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Two pairs of new isobenzofuranone derivative enantiomers, (±)-penicifurans E (1) and (±)-penicifurans F (2), together with four know compounds (3–6) were isolated from the solid fermentation of Penicillium canescens DWS225. The structures of these enantiomers were elucidated by extensive NMR spectroscopic data, and their absolute configurations were assigned by the experimental and calculated ECD data. The neuroprotective effects of all the isolates against oxygen-glucose deprivation/reperfusion injury in pheochromocytoma-12 cells (PC12) were investigated. [ABSTRACT FROM AUTHOR]

Xylanases are used in several industrial applications, such as feed additives, the bleaching of pulp and paper, and the production of bread, food, and drinks. Xylanases are required to remain active after heat treatment at 80–90 °C for 30 s to several minutes due to the conditions of feed pelleting. Also, xylanases need to be active at 60–70 °C for several hours while bleaching of pulp and paper or manufacturing of bread, food, and drinks is performed. Xylanases of the glycoside hydrolase family GH10 are good candidates for application in such processes because of their high thermostability and, in particular, as feed additives because of their insensitivity to protein inhibitors in cereal feeds. In the study, the thermostability of GH10 xylanase E from Penicillium canescens was improved to reach a half-inactivation period of 2 min at 80 °C compared to 21 s for the wild-type enzyme (WT). Enzymatic activity was increased by 22–48 % at 40–70 °C, which improved the action of the enzyme as a feed additive in the gastric system of animals and during bleaching of pulp and paper. Molecular dynamics simulations demonstrated lower flexibility of the tertiary structure of the engineered enzyme at elevated temperatures compared to WT. The residues W113, Q116, W313, and W321 in the (−1) and (−2) subsites for the substrate binding were less flexible. In the simulations, the engineered enzyme had a comparable content of α-helixes, 3 10 -helixes, β-sheets, and β-bridges as WT, but a lower content of coils and a higher content of β-turns. [Display omitted] • Thermostability was improved by six times to reach t 1/2 of 2 min at 80 °C. • Enzymatic activity was increased by 22–48 % at 40–70 °C. • Stability of active center cleft and surface loops and α-helixes was increased. [ABSTRACT FROM AUTHOR]

The interest in peroxidases of the basidiomycete secreted enzyme complex is due to their wide substrate specificity and the ability of these enzymes to participate in the biodegradation of such difficult to degrade biopolymers as lignin. However, due to the difficulty of isolating these enzymes from native sources, their study is difficult. In this work, expression vectors were created that carried the sequence encoding the T. hirsuta LE-BIN072 versatile peroxidase VP2, which was transformed into the genome of the P. canescens strain. Screening of transformants showed the presence of peroxidase activity up to 1 U/mL. Fragments of the target protein in the culture liquids of the selected transformants were identified by mass spectrometric analysis. A new strain, P. canescens pVP2D-6, a producer of the recombinant versatile peroxidase VP2 of T. hirsuta LE-BIN072, was obtained for the first time, and the ability of the enzyme complex secreted by it to modify alkaline lignin was shown. [ABSTRACT FROM AUTHOR]

Objective(s): Salvia abrotanoides is considered as a new source of tanshinone-producing plants in Iran. Symbiosis of endophytic fungi with their host plants is an effective tool to promote the growth and secondary metabolism of medicinal herbs. Therefore, using endophytic fungi as a biotic elicitor is a proper solution to increase the yield of plant products. Materials and Methods: In this study, some endophytic fungi were first isolated from the root of S. abrotanoides, then two of them (Penicillium canescens and Talaromyces sp.) were co-cultivated with the sterile seedling of S. abrotanoides in pot culture. After proving the colonization of these fungi in the root tissues by microscopic studies, their effects on the production of critical medicinal compounds such as tanshinones and phenolic acids were investigated in the vegetation stage (120 days). Results: Our results showed that the content of cryptotanshinone (Cry) and tanshinone IIA (T-IIA) in plants inoculated with P. canescens increased by 77.00% and 19.64%, respectively, compared with non-inoculated plants (control). The contents of mentioned compounds in plants inoculated with Talaromyces sp. increased by 50.00% and 23.00%, respectively. In this case, in plants inoculated with P. canescens, it was found that the level of caffeic acid, rosmarinic acid, and its PAL enzyme activity increased by 64.00%, 69.00%, and 50.00%, respectively, compared with the control.Conclusion: Endophytic fungi have specific modes of action and the ability to provide multiple benefits. Each of the two strains is a highly considerable microbial resource for the growth and accumulation of active compounds of S. abrotanoides.

