Penicillium Canescens Research Articles

Bioactive sesquiterpene, plasticizer, and phenols from the fungal endophytes of Polygonum chinense L.

There is a constant need for novel antibiotic and antioxidant sources due to the ever-increasing resilience of pathogens and the occurrence of chronic diseases. The natural sources of these agents have advantages due to lower production cost, structural variation, and uses of active compounds for pharmaceutical uses. The microbes living in planta termed “endophytes” are alternative sources of host bioactive compounds. In this study, ten endophytic fungi were isolated from Polygonum chinense L. and identified by sequencing of the internal transcribed spacer regions. The fungal strains were fermented and the ethyl acetate extracts were evaluated for antimicrobial and antioxidant capacities. Almost 80% of the endophytes showed antibacterial potency against one or more pathogenic bacteria. Among all strains, Penicillium canescens showed broad-spectrum antimicrobial activity against gram-positive and gram-negative pathogens as well as significant antioxidative and DNA protective capacities. The strain Fusarium chlamydosporum displayed significant anti-radical (126.8 ± 6.7 μg/ml) and ferric reducing (84.7 ± 2.1 mg AA/g dry extract) capacities. The bio-autography, chromatography, and mass spectroscopy analyses of P. canescens extract revealed the presence of sesquiterpene (germacrene), plasticizer (phthalic acid ester) along with phenolic acids, flavonoid (quercetin), and short chain hydrocarbons. The secondary metabolites of F. chlamydosporum were identified with phenolic acids as bioactive compounds by chromatography and mass spectroscopy. This study indicates P. chinense endophytes as potential sources of antimicrobial and antioxidant compounds for novel drug discovery.

Cloning, Isolation, and Properties of a New Homologous Exoarabinase from the Penicillium canescens Fungus

A novel exo-arabinase (GH93, exo-ABN) enzyme produced by the ascomycete Penicillium canescens has been studied. Cloning of the abn1 gene coding for exo-ABN into the recipient P. canescens strain RN3-11-7 yielded recombinant producing strains characterized by a high yield of extracellular exo- ABN production (20–30% of the total amount of extracellular protein). Chromatographic purification yielded a homogenous exo-ABN with a molecular weight of 47 kDa, as shown by SDS-PAGE. The enzyme showed high specific activity towards linear arabinan (117 U/mg) and low specific activity towards branched arabinan and arabinoxylan (4–5 U/mg) and para-nitrophenyl-α-L-arabinofuranoside (0.3 U/mg), whereas arabinogalactan and para-nitrophenyl-α-L-arabinopyranoside, the substrates that contained the pyranose form of arabinose, were not hydrolyzed. Arabinohexaose was the major product of linear arabinan hydrolysis. Exo-ABN had a pH optimum at 5.0 and a temperature optimum at 60°C. The enzyme was stable in a broad pH range (4.0–7.0) and upon heating to 50°C during 180 min. Extensive hydrolysis of linear and branched arabinans by exo- and endo-arabinase mixtures, arabinofuranosidase, and arabinofuran-arabinoxylan hydrolase has been performed. The degree of substrate conversion amounted to 67 and 83% of the maximal possible value, respectively.

Secondary Metabolites from the Root Rot Biocontrol Fungus Phlebiopsis gigantea.

Three cyclopentanoids (phlebiopsin A–C), one glycosylated p-terphenyl (methyl-terfestatin A), and o-orsellinaldehyde were isolated from the biocontrol fungus Phlebiopsis gigantea, and their structures were elucidated by 1D and 2D NMR spectroscopic analysis, as well as by LC-HRMS. The biological activity of the compounds against the root rot fungus Heterobasidion occidentale, as well as against Fusarium oxysporum and Penicillium canescens, was also investigated, but only o-orsellinaldehyde was found to have any antifungal activity in the concentration range tested.

