Preparation of ß-galacto-oligosaccharides using a novel endo-1,4-ß-galactanase from Penicillium oxalicum.

Endo-1,4-ß-galactanase is an indispensable tool for preparing prebiotic ß-galacto-oligosaccharides (ß-GOS) from pectic galactan resources. In the present study, a novel endo-1,4-ß-galactanase (PoßGal53) belonging to glycoside hydrolase family 53 from Penicillium oxalicum sp. 68 was cloned and expressed in Pichia pastoris GS115. Upon purification by affinity chromatography, recombinant PoßGal53 exhibited a single band on SDS-PAGE with a molecular weight of 45.0 kDa. Using potato galactan as substrate, PoßGal53 showed optimal reaction conditions of pH 4.0, 40 °C, and was thermostable, retaining >80 % activity after incubating below 45 °C for 12 h. Significantly, PoßGal53 exhibited relatively conserved substrate specificity for (1 â†’ 4)-ß-D-galactan with an activity of 6244 ± 282 U/mg. In this regard, the enzyme is in effect the most efficient endo-1,4-ß-galactanase identified to date. By using PoßGal53, ß-GOS monomers were prepared from potato galactan and separated using medium pressure liquid chromatography. HPAEC-PAD, MALDI-TOF-MS and ESI-MS/MS analyses demonstrated that these ß-GOS species ranged from 1,4-ß-D-galactobiose to 1,4-ß-D-galactooctaose (DP 2-8) with high purity. This work provides not only a highly active tool for enzymatic degradation of pectic galactan, but an efficient protocol for preparing ß-GOS.

Potential biodegradation of polycyclic aromatic hydrocarbons (PAHs) and petroleum hydrocarbons by indigenous fungi recovered from crude oil-contaminated soil in Iran.

A total of 265 fungal individuals were isolated from soils exposed to heavy oil spills in the Yadavaran oil field in Iran to discover indigenous fungal species with a high potential to biodegrade petroleum hydrocarbon pollutants. Morphological and molecular identification of obtained fungal species led to their assignment into 16 genera and 25 species. Alternaria spp. (78%), Fusarium spp. (5%), and Cladosporium spp. (4%) were the most common genera, along with Penicillium spp., Neocamarosporium spp., Epicoccum sp., Kotlabaea sp., Aspergillus sp., Mortierella sp., and Pleurotus sp. A preliminary screening using the DCPIP indicator revealed that approximately 35% of isolates from Alternaria, Epicoccum, Neocamarosporium, Cladosporium, Fusarium, Stachybotrys, Penicillium, and Stemphylium demonstrated promising tolerance to crude oil. The best-performing isolates (12 fungal individuals) were further investigated for their capacity to mineralize a mixture of four polycyclic aromatic hydrocarbons (PAH) for 47 days, quantified by GC-MS. Eventually, two top-performing isolates, namely 5c-12 (Alternaria tenuissima) and 3b-1 (Epicoccum nigrum), were applied to petroleum-contaminated soil. The GC-MS analysis showed that 60 days after inoculation, these isolates successfully degraded more than 70% of the long-chain hydrocarbons in the soil, including C8-C16 n-alkanes, C36 n-alkane, and Pristane. This study introduces two fungal species (5c-12 and 3b-1) with high potential for biodegrading petroleum compounds and PAHs, offering promising prospects for the decontamination of oil-contaminated soil.

Survey of organochlorine-tolerant culturable mycota from contaminated soils, and 2,4-D removal ability of Penicillium species in synthetic wastewater.

Agrochemical wastewater, which is produced by the extensive use of herbicides, has become a serious environmental pollutant. In this study, culturable mycota were isolated from soils contaminated with herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-chloro-2-methylphenoxyacetic acid (MCPA), and their ability to tolerate and remove 2,4-D was assessed. The mycota were isolated on solid medium supplemented with 10 mmol L-1 of MCPA or 2,4-D. Tolerance and removal assays were performed in synthetic wastewater, and removal was quantified by HPLC-UV and MS/MS. Fusarium spp., Aspergillus spp., and Penicillium spp. were the most frequently isolated genera. Six Penicillium strains were able to tolerate up to 25 mmol L-1 of 2,4-D. Within this group, two P. crustosum strains (RCP4 and RCP13) degraded more than 50% of the 2,4-D in the medium during the first 7 days of incubation. Removal percentages reached 54% for RCP4 and 75% for RCP13 after 14 days. These two strains, therefore, could potentially be considered for the design of bioaugmentation strategies aimed at reducing contamination by 2,4-D in wastewater.