Objective(s): Salvia abrotanoides is considered as a new source of tanshinone-producing plants in Iran. Symbiosis of endophytic fungi with their host plants is an effective tool to promote the growth and secondary metabolism of medicinal herbs. Therefore, using endophytic fungi as a biotic elicitor is a proper solution to increase the yield of plant products. Materials and Methods: In this study, some endophytic fungi were first isolated from the root of S. abrotanoides, then two of them (Penicillium canescens and Talaromyces sp.) were co-cultivated with the sterile seedling of S. abrotanoides in pot culture. After proving the colonization of these fungi in the root tissues by microscopic studies, their effects on the production of critical medicinal compounds such as tanshinones and phenolic acids were investigated in the vegetation stage (120 days). Results: Our results showed that the content of cryptotanshinone (Cry) and tanshinone IIA (T-IIA) in plants inoculated with P. canescens increased by 77.00% and 19.64%, respectively, compared with non-inoculated plants (control). The contents of mentioned compounds in plants inoculated with Talaromyces sp. increased by 50.00% and 23.00%, respectively. In this case, in plants inoculated with P. canescens, it was found that the level of caffeic acid, rosmarinic acid, and its PAL enzyme activity increased by 64.00%, 69.00%, and 50.00%, respectively, compared with the control. Conclusion: Endophytic fungi have specific modes of action and the ability to provide multiple benefits. Each of the two strains is a highly considerable microbial resource for the growth and accumulation of active compounds of S. abrotanoides. [ABSTRACT FROM AUTHOR]

Two undescribed phenylacetate derivatives (compounds 1 and 2) with a known analog were isolated from a soil-derived fungus Penicillium canescens through the “one strain-many compounds” method. The new structures were assigned using extensive 1D and 2D NMR spectra, high-resolution electrospray ionization mass spectrometry, and the comparison of coupling cleavages of known compounds, which are two pairs of racemic mixtures of aromatic polyketides with a terminal butan-2,3-diol group. In the bioassay, the biological screening signifies no cytotoxic activities against several human cancer cell lines (HL-60, A549, SMMC-7721, MCF-7, and SW480) at a concentration of 40.0 µM.

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FPAses have been isolated and studied from psychrotolerant yeast Rhodotorula mucilaginosa BPT1, Penicillium canescens BPF4 and Pseudogymnoascus roseus BPF6. BPT1 showed 100% activity at 4ºC, 30ºC and 50ºC, while that from BPF4 and BPF6 showed maximal activity at 60ºC and 40ºC respectively. The enzyme from BPT1 showed three peak activities, BPF4 and BPF6 showed single peak activity. While BPT1 FPAse showed 100% activity at low temperature i.e. 4ºC, rendering it very useful enzyme. The FPAses from both the other fungi also showed more than 60% residual activity at cold temperatures. The cold-activity of the enzymes makes them potential for application in simultaneous saccharification and fermentation and other industries especially food processing ones. [ABSTRACT FROM AUTHOR]

Abstract Background Recombinant carbohydrases genes are used to produce transgenic woody plants with improved phenotypic traits. However, cultivation of such plants in open field is challenging due to a number of problems. Therefore, additional research is needed to alleviate them. Results Results of successful cultivation of the transgenic aspens (Populus tremula) carrying the recombinant xyloglucanase gene (sp-Xeg) from Penicillium canescens in semi-natural conditions are reported in this paper for the first time. Change of carbohydrate composition of wood was observed in transgenic aspens carrying the sp-Xeg gene. The transformed transgenic line Xeg-2-1b demonstrated accelerated growth and increased content of cellulose in wood of trees growing in both greenhouse and outside in comparison with the control untransformed line Pt. The accelerated growth was observed also in the transgenic line Xeg-1-1c. Thicker cell-wall and longer xylem fiber were also observed in both these transgenic lines. Undescribed earlier considerable reduction in the wood decomposition rate of the transgenic aspen stems was also revealed for the transformed transgenic lines. The decomposition rate was approximately twice as lower for the transgenic line Xeg-2-3b in comparison with the control untransformed line Pt. Conclusion A direct dependence of the phenotypic and biochemical traits on the expression of the recombinant gene sp-Xeg was demonstrated. The higher was the level of the sp-Xeg gene expression, the more pronounced were changes in the phenotypic and biochemical traits. All lines showed phenotypic changes in the leave traits. Our results showed that the plants carrying the recombinant sp-Xeg gene do not demonstrate a decrease in growth parameters in semi-natural conditions. In some transgenic lines, a change in the carbohydrate composition of the wood, an increase in the cell wall thickness, and a decrease in the rate of decomposition of wood were observed.

Investigations of the influence of chemical and physical factors on the conformational and functional properties of enzymes make a significant contribution to the study of the mechanism of action of industrially important proteins. The aim of the work was to evaluate the effect of chemical reagents and UV irradiation on the catalytic properties of Penicillium canescens α-galactosidase. Enzyme activity was assessed with p-nitrophenyl-α-D-galactopyranoside. Studies of the functionally active glycosidase groups were carried out on the basis of inhibitory and kinetic analysis using Dixon and Luinuiver-Burke methods with help of specific chemical reagents. A significant decrease in the activity of α-galactosidase in the presence of carbodiimi­des, diethylpyrocarbonate, the reagents on sulfhydryl groups was shown. A UV-induced decrease in enzyme activi­ty in the dose range of 900-7200 J/m2 was noted. Based on the data obtained, the imidazole group of histidine, carboxyl groups of C-terminal amino acids and the SH-groups of cysteine are assumed to play an important role in the manifestation of the activity of P. canescens α-galactosidase.