A New Enzyme Preparation for Reducing the Viscosity of Whole-Grain Rye Extracts

New recombinant strains based on the lower fungus Penicillium canescens are engineered to produce an extracellular enzyme complex that contains a homologous endo-1,4-β-xylanase E (XylE, EC 3.2.1.8). Expression of the gene encoding XylE is controlled by the promoter of the xylA gene, which encodes endo- 1,4-β-xylanase A. Comparative analysis of enzyme preparations (EPs) isolated from the culture liquid of the host strain (Penicillium canescens RN3-11-7) and the new recombinant P. canescens strains of the XylE series is performed. The specific activity of the EPs against a specific substrate (birch glucuronoxylan) is studied, and the qualitative and quantitative composition of the new EPs XylE-B5 and XylE-C1, as well as the RN3 EP from the host strain, is determined. The decrease in the reduced viscosity of native (not subjected to thermal inactivation) aqueous extracts of rye treated with XylE-B5 EP was double that in the case of treatment with RN3 EP, due to high content of inhibition-resistant XylE in the new EPs.

Antifungal Activity of Natural Dye from Aerial Biomass of Barleria prionitisL. and Dyed Fabrics

Natural dye extracted from aerial parts of Barleria prionitis and different kinds of textile fabrics dyed with the natural dye were investigated for their antifungal activity. Antifungal activity of natural dye and dyed fabrics was assessed against standard strains of five fungi namely Aspergillus flavus, A. niger, A. parasiticus, Fusarium moniliforme and Penicillium canescens using agar-well diffusion method. The MIC was determined by the broth dilution method. Further, the antifungal potency of dyed fabrics (silk, wool, and cotton) against the test fungi was quantitatively evaluated by the reported method. Different treatment doses of natural dye exhibited a varying degree of antifungal activity against the five test fungi. The highest growth reduction in all the test fungi, however, was recorded with 500 mg/ml concentration of natural dye. The antifungal activity at this concentration is found almost at par with the positive control. The Minimum Inhibitory Concentrations (MICs) of natural dye against test fungi were ranged within, 22.50-23.50 mg/mL. Dyed silk, wool, and cotton fabrics also showed remarkable antifungal efficacy against all the test fungi. Dyed silk fabrics exhibited the maximum growth reduction followed by wool and cotton. The study revealed the remarkable antifungal activity of natural dye from B. prionitis aerial biomass and dyed fabrics. Therefore, B. prionitis can be considered as a potential source of natural dye with functional properties and can be used in the protective finishing of different kinds of textile fabrics.

Improvement of the catalytic efficiency of a hyperthermophilic xylanase from Bispora sp. MEY-1.

Extremophilic xylanases have attracted great scientific and industrial interest. In this study, a GH10 xylanase-encoding gene, Xyl10E, was cloned from Bispora sp. MEY-1 and expressed in Pichia pastoris GS115. Deduced Xyl10E shares the highest identities of 62% and 57% with characterized family GH10 xylanases from Talaromyces leycettanus and Penicillium canescens (structure 4F8X), respectively. Xyl10E was most active at 93 to 95°C and pH 4.0, retained more than 75% or 48% of the initial activity when heated at 80°C or 90°C for 30 min, respectively, and hardly lost activity at pH 1.0 to 7.0, but was completely inhibited by SDS. Two residues, A160 and A161, located on loop 4, were identified to play roles in catalysis. Mutants A160D/E demonstrated higher affinity to substrate with lower Km values, while mutants A161D/E mainly displayed elevated Vmax values. All of these mutants had significantly improved catalytic efficiency. According to the molecular dynamics simulation, the mutation of A160E was able to affect the important substrate binding site Y204 and then improve the substrate affinity, and the mutation of A161D was capable of forming a hydrogen bond with the substrate to promote the substrate binding or accelerate the product release. This study introduces a highly thermophilic fungal xylanase and reveals the importance of loop 4 for catalytic efficiency.