Penicillium chrysogenum as a fungal factory for feruloyl esterases.

Plant biomass is a promising substrate for biorefinery, as well as a source of bioactive compounds, platform chemicals, and precursors with multiple industrial applications. These applications depend on the hydrolysis of its recalcitrant structure. However, the effective biological degradation of plant cell walls requires several enzymatic groups acting synergistically, and novel enzymes are needed in order to achieve profitable industrial hydrolysis processes. In the present work, a feruloyl esterase (FAE) activity screening of Penicillium spp. strains revealed a promising candidate (Penicillium rubens Wisconsin 54-1255; previously Penicillium chrysogenum), where two FAE-ORFs were identified and subsequently overexpressed. Enzyme extracts were analyzed, confirming the presence of FAE activity in the respective gene products (PrFaeA and PrFaeB). PrFaeB-enriched enzyme extracts were used to determine the FAE activity optima (pH 5.0 and 50-55 °C) and perform proteome analysis by means of MALDI-TOF/TOF mass spectrometry. The studies were completed with the determination of other lignocellulolytic activities, an untargeted metabolite analysis, and upscaled FAE production in stirred tank reactors. The findings described in this work present P. rubens as a promising lignocellulolytic enzyme producer. KEY POINTS: • Two Penicillium rubens ORFs were first confirmed to have feruloyl esterase activity. • Overexpression of the ORFs produced a novel P. rubens strain with improved activity. • The first in-depth proteomic study of a P. rubens lignocellulolytic extract is shown.

Expression of citrinin biosynthesis gene in Liupao tea and effect of Penicillium citrinum on tea quality.

Similar to Pu-erh tea, Liupao tea is a post-fermented tea that is produced through natural fermentation by microorganisms. Penicillium citrinum is involved in multiple production processes of Liupao tea that can produce citrinin, a secondary metabolite with renal toxicity; however, the effect of P. citrinum on the quality of Liupao tea has not been investigated yet. Citrinin production is regulated by approximately 16 biosynthesis genes. However, little is known about the genetic background of citrinin in the complex Liupao tea system. In the present study, we cultured P. citrinum on potato dextrose agar and Liupao tea powder media and analyzed the changes of its nutritional components in Liupao tea. We selected six citrinin biosynthesis genes identified in Monascus exhibiting homology and high sequence similarity to those in P. citrinum and further analyzed the expression of citrinin biosynthesis genes in Liupao tea and the changes in citrinin yield. The results showed that the changes in nutritional components of Liupao tea were closely related to the growth and metabolism of P. citrinum and the quality of the tea. Decreases in the contents of soluble sugars (from 10.29% to 9.58%), soluble pectins (from 3.71% to 3.13%), free amino acids (from 3.84% to 3.14%), and tea polyphenols (from 22.84% to 18.78%) were noted. The Spearman's correlation analysis indicated that P. citrinum growth can improve the tea quality to some extent. Quantitative real-time PCR demonstrated that ctnA gene was a positive regulator of citrinin production regardless of the culture medium used. ctnA and orf5 expressions greatly influenced the metabolism of citrinin by P. citrinum in Liupao tea. In conclusion, the citrinin biosynthesis genes, ctnA and orf5, may be the promising targets for developing strategies to control P. citrinum infection and citrinin biosynthesis in Liupao tea.

Non-targeted metabolomics reveals the stress response of a cellulase-containing penicillium to uranium.

Human industrial activities have caused environmental uranium (U) pollution, resulting in uranium(VI) had radiotoxicity and chemical toxicity. Here, a cellulase-producing Penicillium fungus was screened and characterized by X-ray fluorescence (XRF), and Fourier transform infrared reflection (FT-IR), as well as by GC/MS metabolomics analysis, to study the response to uranium(VI) stress. The biomass of Penicillium decreased after exposure to 100 mg/L U. Uranium combined with carboxyl groups, amino groups, and phosphate groups to form uranium mineralized deposits on the surface of this fungal strain. The α-activity concentration of uranium in the strain was 2.57×106 Bq/kg, and the ß-activity concentration was 2.27×105 Bq/kg. Metabolomics analysis identified 118 different metabolites, as well as metabolic disruption of organic acids and derivatives. Further analysis showed that uranium significantly affected the metabolism of 9 amino acids in Penicillium. These amino acids were related to the TCA cycle and ABC transporter. At the same time, uranium exhibited nucleotide metabolism toxicity to Penicillium. This study provides an in-depth understanding of the uranium tolerance mechanism of Penicillium and provides a theoretical basis for Penicillium to degrade hyper-enriched plants.