Background: Recombinant carbohydrases genes are used to produce transgenic woody plants with improved phenotypic traits. However, cultivation of such plants in open field is challenging due to a number of problems. Therefore, additional research is needed to alleviate them. Results: Results of successful cultivation of the transgenic aspens (Populus tremula) carrying the recombinant xyloglucanase gene (sp-Xeg) from Penicillium canescens in semi-natural conditions are reported in this paper for the first time. Change of carbohydrate composition of wood was observed in transgenic aspens carrying the sp-Xeg gene. The transformed transgenic line Xeg-2-1b demonstrated accelerated growth and increased content of cellulose in wood of trees growing in both greenhouse and outside in comparison with the control untransformed line Pt. The accelerated growth was observed also in the transgenic line Xeg-1-1c. Thicker cell-wall and longer xylem fiber were also observed in both these transgenic lines. Undescribed earlier considerable reduction in the wood decomposition rate of the transgenic aspen stems was also revealed for the transformed transgenic lines. The decomposition rate was approximately twice as lower for the transgenic line Xeg-2-3b in comparison with the control untransformed line Pt. Conclusion: A direct dependence of the phenotypic and biochemical traits on the expression of the recombinant gene sp-Xeg was demonstrated. The higher was the level of the sp-Xeg gene expression, the more pronounced were changes in the phenotypic and biochemical traits. All lines showed phenotypic changes in the leave traits. Our results showed that the plants carrying the recombinant sp-Xeg gene do not demonstrate a decrease in growth parameters in semi-natural conditions. In some transgenic lines, a change in the carbohydrate composition of the wood, an increase in the cell wall thickness, and a decrease in the rate of decomposition of wood were observed. [ABSTRACT FROM AUTHOR]

A new isopentylated dibenzodioxocinone, canescenin A (1), and a new isopentylated pyran-3,5-dione derivative, canescenin B (2), were isolated from an extract of the deep-sea-derived fungus Penicillium canescens SCSIO z053. Their structures were elucidated by spectroscopic analysis. It was rare to obtain pyran-3,5-dione derivatives from nature. Antibacterial, cytotoxic, and antiviral activities of 1 and 2 were also evaluated. [ABSTRACT FROM AUTHOR]

Laccases (EC 1.10.3.2) are multicopper polyphenol oxidases found in various organisms, in particular, in fungi. Fungal laccases are encoded by multigene families, which can contain up to 17 genes. However, not all isozymes can be obtained from native producers. Previous studies have shown that the filamentous fungus Penicillium canescens is a promising object for the heterologous expression of various laccase isozymes of Trametes hirsuta 072. In this work, the cultivation conditions of P. canescens strains, recombinant producers of T. hirsuta heterologous minor laccase, are optimized. The optimization of the rLacD and rLacF purification method increased their yield and specific activity of rLacD. In addition, the melting points of minor laccase isoenzymes are measured and the glycosylation of isoenzymes is studied. [ABSTRACT FROM AUTHOR]

The aim of this work was the study of native and galactosidases from Penicillium canescens under denaturing conditions caused by guanidine hydrochloride. Calculation of kinetics and constants of enzymes inactivation was carried out on using experimental kinetic curves of enzyme denaturation. We observed significant differences in the kinetics of inactivation of native and deglycosylated α-galactosidases from P. canescens caused by guanidine hydrochloride. Native enzyme was stable within the selected range of guanidine hydrochloride concentrations (from 0.1 to 3.0 M), retaining no less than 50% of the initial enzyme activity for 3 days. Deglycosylated enzyme preparations were less stable and they lost their activity within 5–30 minutes, when they were treated with guanidine hydrochloride in concentrations above 1 M. Dissociation rate constant of native and deglycosylated forms of the enzyme differed by 10 to 100 folds. It was shown that subunit interactions play a major role in the process of inactivation of the enzyme, and the carbohydrate component is essential for stabilizing of subunit bonds and maintaining conformational stability of the enzyme under denaturing conditions of chemical agents.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The effect of the glycosylation inhibitors (tunicamycin and 2-deoxy-D-glucose) on the activity, stabili­ty and production of fungal glycosidases has been studied. It was shown that inhibition of N-glycosylation sites did not affect the secretion of Aspergillus niger α-galactosidase, however reduced yield of Cladosporium cladosporioides and Penicillium canescens α-galactosidases. Changes in the level of O-glycosylation resulted in a significant reduction in the activity and stability of α-galactosidases of all three producers tested. Activity of the modified enzymes was significantly lower than that of the native ones, and was 2.6 and 0.33 U/mg for A. niger α-galactosidase, 3.3 and 32.5 U/mg for C. cladosporioides α-galactosidase, 11.66 and 31.1 U/mg for P. canescens α-galactosidase, respectively. A. niger α-galactosidase completely lost activity during purification and storage. The decrease of thermal stability at 55 °C by 20% was shown for C. cladosporioides and P. canescens α-galactosidases. It was also noted that O-deglycosylation led to a decrease in resistance of these enzymes to the action of proteases.