Properties of two laccases from the Trametes hirsuta 072 multigene family: Twins with different faces

Utilization of laccases in biotechnology and bioremediation has created a strong demand for the characterization of new enzymes and an increase in production of known laccases. Thus, additional research into these enzymes is critically needed.In this study, we report a comparative study of the biochemical and transcriptional properties of two different laccase isozymes from Trametes hirsuta 072 – the constitutive and inducible forms. A recombinant LacC enzyme was expressed in Penicillium canescens to characterize its properties. LacC is single-purpose enzyme, unlike LacA, which can operate efficiently under a wide range of temperatures and pHs (55–70 °C and pH 3–5, respectively). LacC has a lower RedOx potential than LacA and does not oxidize substrates containing amine groups. Expression of the lacC gene was selective compared to that of the lacA gene and increased significantly in the presence of complex synthetic compounds such as dyes and xenobiotics.This study shows that laccases from the multigene families of basidiomycetes differ significantly in their properties, thus providing a complementary effect during lignin degradation.

Site-directed mutagenesis of GH10 xylanase A from Penicillium canescens for determining factors affecting the enzyme thermostability

In order to investigate factors affecting the thermostability of GH10 xylanase A from Penicillium canescens (PcXylA) and to obtain its more stable variant, the wild-type (wt) enzyme and its mutant forms, carrying single amino acid substitutions, were cloned and expressed in Penicillium verruculosum B1-537 (niaD-) auxotrophic strain under the control of the cbh1 gene promoter. The recombinant PcXylA-wt and I6V, I6L, L18F, N77D, Y125R, H191R, S246P, A293P mutants were successfully expressed and purified for characterization. The mutations did not affect the enzyme specific activity against xylan from wheat as well as its pH-optimum of activity. One mutant (L18F) displayed a higher thermostability relative to the wild-type enzyme; its half-life time at 50–60°C was 2–2.5–fold longer than that for the PcXylA-wt, and the melting temperature was 60.0 and 56.1°C, respectively. Most of other mutations led to decrease in the enzyme thermostability. This study, together with data of other researchers, suggests that multiple mutations should be introduced into GH10 xylanases in order to dramatically improve their stability.

Antimicrobial activity of endophytic fungi from olive tree leaves.

In this study, the antimicrobial potential of three fungal endophytes from leaves of Olea europaea L. was evaluated and the host plant extract effect in the antimicrobial activity was examined. The volatile compounds produced by endophytes were identified by GC/MS and further correlated with the antimicrobial activity. In potato dextrose agar, both Penicillium commune and Penicillium canescens were the most effective inhibiting Gram-positive and -negative bacteria (up to 2.7-fold compared to 30µg/mL chloramphenicol), whereas Alternaria alternata was most effective inhibiting yeasts (up to 8.0-fold compared to 25μg/mL fluconazole). The presence of aqueous leaf extract in culture medium showed to induce or repress the antimicrobial activity, depending on the endophytic species. In the next step, various organic extracts from both A. alternata mycelium and cultured broth were prepared; being ethyl acetate extracts displayed the widest spectrum of anti-microorganisms at a minimum inhibitory concentration ≤0.095mg/mL. The volatile composition of the fungi that displayed the highest (A. alternata) and the lowest (P. canescens) antimicrobial activity against yeasts revealed the presence of six volatiles, being the most abundant components (3-methyl-1-butanol and phenylethyl alcohol) ascribed with antimicrobial potentialities. Overall the results highlighted for the first time the antimicrobial potential of endophytic fungi from O. europaea and the possibility to be exploited for their antimicrobial agents.

Optimization of Culture Condition for Enhanced Decolorization of Direct blue Dye by Aspergillus flavus and Penicillium canescens

Induction and enhancement of the novel marine-derived Alternaria tenuissima KM651985 laccase enzyme using response surface methodology: Application to Azo and Triphenylmethane dyes decolorizationAbeer A. Abd El Aty, Eman R. Hamed, Ahmed A. El-Beih, Ahmed I. El-Diwany

Improving the efficiency of the bioconversion of plant raw materials with mutant cellulases of Penicillium verruculosum

