Bangkok in central Thailand were studied through use of single-nucleotide primers (GACA)₄ and the phage M13 core sequence. Discrimination of fingerprint patterns was based on differences in the number of major bands. The P. marneffei isolates were divided into four types, i.e., A, B, C, and D. Type A was found in two isolates from Chiang Rai (6.7%). Types B and C respectively were found in two (6.7%) and one (3.3%) isolates from Bangkok. The predominate type D (83.3%) was found in isolates obtained from Chiang Rai and Bangkok. The PCR fingerprinting method was found to be useful for the epidemiological study of P. marneffei, a dimorphic opportunistic fungus and an emerging pathogen in the HIV pandemic. In vitro drug susceptibility testing by broth macrodilution to four antifungal agents against the yeast form ofP. marneffei was performed. The MIC ranges for amphotericin B, fluconazole, itraconazole, and ketoconazole were 0.125–0.5, 4.0–8.0, <0.032, and <0.125 μg/ml respectively. This revised version was published online in June 2006 with corrections to the Cover Date.     

Novel cellulases from an extremophilic filamentous fungi <Emphasis Type="Italic">Penicillium citrinum</Emphasis>: production and characterization

The enzymatic hydrolysis of cellulose has potential economical and environment-friendly applications. Therefore, discovery of new extremophilic cellulases is essential to meet the requirements of industry. Penicillium citrinum (MTCC 6489) that was previously isolated from soil in our laboratory, produced alkali tolerant and thermostable cellulases. Endoglucanase and filter paper activity hydrolase (FPAse) production of P. citrinum were studied using wheat bran substrate in solid state and submerged culture. Zymogram analysis of endoglucanase revealed the presence of two isoforms differing in molecular weight. One of them was 90 kDa and other one was 38 kDa. Partially purified endoglucanase showed two different peaks at pH 5.5 and 8.0, respectively, in its pH optima curve. But FPase showed only one peak (at pH 6.5) in its pH optima curve. Cellulase of P. citrinum is thermostable in nature. The present work reports for the first time, the alkali stable cellulase from alkali tolerant fungus Penicillium citrinum. Thermostable endoglucanase from P. citrinum may have potential effectiveness as additives to laundry detergents.     

Effect of triton X-100 on compactin production from<Emphasis Type="Italic">Penicillium citrinum</Emphasis>

Glucose alone was found to be the most effective carbon source for producing compactin. An initial glucose concentration of 40 g/L gave the highest compactin concentration of 250 mg/L. Among the various nitrogen sources, when 5 g/L of pharmamedia and soybean meal as the sole nitrogen source were used, respectively, the compactin concentration was higher than 250 mg/L. Especially, in the case of the mixture of 6 g/L of pharmamedia and 8 g/L of soybean meal, the compactin concentration was 400 mg/L. To select the best surfactant for effective compactin production, various surfactants were investigated. When Triton X-100 was used, the maximum compactin concentration was 445 mg/L. With the initial concentration ranging from 1.5 to 2.0 g/L, the compactin concentration was the highest at 465–450 g/L. The cell concentration was similar to that of the control without the addition of Triton X-100. On the other hand, when the above 4.0 g/L of Triton X-100 were used, the cell concentration decreased. Using the based results the continuous fed-batch cultures by adding the Triton X-100 were carried out for 10 days in an air-lift bioreactor. When 1.5 g/L of Triton X-100 was added to the culture broth at 0, 4, and 8 days of culture, respectively, the compactin production was increased with the increase of culture time. The maximum compactin concentration after 10 days of culture was 1,200 mg/L, which was about 2.0-fold higher than that of the control without the addition of Triton X-100.     

马齿苋内生菌橘青霉和波兰青霉中抗青枯菌的活性物质

为了挖掘农作物病害生物防治新资源,以药用植物马齿苋(Portulaca oleracea)为材料,通过培养基种植法分离和纯化其根、茎、叶中的内生菌,以青枯菌(Ralstonia solanacearum)的抑菌试验评价其活性,采用菌落形态观察和ITS序列分析鉴定菌种。结果表明,从马齿苋筛选出2种具有抑制青枯菌的内生菌橘青霉(Penicillium citrinum)和波兰青霉(P. polonicum),采用液相与四极杆飞行时间串联质谱(UPLC-QTOF-MS)鉴定2种内生菌的主要活性物质为橘霉素,其对青枯菌的抑制效果比链霉素更好。因此,这为植物青枯病的生物防治提供科学依据。     

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.     