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. [ABSTRACT FROM AUTHOR]

A 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. [ABSTRACT FROM AUTHOR]

Microbial aspects of endo-β-1,4-xylanase production in solid-state fermentation by Penicillia: the case of Penicillium canescens. Production of xylanases by Penicillium canescens 10-10c is the research object in Walloon Center of Industrial Biology. Previous works used submerged or liquid fermentation. The actual works are oriented more and more towards solid fermentation from agricultural or agro-alimentary residues. In addition to the valorization of these residues, solid-state fermentation reaches an increasingly significant interest in various other fields like the biological breakdown of the solid residues, the bioremediation of the organic pollutants in the grounds and the reduction of the air pollution by the biofiltration. Xylanase is an industrial enzyme used in general in extraction and clarification processes. P. canescens can produce an activity of it, particularly in its balanced forms of xylanases, beta-xylosidase and arabinosidase, and not contaminated by cellulolytic and amylolytic activities. It is a hyper producing strain of xylanase. The production rate is one of the highest in literature (535 U.ml-1 and 9,632 U.g-1 in Erlenmeyer flasks, in submerged and solid state fermentation, respectively). The biobleaching activity of the cellulose pulp by the purified enzyme is higher than a commercial preparation of xylanases from Trichoderma longibrachiatum used industrially. It has a complete hydrolysis degree of 40% (on glucuronoxylan) and 35% (on arabinoxylan) at 55°C and at pH of 5.9. These characteristics lead to many industrial applications of this enzyme. That is why the optimization of its production by the solid-state fermentation at the laboratory scale in order to define a policy for the industrial transposition later is carried out. This article presents a summary of the scientific literature on this subject.

Two undescribed polyketides canecines A-B, one unreported cyclopentenone canecine C, together with 12 known compounds were isolated from an extract of the fungus Penicillium canescens DJJ-1. Their structures were elucidated by detailed analysis of spectroscopic data, NMR calculations with d J -DP4 or DP4+, and their absolute configurations were further determined by quantum chemical calculations of ECD spectra or X-crystallography. Canecine A was a grisan polyketide featuring a dimethyltetrahydro-4 H -furo[2,3- b ]pyran. Canecine A exhibited significant inhibitory activity against Candida albicans with an MIC value of 1 μg/mL and showed inhibitory effect on nitric oxide production in LPS-activated RAW264.7 macrophages. These results enrich the structural diversities of polyketides from endophytic fungi. Polyketides with antimicrobial activities from Penicillium canescens DJJ-1 of Aconitum brevicalcaratum Diels (Ranunculaceae). [Display omitted] • Three undescribed polyketides were isolated from Penicillium canescens DJJ-1. • One compound showed antimicrobial activities. • One compound showed inhibitory effects on LPS-induced NO production. [ABSTRACT FROM AUTHOR]

Objectives: Salvia abrotanoides is considered as a new source of tanshinone-producing plants in Iran. Symbiosis of endophytic fungi with their host plants is an effective tool to promote the growth and secondary metabolism of medicinal herbs. Therefore, using endophytic fungi as a biotic elicitor is a proper solution to increase the yield of plant products., Materials and Methods: In this study, some endophytic fungi were first isolated from the root of S. abrotanoides , then two of them ( Penicillium canescens and Talaromyces sp.) were co-cultivated with the sterile seedling of S. abrotanoides in pot culture. After proving the colonization of these fungi in the root tissues by microscopic studies, their effects on the production of critical medicinal compounds such as tanshinones and phenolic acids were investigated in the vegetation stage (120 days)., Results: Our results showed that the content of cryptotanshinone (Cry) and tanshinone IIA (T-IIA) in plants inoculated with P. canescens increased by 77.00% and 19.64%, respectively, compared with non-inoculated plants (control). The contents of mentioned compounds in plants inoculated with Talaromyces sp . increased by 50.00% and 23.00%, respectively. In this case, in plants inoculated with P. canescens , it was found that the level of caffeic acid, rosmarinic acid, and its PAL enzyme activity increased by 64.00%, 69.00%, and 50.00%, respectively, compared with the control., Conclusion: Endophytic fungi have specific modes of action and the ability to provide multiple benefits. Each of the two strains is a highly considerable microbial resource for the growth and accumulation of active compounds of S. abrotanoides., Competing Interests: The authors declare that they have no competing interests.

The producer of fungal penicillopepsin, an aspartate protease, has been created by genetic engineering. The biochemical and physicochemical properties of the penicillopepsin enzyme preparation obtained from the culture liquid of the producer were studied. Properties of the new enzyme preparation and the commercially available aspergillopepsin were compared. Their proteolytic activities were found to be 670-680 U/g of the preparation. The soluble protein yield upon the wheat flour hydrolysis with penicillopepsin was 2.7 times higher than with aspergillopepsin. It is probably caused by the presence of the xylanase activity in the penicillopepsin preparation. [ABSTRACT FROM AUTHOR]

Two xylanases (XylA and XylE) of glycoside hydrolase family 10 are isolated from an enzyme preparation produced by Penicillium canescens fungi. The kinetics of the hydrolysis of glucuronoxylan and arabinoxylan by the purified enzymes and the effect of proteinaceous (XIP-like) inhibitors from rye on the viscometric activity of the xylanases are studied. XylA provides a more complete conversion of glucuronoxylan than XylE, while XylE is more effective in the arabinoxylan hydrolysis. Unlike XylA, XylE is resistant to the proteinaceous inhibitors from rye-this property is rarely found in the enzymes of family 10. Thus, XylE is a promising enzyme for use as a cereal feed additive, while XylA may potentially be used for the biobleaching of cellulose from hardwoods, which contain glucuronoxylan as one of the major components. [ABSTRACT FROM AUTHOR]