Plant biomass is the main type of organic material on Earth. The efficiency of biocatalytic conversion of plant raw materials determines the cost of their biotechnological processing to produce commercially valuable products such as organic alcohols and acids, carbohydrates, and hydrocarbons. New recombinant Penicillium canescens strains that produce not only their own enzyme complex but also heterologous cellulases (i.e., mutant and wild-type cellobiohydrolase I (CBH I) and endoglucanase II (EG II) of P. verruculosum) are constructed. Enzymatic agents (EAs) prepared on the basis of recombinant strains of P. canescens are found to be more active in the hydrolysis of crushed aspen wood. Yields of glucose and reducing sugars are observed 24–72 h after hydrolysis with EAs prepared in recombinant strains to be from 48 to 52 and 60 to 64%, respectively, higher than those for hydrolysis with EAs prepared in the initial recipient strain. The presence of N45A and N194A site-specific mutations introduced to reduce surface glycosilation thus results in a substantial increase in the yields of desired CBH I and EG II.

Decomposition of Leaves, Stems and Roots of Transgenic Aspen with the Xyloglucanase (sp-Xeg) Gene under Laboratory Microcosm Conditions

The genetic transformation of trees by wood modification genes for the improvement of forest plantations results in shifts in plant litter quality. These alterations in plant chemistry lead to changes in decomposition rates, thus affecting the carbon and nitrogen cycling in ecosystems and nutrient availability for plants. To assess the environmental impacts of transgenic trees, we studied the decomposition of plant litter from aspen plants (Populus tremula L.) transformed with the xyloglucanase gene from Penicillium canescens. Mass, carbon and nitrogen losses in the leaves, stems and roots of greenhouse-grown plants were evaluated during incubation in laboratory microcosms. After 12 months of the decomposition experiment, leaves, stems, and roots lost on average 51%, 46%, and 37% of initial mass, respectively. Decomposition of the transgenic stems was not different from wild-type aspen, but we observed significant differences for the leaves (only at the end of the experiment) and the roots (at the early stage). These differences may be related to the nitrogen content and the C/N ratio in the initial samples. Since the litter decomposability determines the availability of nutrients, such alterations should be taken into consideration when cultivating transgenic trees.

Deacetylation biocatalysis and elicitation by immobilized Penicillium canescens in Astragalus membranaceus hairy root cultures: towards the enhanced and sustainable production of astragaloside IV.

SummaryA novel biotechnology approach by combining deacetylation biocatalysis with elicitation of immobilized Penicillium canescens (IPC) in Astragalus membranaceus hairy root cultures (AMHRCs) was proposed for the elevated production of astragaloside IV (AG IV). The highest AG IV accumulation was achieved in 36‐day‐old AMHRCs co‐cultured with IPC for 60 h, which resulted in the enhanced production of AG IV by 14.59‐fold in comparison with that in control (0.193 ± 0.007 mg/g DW). Meanwhile, AG IV precursors were almost transformed to AG IV by IPC deacetylation. Moreover, expression of genes involved in AG IV biosynthetic pathway was significantly up‐regulated in response to IPC elicitation. Also, FTIR and SEM showed that cell wall lignification was enhanced following IPC treatment and root surface was likely to be IPC deacetylation site. Overall, dual roles of IPC (biocatalyst and elicitor) offered an effective and sustainable way for the mass production of AG IV in AMHRCs.

Automated Detection of Natural Halogenated Compounds from LC-MS Profiles-Application to the Isolation of Bioactive Chlorinated Compounds from Marine-Derived Fungi.

A collection of culture extracts obtained from several marine-derived fungal strains collected on the French Atlantic coast was investigated by high performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) in order to prospect for halogenated compounds and to identify potentially new ones. To achieve a fast, automated, and efficient data analysis, a bioinformatics tool named MeHaloCoA (Marine Halogenated Compound Analysis) was developed and included into R. After extraction of all the peaks from the metabolic fingerprints and their associated mass spectra, a mathematical filter based on mass isotopic profiles allowed the selective detection of halogenated (Cl and Br) molecules. Integrating MeHaloCoA into a dereplication approach allowed the identification of known and new halogenated compounds in a competitive amount of time. Subsequent targeted purification led to the isolation of several chlorinated metabolites, including two new natural products with bioactive potential, griseophenone I and chlorogriseofulvin, from a marine-derived Penicillium canescens strain.