Bioleaching Performance of Titanium from Bauxite Residue Under a Continuous Mode Using Penicillium Tricolor.

The present study performed a continuous mode of bioleaching to investigate the leaching efficiency of Titanium (Ti) from bauxite residue using Penicillium Tricolor at between 4% and 12% pulp densities during a 120-day running. Obtained results of the current study showed that increased pulp density led to a decrease in biomass, dissolved oxygen, and amount of leaching Ti as well as an increase in pH value. Further, it was found that efficiency of bioleaching can be enhanced by increasing the rate of aeration, retention time, and concentration of carbon source. However, it was also evident that, at high pulp density, excessive agitation did not give an expected leaching efficiency but a collapse of biomass. In addition, results of the present study showed that the maximum leaching amount of Ti was 3202 mg/L with a corresponding leaching ratio of 50.35% during the whole bioleaching process. Moreover, it was noted that the biomass showed a significant negative correlation with the pH value and dissolved oxygen. However, the biomass showed a significant positive correlation with leaching amount of Ti and thus indicate that microbial metabolic activities are the uppermost factor affecting the continuous leaching performance.

Succession of the wheat seed-associated microbiome as affected by soil fertility level and introduction of Penicillium and Bacillus inoculants in the field.

During germination, the seed releases nutrient-rich exudates into the spermosphere, thereby fostering competition between resident microorganisms. However, insight into the composition and temporal dynamics of seed-associated bacterial communities under field conditions is currently lacking. This field study determined the temporal changes from 11 to 31 days after sowing in the composition of seed-associated bacterial communities of winter wheat as affected by long-term soil fertilization history, and by introduction of the plant growth-promoting microbial inoculants Penicillium bilaiae and Bacillus simplex. The temporal dynamics were the most important factor affecting the composition of the seed-associated communities. An increase in the relative abundance of genes involved in organic nitrogen metabolism (ureC and gdhA), and in ammonium oxidation (amoA), suggested increased mineralization of plant-derived nitrogen compounds over time. Dynamics of the phosphorus cycling genes ppt, ppx and cphy indicated inorganic phosphorus and polyphosphate cycling, as well as phytate hydrolysis by the seed-associated bacteria early after germination. Later, an increase in genes for utilization of organic phosphorus sources (phoD, phoX and phnK) indicated phosphorus limitation. The results indicate that community temporal dynamics are partly driven by changed availability of major nutrients, and reveal no functional consequences of the added inoculants during seed germination.

Endophytic fungi stimulate the concentration of medicinal secondary metabolites in houttuynia cordata thunb.

Endophytic fungi usually establish a symbiotic relationship with the host plant and affect its growth. In order to evaluate the impact of endophytic fungi on the Chinese herbal medicinal plant Houttuynia cordata Thunb., three endophytes isolated from the rhizomes of H. cordata, namely Ilyonectria liriodendra (IL), unidentified fungal sp. (UF), and Penicillium citrinum (PC), were co-cultured individually with H. cordata in sterile soil for 60 days. Analysis of the results showed that the endophytes stimulated the host plant in different ways: IL increased the growth of rhizomes and the accumulation of most of the phenolics and volatiles, UF promoted the accumulation of the medicinal compounds afzelin, decanal, 2-undecanone, and borneol without influencing host plant growth, and PC increased the fresh weight, total leaf area and height of the plants, as well as the growth of the rhizomes, but had only a small effect on the concentration of major secondary metabolites. Our results proved that the endophytic fungi had potential practical value in terms of the production of Chinese herbal medicines, having the ability to improve the yield and accumulation of medicinal metabolites.

Metabolite fingerprinting, in vitro antimicrobial and antioxidant activities and in-silico docking in Alloteropsis cimicina and its endophytic fungus Penicillium pinophilum.