Recombinant strains of Penicillium canescens producing homologous pectin lyase A and heterologous endo-1,5-α-arabinase A and endo-1,4-α-polygalacturonase, as well as enzymes of the host strain (α-L-arabinofuranosidases, xylanases, and others), were obtained by genetic engineering. The enzyme preparations (EPs) obtained from the cultural medium of recombinant P. canescens strains efficiently hydrolyzed raw plant material with a high content of pectin compounds. It was shown that the yield of reducing sugars and arabinose increased 16 and 22% in comparison with the control EP based on the host strain when one of the obtained EPs was used for beet pulp hydrolysis. It was established that the most active EP consisted of pectin lyase (10%), endo-1,5-arabinase (26%),α-L-arabinofuranosidase and arabinoxylan-arabinofuranohydrolase (12%), and xylanase (10%). The activities of pectin lyase, polygalacturonase, and arabinase of the EP in reactions with various substrates were determined. The specificity, pH and T-optima, and thermal stability of the homogenous recombinant endo-1,5-α-arabinase were investigated. The kinetic parameters ( K, k) of the linear arabinan hydrolysis were determined. [ABSTRACT FROM AUTHOR]

Penicillium canescens is a filamentous fungus that normally does not secrete notable levels of cellulase activity. Cellobiohydrolase I of P. canescens (PcCel7A) was homologously cloned into a host strain RN3-11-7 ( niaD- ) and then expressed under the control of a strong xylA promoter. Using three steps of chromatography, PcCel7A was purified. The enzyme displayed maximum activity at pH 4.0–4.5. PcCel7A was stable at 50 °C and pH 4.5 at least for 3 h, while at 60 °C it lost 45% of activity after 30 min of incubation. When equalized by protein concentration, PcCel7A demonstrated a higher performance in prolonged hydrolysis of Avicel and milled aspen wood than CBH I (Cel7A) from Trichoderma reesei , the most industrially utilized cellulase at this moment. The high catalytic efficiency of the PcCel7A makes it a potential candidate for industrial applications. [ABSTRACT FROM AUTHOR]

Highlights: [•] The novel biotransformation was environmentally friendly and high-efficiency. [•] Deacetylation of the fungal endophyte was significant. [•] The fungal endophyte was firstly isolated from pigeon pea. [•] Content of astragaloside IV was 5.51-fold to untreated one after biotransformation. [•] An alternative for the production of astragaloside IV in commercial process. [ABSTRACT FROM AUTHOR]

Highlights: [•] N-Glycosylation of two Penicillium canescens α-l-arabinofuranosidases was studied. [•] Enzymes belong to the glycoside hydrolase families 51 and 54 (Abf51A and Abf54A). [•] N-Linked glycans were found at 5 out of 8 potential N-glycosylation sites in Abf51A. [•] One out of 3 potential N-glycosylation sites in Abf54A was glycosylated. [•] Formula of N-linked glycans: (Man) x (GlcNAc)2, where x varied from 7 to 0. [Copyright &y& Elsevier]

Here we report the first isolation to homogeneous forms of two glucoamylases from the fungus Penicillium verruculosum and their study in comparison with known glucoamylases from Aspergillus awamori and Aspergillus niger. Genes that encode glucoamylases from P. verruculosum were cloned and expressed in the fungus Penicillium canescens, and the recombinant glucoamylases were obtained with subsequent study of their molecular weights, isoelectric points, optimal temperature and pH values, and stability. The catalytic activities of the recombinant glucoamylases were determined in relation to soluble potato starch. Changes in molecular mass distribution and content of low molecular weight products during starch hydrolysis by glucoamylases from P. verruculosum, A. awamori, and A. niger were studied. An exo-depolymerization mechanism was established to be the pathway for destruction of starch by the glucoamylases. [ABSTRACT FROM AUTHOR]

The gene xylE encoding endo-1,4-β-xylanase from the 10th family of glycosyl hydrolases produced by the mycelial fungus Penicillium canescens has been expressed under the control of the strong promoter of the bgaS gene encoding β-galactosidase from P. canescens. As a result, a strain-producer of endoxylanase XylE was developed. The recombinant enzyme was isolated and purified to homogeneity with specific activity of 50 U/mg. The physicochemical and biochemical properties of the endoxylanase were studied. The maximal enzymatic activity was observed at pH 6.0 and 70°C. Endoxylanase XylE was shown to be a highly thermostable enzyme with half-inactivation period τ of 7 h at 60°C. The kinetic parameters were 0.52 mg/ml ( K) and 75 μmol/min per mg ( V) using birch xylan as the substrate. Crystals of endoxylonase XylE were obtained, and the 3D structure was solved at 1.47 Å resolution. The 3D structure of an endo-1,4-β-xylanase from the 10th family containing carbohydrate and unique cyclic structure located at the C-terminus of the polypeptide chain was obtained for the first time. [ABSTRACT FROM AUTHOR]