Ultrastructural evidence for an osmoregulatory effect of nikkomycin in the mite Tetranychus urticae

Histological effects of the microbial metabolite and chitin synthesis inhibitor complex Nikkomycin (AMS 0896 Bayer Leverkusen) on osmoregulatory organs of all developmental stages of Tetranychus urticae are described. The metabolite, in a concentration of 100 ppm, was applied via the nutritive plant. Mites fed for 2 to 14 days, and then were collected and immediately fixed. Two osmoregulatory organs occur in T. urticae. The Malpighian complex, differentiated only in females, shows an increased number of apical microvilli in the epithelium of the distal regions after metabolite application, thus resulting in an enlargement of the surface area. Changes in the second osmoregulatory organ, the coxal organ, after Nikkomycin application include depositions of membranous bodies in the lumen as well as in cytoplasmic vacuoles of the proximal tubule. Additionally, an increase in the luminal diameter occurs. Numerous vacuoles of different contents are observed in the cytoplasm of the distal tubule. Consequences of histological alterations in osmoregulatory organs after Nikkomycin application are discussed with special reference to the composition of salivary secretions.     

Improvement of compactin (ML-236B) production by genetic engineering in compactin high-producing <Emphasis Type="Italic">Penicillium citrinum</Emphasis>

An increase in compactin (ML-236B) production was achieved by introducing a whole compactin biosynthetic gene cluster or the regulatory gene mlcR into compactin high-producing Penicillium citrinum. In the previous report, we introduced mlcR encoding the positive regulator of compactin biosynthetic genes into compactin high-producing strain no. 41520, and most of the transformants produced higher amounts of compactin. Here, we characterize one of the resulting high producers (strain TIR-35, which produced 50% more compactin) and reveal that TIR-35 contained five copies of mlcR and that early, enhanced expression of mlcR caused compactin overproduction. Similarly, the introduction of mlcR into strain T48.19, which was created previously from strain no. 41520 by introducing a partial compactin biosynthetic gene cluster, enhanced compactin production further. Our results indicated that genetic engineering is an effective tool to improve compactin production, even in compactin high producers.     

Production and partial purification of naringinase by Penicillium decumbens PTCC 5248

Penicillium decumbens PTCC 5248 produced naringinase when grown in a medium contained naringin as a source of carbon. Rhamnose also induced production of naringinase. Prunin disappeared as the time of enzymatic reaction increased. On fractionation with isopropanol 24-fold purification was achieved. Optimum pH and temperature for naringinase activity were determined to be 4.5 and 55 °C respectively. The K_m value of the enzyme with respect to naringin was found to be 1.7 mM. Citric acid, glucose, Ca²⁺, Mg²⁺, Zn²⁺ all inhibited naringinase activity.     

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.     

Purification and properties of an intracellular leucine aminopeptidase from the fungus,Penicillium citrinum strain IFO 6352

An intracellular leucine aminopeptidase (LAP) fromPenicillium citrinum (IFO 6352) was purified to homogeneity using three successive purification steps. The enzyme has a native molecular mass of 63 kDa using HPLC gel filtration analysis and a molecular mass of 65 kDa when using SDS-polyacrylamide gel electrophoresis. This monomeric aminopeptidase showed maximum enzyme activity at pH 8.5. An optimum temperature was 45–50°C whenl-Leu-p-nitroanilide (pNA) was the substrate, and enzyme activity drastically decreased above 60°C. The Michaelis-Menten constants forl-Leu-pNA andl-Met-pNA were 2.7 mM and 1.8 mM, respectively. When the enzyme reacted with biosynthetic methionyl human growth hormone, it showed high specificity for N-terminal methionine residue and recognized a stop sequence (Xaa-Pro). The aminopeptidase was inactivated by EDTA or 1,10-phenanthroline, indicating that it is a metallo-exoprotease. Enzyme activity was restored to 90% of maximal activity by addition of Co²⁺ ions. The activity of EDTA-treated enzyme was restored by addition of Zn²⁺, but reconstitution with Ca²⁺, Mg²⁺ or Mn²⁺ restored some enzyme activity. It is likely that Co²⁺ ions play an important role in the catalysis or stability of thePenicillium citrinum aminopeptidase, as zinc plays a similar function in other leucine aminopeptidases.     