Effect of N-linked glycosylation on the activity and other properties of recombinant endoglucanase IIa (Cel5A) from Penicillium verruculosum.

Endoglucanase IIa from Penicillium verruculosum (PvCel5A) has three potential N-glycosylation sites: Asn19, Asn42 and Asn194. In order to study the role of N-glycosylation, the wild type (wt) PvCel5A and its mutant forms, carrying Asn to Ala substitutions, were cloned into Penicillium canescens. All forms of the rPvCel5A were successfully expressed and purified for characterization. The MALDI-TOF mass spectrometry peptide fingerprinting showed that N-glycans linked to Asn42 and Asn194 represent variable oligosaccharides, according to the formula (Man)1-9(GlcNAc)2. No evidence for Asn19 glycosylation was found. Mutations had no notable effect on the enzyme thermostability; however, the N-linked glycans stabilized the enzyme against proteolytic attack. For N42A and N194A mutants, a slight shift of pH-optimum to pH 5.0 was observed (from pH-optimum of 4.5 for the native enzyme, rPvCel5A-wt and N19A mutant). The N19A mutation led to a notable decrease in the specific activity against carboxymethylcellulose and barley β-glucan (by 26% and 12% relative to the rPvCel5A-wt), while the N42A and N194A mutants displayed 12-13% and 32-35% increase in the activities. Similar effects of the mutations were observed in prolonged hydrolysis of β-glucan and milled aspen wood by rPvCel5A forms in the presence of purified β-glucosidase.

Composition and microstructure alteration of triticale grain surface after processing by enzymes of cellulase complex

It is found that the pericarp tissue of grain have considerable strength and stiffness, that has an adverse effect on quality of whole-grain bread. Thereby, there exists the need for preliminary chemical and biochemical processing of durable cell walls before industrial use. Increasingly used in the production of bread finds an artificial hybrid of the traditional grain crops of wheat and rye - triticale, grain which has high nutritional value. The purpose of this research was to evaluate the influence of cellulose complex (Penicillium canescens) enzymes on composition and microstructure alteration of triticale grain surface, for grain used in baking. Triticale grain was processed by cellulolytic enzyme preparations with different composition (producer is Penicillium canescens). During experiment it is found that triticale grain processing by enzymes of cellulase complex leads to an increase in the content of water-soluble pentosans by 36.3 - 39.2%. The total amount of low molecular sugars increased by 3.8 - 10.5 %. Studies show that under the influence of enzymes the microstructure of the triticale grain surface is changing. Microphotographs characterizing grain surface structure alteration in dynamic (every 2 hours) during 10 hours of substrate hydrolysis are shown. It is found that the depth and direction of destruction process for non-starch polysaccharides of grain integument are determined by the composition of the enzyme complex preparation and duration of exposure. It is found, that xylanase involved in the modification of hemicelluloses fiber having both longitudinal and radial orientation. Hydrolysis of non-starch polysaccharides from grain shells led to increase of antioxidant activity. Ferulic acid was identified in alcoholic extract of triticale grain after enzymatic hydrolysis under the influence of complex preparation containing cellulase, xylanase and β-glucanase. Grain processing by independent enzymes containing in complex preparation (xylanase and β-glucanase) shows that more significant role in polysaccharide complex composition and grain surface microstructure alteration belongs to xylanase. Grain processing by independent of cellulolytic enzymes may decrease the strength of pericarp tissue of grain and improved sensory characteristics of the bread.

Redistribution of mineral elements in wheat grain when applying the complex enzyme preparations based on phytase