Plant xyloglucans play an important role in the processes of cell wall extension, determine their mechanical properties, thus affecting growth and morphology of individual cells and whole organs. Being one of the main components of hemicellulose, xyloglucans play a particular physiological role in woody plants. To study xyloglucan physiological role, transgenic aspen ( Populus tremula L.) plants with a recombinant sp-Xeg gene from the fungus Penicillium canescens were produced. Constitutive expression of this gene in the heterologous surrounding was confirmed by RT-PCR method. The analysis of protein extracts from the leaves of greenhouse-grown plants and microshoots grown in vitro showed activation of xylogluconase in transgenic lines. The strongest activation (1.6-fold) was observed in the leaf extracts (clone PtXVXeg1b) and in vitro microshoots (clone PtXVXeg1c). In transgenic plants, the relative content of pentosans in the wood was declined. In control plants (Pt genotype), it was equal to 148 mg/g dry wt, whereas in tested clones (PtXVXeg1a, PtXVXeg1b, and PtXVXeg1c), it varied from 100 to 140 mg/g dry wt. The strongest decrease (by 31%) in the content of pentosans was observed for the line PtXVXeg1c; the content was equal to 102.1 ± 1.5 mg/g dry wt. A comparative analysis of leaf morphology revealed an increase in the length of petiole and a decrease in the length of the main vein in transgenic lines. In control plants, the ratio of the petiole length to the length of the main vein was equal to 0.49, whereas in transgenic plants, it varied from 0.51 to 0.66. A significant increase of this index was observed in 12 from 14 transgenic lines. [ABSTRACT FROM AUTHOR]

The genes inuA and inu1, encoding two inulinases (32nd glycosyl hydrolase family) from filamentous fungi Aspergillus niger and A. awamori, were cloned into Penicillium canescens recombinant strain. Using chromatographic techniques, endoinulinase InuA (56 kDa, p I 3) and exoinulinase Inu1 (60 kDa, p I 4.3) were purified to homogeneity from the enzymatic complexes of P. canescens new transformants. The properties, such as substrate specificity, pH- and T-optima of activity, stability at different temperatures, influence of cations and anions on the catalytic activity, etc., of both recombinant inulinases were studied. [ABSTRACT FROM AUTHOR]

Penicillium canescens strain F178 is a natural producer of β-galactosidase and endo-1,4-β-xylanase. Transcription of bgaS and xylA genes coding these proteins is subject to carbon catabolite repression. A system of direct selection of P. canescens regulatory mutants has been developed. Two mutant strains from the obtained collection have been studied in detail. Both mutations have been shown to be complemented by the creA gene coding global regulator of carbon catabolite repression in filamentous fungi. Also, creA alleles contain frameshift mutations in the CreA C-domain. It has been found that the xylA gene is derepressed in mutants at the transcription level in the presence of D-glucose. The transcription of the creA gene in mutants is also derepressed proving the effect of autoregulation for this gene. [ABSTRACT FROM AUTHOR]

The F178 strain of Penicillium canescens is a natural producer of beta-galactosidase and endo-1,4-beta-xylanase. Transcription of the genes bgaS and xylA encoding these proteins is affected by the carbon catabolite repression, which occurs in the filamentous fungi, primarily by the transcriptional repressor CreA. The creA gene of P. canescens was cloned and it was demonstrated that its own transcription is influenced by the carbon catabolic repression. Interestingly, the CreA protein remains to be nuclear localized irrespectively of the nature of the carbon source and glucose concentration in the medium. In vitro experiments revealed four CreA binding sites localized within promoter of the bgaS gene, four sites in the xylA gene promoter, and one site in the creA gene promoter. [ABSTRACT FROM AUTHOR]

Using chromatographic technique, xyloglucanase (XG) A (25 kDa, p I 3.5, 12th glycosyl hydrolase family) was isolated from the enzyme complex secreted by the mycelial fungus Penicillium canescens, and xyloglucanases XG 25 (25 kDa, p I 4.1, 12th glycosyl hydrolase family) and XG 70 (70 kDa, p I 3.5, 74th glycosyl hydrolase family) were isolated from the enzyme complex of Penicillium verruculosum. Properties of the isolated enzymes (substrate specificity, optimal ranges of pH and temperature for enzyme activity and stability, effect of metal ions on catalytic activity) were compared with the properties of xyloglucanases XG 32 of Aspergillus japonicus, XG 78 of Chrysosporium lucknowense, and XG of Trichoderma reesei. The gene xegA encoding XG A of P. canescens was isolated, and the amino acid sequence of the corresponding protein was determined. [ABSTRACT FROM AUTHOR]

Gene egl2 of secreted endo-(1–4)-β-glucanase of glycosyl hydrolase family 5 of the mycelial fungus Penicillium canescens was cloned. The gene was expressed in P. canescens under control of a strong promoter of the bgaS gene encoding β-galactosidase of P. canescens, and endoglucanase producing strains were obtained. Chromatographically purified recombinant 48 kDa protein had pH and temperature optima 3.4 and 60°C, respectively, exhibited specific activity of 33 IU, and had Km and Vmax in CM-cellulose hydrolysis of 10.28 g/liter and 0.26 μmol/sec per mg, respectively. [ABSTRACT FROM AUTHOR]