Endophytic fungal interaction with medicinal plant resulting in the production of bioactive compounds influenced the present study. Endophytic fungus Penicillium pinophilum Hedgc. from Alloteropsis cimicina with high incidence was isolated by incubation methods and characterized by morphological and molecular (ITS rDNA region) methods. Penicillium pinophilum was cultured on PD broth and metabolites of host and endophyte were extracted with ethyl acetate and methanol. Metabolites were assayed for antimicrobial potential by well diffusion and scavenging ability by spectrophotometric and electrochemical methods. Metabolite profiling by Orbitrap High-Resolution Liquid Chromatography-Mass Spectrometry and Fourier-transform infrared spectroscopy and by validation of antimicrobial and antioxidant activities in-silico drug-likeness of spectral compound prediction and molecular docking were performed. Host and P. pinophilum extracts showed strong antimicrobial potential against certain clinical bacterial strains and Fusarium oxysporum. Fungal extracts exhibited higher antioxidant activity than A. cimicina extract. Metabolite profiling indicated 14 and 21 antimicrobial, 10 and 13 antioxidant compounds in A. cimicina and P. pinophilum extracts, respectively. There were eight spectral compounds common to endophyte and host with high binding affinity towards receptors. The present study revealed that P. pinophilum and A. cimicina are natural reservoirs of novel bioactive compounds with antimicrobial and antioxidant properties.

Comparison of characteristic components in tea-leaves fermented by Aspergillus pallidofulvus PT-3, Aspergillus sesamicola PT-4 and Penicillium manginii PT-5 using LC-MS metabolomics and HPLC analysis.

Microbiota influenced quality formation of ripened Pu-erh tea. To understand the effect of each tea-derived fungal strain, tea-leaves were fermented by Aspergillus pallidofulvus PT-3 (ApaPT), Aspergillus sesamicola PT-4 (AsePT) and Penicillium manginii PT-5 (PmaPT), respectively. 14 Phenolic compounds, 3 purine alkaloids, 19 free amino acids and γ-aminobutyric acid contents were determined by HPLC and amino acid analyzer analysis. Additionally, UHPLC-Q-TOF/MS method was developed for LC-MS metabolomics analysis. Multivariate statistical analyses, such as PCA and HCA, exhibited that the chemical profile of PmaPT fermentation was similar to biocidal treatment, but had significant differences with ApaPT and AsePT fermentation. The differentiated metabolites (VIP > 1, p < 0.05 and FC > 1.50 or < 0.66) and one-way ANOVA revealed the impact of three fungal strains in tea-leaves fermentation. APaPT and AsePT contributed to biosynthesis of gallic acid and several flavonoids, such as kaempferol, quercetin and myricetin in the metabolism of phenolic compounds.

Capability of Penicillium oxalicum y2 to release phosphate from different insoluble phosphorus sources and soil.

Due to insufficient amount of soluble phosphate and poor persistence of traditional chemical phosphate fertilizers in agricultural soils, the eco-friendly and sustainable phosphorus sources for crops are urgently required. The efficient phosphate-releasing fungal strain designated y2 was isolated and identified by the internal transcribed spacer of rDNA as Penicillium oxalicum y2. When lecithin, Ca3(PO4)2, or ground phosphate rock were separately used as sole phosphorus source, different phosphate-releasing modes were observed. The strain y2 was able to release as high as 2090 mg/L soluble phosphate within 12 days of incubation with Ca3(PO4)2 as sole phosphorus source. In the culture solution, high concentration of oxalic, citric, and malic acids and high phosphatase activity were detected. The organic acids contributed to solubilizing inorganic phosphate sources, while phosphatase was in charge of the mineralization of organic phosphorus lecithin. Afterwards, the fungus culture was applied to the soil with rape growing. During 50 days of incubation, the soil's available phosphate concentration increased by three times compared with the control, the dry weight of rape increased by 78.73%, and the root length increased by 38.79%. The results illustrated that P. oxalicum y2 possessed both abilities of solubilizing inorganic phosphorus and mineralizing organic phosphorus, which have great potential application in providing biofertilizer for modern agriculture.

Enhanced bio-recovery of aluminum from low-grade bauxite using adapted fungal strains.