Engineering of NADPH-dependent aldo-keto reductase from <Emphasis Type="Italic">Penicillium citrinum</Emphasis> by directed evolution to improve thermostability and enantioselectivity

Penicillium citrinum β-keto ester reductase (KER) can catalyze the reduction of methyl 4-bromo-3-oxobutyrate (BAM) to methyl (S)-4-bromo-3-hydroxybutyrate with high optical purity. To improve the thermostability of KER, protein engineering was performed using error-prone polymerase chain reaction-based random mutagenesis. Variants with the highest levels of thermostability contained the single amino acid substitutions L54Q, K245R, and N271D. The engineered L54Q variant of KER retained 62% of its initial activity after heat treatment at 30°C for 6 h, whereas wild-type KER showed only 15% activity. The L54Q substitution also conferred improved enantioselectivity by KER. An Escherichia coli cell biocatalyst that overproduced the L54Q mutant of KER and glucose dehydrogenase as a cofactor regeneration enzyme showed the highest level of BAM reduction in a water/butyl acetate two-phase system.     

Phosphatase activity ofPenicillium citrinum submerged batch cultures and its relationship to fungal activity

Acid and alkaline phosphatase activities were evaluated using batch fermenter cultues ofPenicillium citrinum, an organism used in studies of fungal functioning in soil. Fungal activity was assessed by monitoring rates of O₂ utilization, glucose utilization, dry weight changes over time, and lengths of FDA-stained hyphae. At low growth rates (7 g dry wt increases·h^–1·ml^–1) and low culture activity, phosphatase activity at both pH 8.5 and 5.5 tended to decrease with culture age, with the exception that phosphatase activity at pH 8.5 peaked during early stationary phase. At higher growth rates (25 g dry wt increase·h^–1·ml^–1) and high culture activity, phosphatase activity tended to remain constant throughout the course of the experiment. The relationship between phosphatase activity and other measures of fungal activity was consistent only at low growth rates for acid phosphatase. These results suggest that phosphatase measurements will be of limited utility in assessing activity, except at low growth rates.     

The influence of culture conditions on the biosynthesis of secondary metabolites by Penicillium verrucosum Dierck

A Brazilian strain of Penicillium verrucosum was cultivated under different conditions in a two-step process, in order to verify the influence of nutrients, and of time periods of pre-fermentative and fermentative steps on the biosynthesis of metabolites. Extracellular and intracellular extracts were obtained from each culture in the four different production media used. Chemical profiles of the extracts were obtained by HPLC. Extract trypanocidal activities against trypomastigote forms of Trypanosoma cruzi were evaluated. The time period of incubation in the pre-fermentative and fermentative media, as well as the different nutrients tested, qualitatively and quantitatively modified the production of secondary metabolites by P. verrucosum, and the extract trypanocidal activities.     

Purification and characterization of pectin lyase secreted by <Emphasis Type="Italic">Penicillium citrinum</Emphasis>

The importance of various parameters such as sugarcane juice concentration, pH of the medium, and effects of different solid supports for maximum secretion of pectin lyase from Penicillium citrinum MTCC 8897 has been studied. The enzyme was purified to homogeneity by Sephadex G-100 and DEAE-cellulose chromatography. The molecular mass determined by SDS-PAGE was 31 kDa. The K _m and k _cat values were found to be 1 mg/ml and 76 sec⁻¹, respectively. The optimum pH of the purified pectin lyase was 9.0, though it retains activity in the pH 9.0–12.0 range when exposed for 24 h. The optimum temperature was 50°C, and the pectin lyase was found to be completely stable up to 40°C when exposed for 1 h. The purified pectin lyase was found efficient in retting of Linum usitatissimum, Cannabis sativa, and Crotalaria juncea. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 7, pp. 985–992.