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Under OSMAC strategy, seven unreported multioxidized aromatic polyketides, penicanesins A‒G, were discovered from a soil-derived fungus Penicillium canescens along with seven known compounds. Their structures were assigned by extensive 1D and 2D NMR spectra in combination with HRESIMS and single crystal X-ray diffraction. Absolute stereochemistry of penicanesins A and D were elucidated by theoretical ECD calculation. (±)-Penicanesins A and B are two pairs of racemic aromatic polyketides with an unusual 6/6/6/6 heterotetracyclic ring core. In bioassay, (−)-penicanesin A shows potential cytotoxicity against human cancer cell lines HL-60 and SW480 with IC 50 values at 13.8 ± 0.6 and 12.5 ± 0.9 μ M, respectively, whereas the enantiomer (+)-penicanesin A is less active. [Display omitted] • (±)-Penicanesins A and B, with a 6/6/6/6 tetracyclic core, isolated from Penicillium canescens. • Absolute stereochemistry was determined by theoretical ECD calculation and X-ray diffraction. • (−)-Penicanesin A shows potential cytotoxicity against HL-60 and SW480 cells. Seven unreported multioxidized aromatic polyketides were discovered from a soil-derived fungus Penicillium canescens along with seven known compounds. (−)-Penicanesin A shows potential cytotoxicity against human cancer cell lines HL-60 and SW480 with IC50 values at 13.8 ± 0.6 and 12.5 ± 0.9 μ M respectively, whereas the enantiomer (+)-penicanesin A is less active. [ABSTRACT FROM AUTHOR]

Two α-galactosidases were purified to homogeneity from the enzymatic complex of the mycelial fungus Penicillium canescens using chromatography on different sorbents. Substrate specificity, pH-and temperature optima of activity, stability under different pH and temperature conditions, and the influence of effectors on the catalytic properties of both enzymes were investigated. Genes aglA and aglC encoding α-galactosidases from P. canescens were isolated, and amino acid sequences of the proteins were predicted. In vitro feed testing (with soybean meal and soybean byproducts enriched with galactooligosaccharides as substrates) demonstrated that both α-galactosidases from P. canescens could be successfully used as feed additives. α-Galactosidase A belonging to the 27th glycosyl hydrolase family hydrolyzed galactopolysaccharides (galactomannans) and α-galactosidase C belonging to the 36th glycosyl hydrolase family hydrolyzed galactooligosaccharides (stachyose, raffinose, etc.) of soybean with good efficiency, thus improving the digestibility of fodder. [ABSTRACT FROM AUTHOR]

Pectin lyase A (molecular weight 38 kD by SDS-PAGE, p I 6.7) was purified to homogeneity from culture broth of the myoelial fungus Penicillium canescens using chromatographic techniques. During genomic library screening, the gene encoding pectin lyase A from P. canescens ( pelA) was isolated and sequenced, and the amino acid sequence was generated by applying the multiple alignment procedure (360 residues). A theoretical model for the three dimensional structure of the protein molecule was also proposed. Different properties of pectin lyase A were investigated: substrate specificity, pH-and temperature optimum of activity, stability under different pH and temperature conditions, and the effect of Ca2+ on enzyme activity. In the course of the laboratory trials, it was demonstrated that pectin lyase A from P. canescens could be successfully applied to production and clarification of juice. [ABSTRACT FROM AUTHOR]

The composition of the enzyme complex secreted by Penicillium canescens was investigated. A scheme for purification of the main components of the complex by chromatofocusing on a Mono P column was developed. It was found that along with β-galactosidase, the major components of the complex were endo-β-1,4-xylanase (31 kD, pI 8.2-9.3), α-L-arabinofuranosidase (60 kD, pI 7.6), arabinoxylan-arabinofuranohydrolase (70 kD, pI 3.8-4.0), and endo-β-1,3/1,4-glucanase (40 kD, pI 4.4). The substrate specificity, pH and temperature activity optima, adsorbability, thermal stability, and ability for synergic interaction of the isolated enzymes were studied. [ABSTRACT FROM AUTHOR]

Penicillium canescens has demonstrated the ability to bind high amount of chromium(VI) from aqueous solutions. Cr(VI) adsorption capacity increases with the time during the first 4h and then levels off toward the equilibrium adsorption capacity. Biosorption of Cr(VI) ions reached equilibrium in 4h. Cr(VI) ions binding Penicillium canescens was clearly pH dependent. Cr(VI) loading capacity increased with increasing pH under acidic conditions, presumably as a function of Cr(VI) speciation and due to the H[sup+] competition at same binding sites. The adsorption of Cr(VI) ions reached a plateau value at around pH 6.0 The maximum adsorption capacity of Cr(VI) ions onto the fungal biomass was 34.8mg/g. Elution of Cr(VI) ions was performed using 0.5M HCI. The fungus Penicillium canescens could be used for six cycles for biosorption. © 2003 SDU. All rights reserved. [ABSTRACT FROM AUTHOR]