Filamentous fungi have been proved to have a pronounced capability to recover metals from mineral ores. However, the metal recovery yield is reduced due to toxic effects triggered by various heavy metals present in the ore. The current study highlights the fungal adaptations to the toxic effects of metals at higher pulp densities for the enhanced bio-recovery of aluminum from low-grade bauxite. In the previous studies, a drastic decrease in the aluminum dissolution was observed when the bauxite pulp density was increased from 1 to 10% (w/v) due to the high metal toxicity and low tolerance of Aspergillus niger and Penicillium simplicissium to heavy metals. These fungi were adapted in order to increase heavy metal tolerance of these fungal strains and also to get maximum Al dissolution. A novel approach was employed for the adaptation of fungal strains using a liquid growth medium containing 5% bauxite pulp density supplemented with molasses as an energy source. The mycelia of adapted strains were harvested and subsequently cultured in a low-cost oat-agar medium. Batch experiments were performed to compare the aluminum leaching efficiencies in the direct one-step and the direct two-step bioleaching processes. FE-SEM analysis revealed the direct destructive and corrosive action by the bauxite-tolerant strains due to the extension and penetration of the vegetative mycelium filaments into the bauxite matrix. XRD analysis of the bioleached bauxite samples showed a considerable decline in oxide minerals such as corundum and gibbsite. Results showed a high amount of total Al (≥ 98%) was successfully bioleached and solubilized from low-grade bauxite by the adapted fungal strains grown in the presence of 5% pulp density and molasses as a low-cost substrate. Graphical abstract.

Bioremediation of water contaminated with uranium using Penicillium piscarium.

Penicillium piscarium can be indicated as promising in the treatment of sites contaminated with uranium. Thus, this research aimed to analyze the P. piscarium dead biomass in uranium biosorption. This fungus was previously isolated from a highly contaminated uranium mine located in Brazil. Biosorption tests were carried out at pH 3.5 and 5.5 in solutions contaminated with concentrations of 1 to 100 mg/L of uranium nitrate. Our results showed that the dead biomass of P. piscarium was able to remove between 93.2 and 97.5% uranium from solutions at pH 3.5, at the end of the experiment, the pH of the solution increased to values above 5.6. Regarding the experiments carried out in solutions with pH 5.5, the dead biomass of the fungus was also able to remove between 38 and 92% uranium from the solution, at the end of the experiment, the pH of the solution increased to levels above 6.5. The analysis of electron microscopy, Energy-dispersive spectroscopy, and X-ray fluorescence demonstrated the high concentration of uranium precipitated on the surface of the fungal biomass. These results were impressive and demonstrate that the dead biomass of P. piscarium can be an important alternative to conventional processes for treating water contaminated with heavy metals, and we hope that these ecofriendly, inexpensive, and effective technologies be encouraged for the safe discharge of water from industrial activities.

Biotransformation of Erythrodiol for New Food Supplements with Anti-Inflammatory Properties.

Erythrodiol, a typical pentacyclic triterpenic diol in olive oil and its byproduct, olive pomace, frequently appears in food additives for the prevention of cardiovascular diseases because of its antioxidation, anti-inflammatory, and antitumor activities. To develop new derivatives of erythrodiol (1), preparative biotransformations were investigated through Streptomyces griseus ATCC 13273, Penicilium griseofulvum CICC 40293, and Bacillus subtilis ATCC 6633, and ten new (1a-1j) and one known metabolites were isolated. Their structures were elucidated by high resolution electrospray ionization mass spectrometry (HR-ESI-MS) and one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy. Furthermore, relative to 1, most metabolites exhibited lower toxicity and more potent inhibitory activities against nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. In particular, the glycosylated metabolite 1k showed a dramatically increased inhibitory effect with an IC50 value of 2.40 µM, which is even lower than that of quercetin. Thus, biotransformation of erythrodiol is a viable strategy for discovering new triterpenes as food supplements with anti-inflammatory properties.

[Fermentation of cellulase with multiple types of Salvia miltiorrhiza residues and other solid wastes from Chinese materia medica industrialization].

The solid wastes of Chinese materia dedica industrialization represented by Salvia miltiorrhiza residues have a strong small-molecule bio-recalcitrance in the process of high-value utilization of biotransformation. Highly tolerant strains were bred to break bio-recalcitrance of Salvia miltiorrhiza residues and produce high-value added cellulose, which has a significant significance for recycling and industrial utilization of solid waste. In this study, a strain of fungus, Penicillium expansum SZ13, was found with small-molecule antibacterial substance tanshinone contained in Salvia miltiorrhiza residues by a biological method. The optimal enzyme production process and peak period of SZ13 were determined. It was found that SZ13 could maintain peak enzyme production for 5 days by degrading residues under the conditions of temperature 35 ℃, rotation speed 180 r·min~(-1), 5% of residues addition, and 5% seed solution addition. Meanwhile, the ability of SZ13 to degrade the enzyme production of multiple types of residues was explored. The results showed a high enzyme activity and stable enzyme production of SZ13 in the process of degrading residues. SZ13 could efficiently utilize various types of Chinese medicine residues, such as Salvia miltiorrhiza residues, to realize the high-value utilization of cellulose in multiple types of residues.