Biogenic minerals play an important role in the whole human nutrition, but they are included in the grain of the phytates that reduces their bioavailability. Whole wheat bread is generally considered a healthy food, but the presence of mineral elements in it is insignificant, because of weak phytate degradation. From all sources of exogenous phytase the most productive are microscopic fungi. To accelerate the process of transition hard mineral elements are mobilized to implement integrated cellulolytic enzyme preparation based on the actions of phytase (producer is Penicillium canescens). Phytase activity was assessed indirectly by the rate of release of phosphate from the substrate. It has been established that the release rate of the phosphoric acid substrate is dependent on the composition of the drug and the enzyme complex is determined by the presence of xylanase. The presented experimental data shows that a cellulase treatment of the grain in conjunction with the β-glucanase or xylanase leading to an increase in phytase activity could be 1.4 - 2.3 times as compared with the individual enzymes. As a result of concerted action of enzymes complex preparation varies topography grain, increase the pore sizes in seed and fruit shells that facilitate the penetration of the enzyme phytase in the aleurone layer to the site of phytin hydrolysis and leads to an increase in phytase activity. In terms of rational parameters of enzymatic hydrolysis, the distribution of mineral elements in the anatomical parts of the grain after processing complex enzyme preparation with the help of X-ray detector EMF miniCup system in a scanning electron microscope JEOL JSM 6390 were investigated. When processing enzyme preparation wheat trend in the distribution of mineral elements, characteristic of grain - the proportion of these elements in the aleurone layer decreases, and in the endosperm increases. Because dietary fiber and phytate found together in the peripheral layers of fiber-rich grains, it is difficult to separate the effects of degradation processes nonstarch polysaccharides and fiberphytate redistribution of polyvalent metal ions. However, studies have shown that phytase - an effective mechanism for regulating mineral nutrient diet. Application of phytase in grain bakery technology will increase the biological value of the product.

Correction: Genome mining and molecular characterization of the biosynthetic gene cluster of a diterpenic meroterpenoid, 15-deoxyoxalicine B, in Penicillium canescens.

Correction for ‘Genome mining and molecular characterization of the biosynthetic gene cluster of a diterpenic meroterpenoid, 15-deoxyoxalicine B, in Penicillium canescens’ by Junko Yaegashi et al., Chem. Sci., 2015, 6, 6537–6544.

Sodium Chloride Stress Induced Morphological Changes in Some Halotolerant Fungi

Materials and methods: Nine fungal isolates namely Emericill anidulans, Mucor racemosus , Alternaria pluriseptata Penicillium canescens, Syncephalastrum racemosum, Aspergillus fumigatus, Alternaria chlamydospora, Aspergillus parasiticus and Ulocladium atrum were isolated from AL – SHEGA area at AL- QASSIM region. Results: The influence of different sodium chloride concentrations on the growth rate, morphological and ultrastructure were studied. Considerable differences in their growth rate and morphology were detected on medium containing different concentrations of sodium chloride (NaCl). Low growth rates were obseved on high NaCl concentrations . At 15 % NaCl, low growth of Emericill anidulans, Penicillium canescens, Syncephalastrum racemosum, Aspergillus parasiticus and Mucor racemosus was detected , whereas all fungal isolates were failed to grow at 20% NaCl. Scanning Electron Microscope (SEM) revealed that all fungal asexual reproduction organs were metamorphosed at higher NaCl concentration, fungal heads and sporangia were speculated or elongated. Sporangiophores and conidiophores were shortened and dwarfed ,little number of conidia or spores were detected.

Improvement of the Efficiency of Bioconversion of Plant Materials under the Action of Mutant Strains of Cellulases Penicillium verruculosum

Plant biomasses are the predominant organic material on Earth. The efficiency of biocatalytic conversion of the plant materials is determined by the cost of their biotechnological processing to valuable commercial products (organic alcohols and acids, carbohydrates and hycrocarbons). The obtained recombinant strains Penicillium canescens produce, apart from their own enzymatic complex, heterological cellulases (mutant and non-mutant cellobiohydrolase I (CBHI) and endoglucanase II (EGII) P. verruculosum ). Enzymatic agents (EA) prepared from recombinant strains P. canescens were more active to hydrolysis of crushed aspen wood. The yields of glucose and reducing sugars after 24–72 hour hydrolysis under the action of EA prepared from the recombinant strains were higher by на 48–52 % and 60–64 %, respectively, than under the action of EA prepared from the initial strainrecipient. Thus, introduction of site-specific mutations N45A and N194A for partial inhibition of the surface glycosilation led to a considerable increase in the yields of target CBHI and EGII.