The effects of a new axial impeller (HTPG4) on oxygen volumetric transfer coefficient, K L a, and xylanase production by Penicillium canescens 10-10c were studied and compared for dual-impeller systems, one with one DT4 impeller below and one HTPG4 above (DT4-HTPG4) and one with two DT4 (DT4-DT4) impellers, in a 5-L bioreactor. The volumetric coefficient of oxygen transfer was measured in culture medium using a gassing-out method at different gassing rates and agitation speeds. We observed that the DT4-HTPG4 combination provided better K L a performance than the DT4-DT4 combination. The two combinations were also tested for their influence on xylanase production by a filamentous microorganism; P. canescens 10-10c. These experiments demonstrated that the DT4-HTPG4 combination impeller enhanced enzyme production up to 23% compared with the DT4-DT4 combination at an aeration rate of 1 vvm and an agitation speed of 600 rpm. The main cause for this difference is thought to be a higher shear stress generated by the DT4-DT4 combination, which damages the mycelium of P. canescens and decreases xylanase production. [ABSTRACT FROM AUTHOR]

The functional groups of Penicillium canescens F-436 b-galactosidase have been identified. The p K values and heats of ionization of these groups and photoinactivation of the enzyme with methylene blue indicate that the active site contains carboxyl and imidazole groups. A mechanism for the participation of these groups in the cleavage of the glycoside bond in lactose is proposed. [ABSTRACT FROM AUTHOR]

Hydrolytic enzymes are highly demanded in the industry. Thermostability is an important property of enzymes that affects the economic costs of the industrial processes. The rational design of GH10 xylanase E (XylE) Penicillium canescens for the thermostability improvement was directed by ΔΔG calculations and structure analysis. Amino acid substitutions with stabilizing values of ΔΔG and providing an increase in side-chain volume of buried residues were performed experimentally. From the six designed substitutions, four substitutions appeared to be stabilizing, one – destabilizing, and one – neutral. For the improved XylE variants, values of T m were increased by 1.1–3.1 °C, and times of half-life at 70 °C were increased in 1.3–1.7-times. Three of the four stabilizing substitutions were located in the N- or the C-terminus region. This highlights the importance of N- and C-terminus for the thermostability of GH10 xylanases and also enzymes with (β/α) 8 TIM barrel type of structure. The criteria of stabilizing values of ΔΔG and increased side-chain volume of buried residues for selection of substitutions may be applied in the rational design for thermostability improvement. [Display omitted] • Substitutions were selected due to stabilizing values of ΔΔG and structure changes. • Buried residues were substituted with residues with increased side-chain volume. • Substitutions in the N- and C-terminus region provided thermostabilization. • T m was increased by 1.1–3.1 °C, t 1/2 at 70 °C was increased in 1.3–1.7-times. [ABSTRACT FROM AUTHOR]

To improve xylanase productivity from Penicillium canescens 10–10c culture, an optimization of oxygen supply is required. Because the strain is sensitive to shear forces, leading to lower xylanase productivity as to morphological alteration, vigorous mixing is not desired. The influence of turbine design, agitation speed, and air flow rate on K1a (global mass transfer coefficient, h-1) and enzyme production is discussed. K1a values increased with agitation speed and air flow rate, whatever the impeller, in our assay conditions. Agitation had more influence on K1a values than air flow, when a disk-mounted blade’s impeller (DT) is used; an opposite result was obtained with a hub-mounted pitched blade’s impeller (PBT). Xylanase production appeared as a function of specific power (W/m3), and an optimum was found in 20 and 100 L STRs fitted with DT impellers. On the other hand, the use of a hub-mounted pitched blade impeller (PBT8), instead of a disk-mounted blade impeller (DT4), reduced the lag time of hemicellulase production and increased xylanase productivity 1.3-fold. [ABSTRACT FROM AUTHOR]

Worldwide, 463 million people are affected by diabetes of which the majority is diagnosed with Type 2 Diabetes (T2D). T2D can ultimately lead to retinopathy, nephropathy, nerve damage, and amputation of the lower extremities. α-Glucosidase, responsible for converting starch to monosaccharides, is a key therapeutic target for the management of T2D. However, due to substantial side effects of currently marketed drugs, there is an urgent need for the discovery of new α-glucosidase inhibitors. In our ongoing efforts to identify novel α-glucosidase inhibitors from Nature, we are investigating the potential of endophytic filamentous fungi as sustainable sources of hits and/or leads for future antihyperglycemic drugs. Here we report one previously unreported xanthone (5) and two known xanthones (7 and 11) as α-glucosidase inhibitors, isolated from an endophytic Penicillium canescens , recovered from fruits of Juniperus polycarpos. The three xanthones 5 , 7 , and 11 showed inhibitory activities against α-glucosidase with IC 50 values of 38.80 ± 1.01 μM, 32.32 ± 1.01 μM, and 75.20 ± 1.02 μM, respectively. Further pharmacological characterization revealed a mixed-mode inhibition for 5 , a competitive inhibition for 7 , while 11 acted as a non-competitive inhibitor. Unlabelled Image [ABSTRACT FROM AUTHOR]