Different Effects of Soil Fertilization on Bacterial Community Composition in the Penicillium canescens Hyphosphere and in Bulk Soil.

This study investigated the effects of long-term soil fertilization on the composition and potential for phosphorus (P) and nitrogen (N) cycling of bacterial communities associated with hyphae of the P-solubilizing fungus Penicillium canescens Using a baiting approach, hyphosphere bacterial communities were recovered from three soils that had received long-term amendment in the field with mineral or mineral plus organic fertilizers. P. canescens hyphae recruited bacterial communities with a decreased diversity and an increased abundance of Proteobacteria relative to what was observed in soil communities. As core bacterial taxa, Delftia and Pseudomonas spp. were present in all hyphosphere samples irrespective of soil fertilization. However, the type of fertilization showed significant impacts on the diversity, composition, and distinctive taxa/operational taxonomic units (OTUs) of hyphosphere communities. The soil factors P (Olsen method), exchangeable Mg, exchangeable K, and pH were important for shaping soil and hyphosphere bacterial community compositions. An increased relative abundance of organic P metabolism genes was found in hyphosphere communities from soil that had not received P fertilizers, which could indicate P limitation near the fungal hyphae. Additionally, P. canescens hyphae recruited bacterial communities with a higher abundance of N fixation genes than found in soil communities, which might imply a role of hyphosphere communities for fungal N nutrition. Furthermore, the relative abundances of denitrification genes were greater in several hyphosphere communities, indicating an at least partly anoxic microenvironment with a high carbon-to-N ratio around the hyphae. In conclusion, soil fertilization legacy shapes P. canescens hyphosphere microbiomes and their functional potential related to P and N cycling.IMPORTANCE P-solubilizing Penicillium strains are introduced as biofertilizers to agricultural soils to improve plant P nutrition. Currently, little is known about the ecology of these biofertilizers, including their interactions with other soil microorganisms. This study shows that communities dominated by Betaproteobacteria and Gammaproteobacteria colonize P. canescens hyphae in soil and that the compositions of these communities depend on the soil conditions. The potential of these communities for N and organic P cycling is generally higher than that of soil communities. The high potential for organic P metabolism might complement the ability of the fungus to solubilize inorganic P, and it points to the hyphosphere as a hot spot for P metabolism. Furthermore, the high potential for N fixation could indicate that P. canescens recruits bacteria that are able to improve its N nutrition. Hence, this community study identifies functional groups relevant for the future optimization of next-generation biofertilizer consortia for applications in soil.

Biotechnological potential of an exo-polygalacturonase of the new strain Penicillium janthinellum VI2R3M: biochemical characterization and clarification of fruit juices.

AIMS: The aim of this work was to characterize and apply a polygalacturonase of Penicillium janthinellum new strain VI2R3M. METHODS AND RESULTS: The polygalacturonase obtained from P. janthinellum VI2R3M was incubated in cultures of passion fruit peel and was partially purified by ion-exchange chromatography and gel filtration. The enzyme showed a relative molecular mass of 102·0 kDa, maximum activity at pH 5·0, temperature of 50°C, 100% stablity at 50°C and 80% stablity at pH 3·0-5·0. The apparent Km , Vmax and Kcat values for hydrolyzing polygalacturonic acid were 2·56 mg ml-1 , 163·1 U mg-1 and 277 s-1 respectively. The polygalacturonase presented exo activity and was activated by Mg2+ . The juices treated with polygalacturonase presented increases in transmittance with reduction in colour. CONCLUSIONS: The results suggest that the new lineage P. janthinellum VI2R3M presents a high yield of an exo-polygalacturonase induced by agro-industrial residues, with excellent activity and stability in acidic pH and at 50°C. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of agro-industrial residue to obtain the polygalacturonase can contribute to a decrease enzyme production cost. The results of the activity, stability to acidic pH and excellent performance in the clarification of juices show that the enzyme is promising for industrial application.

Indole Diterpenoids from an Endophytic Penicillium sp.

A chemical investigation of the endophyte Penicillium sp. (strain ZO-R1-1), isolated from roots of the medicinal plant Zingiber officinale, yielded nine new indole diterpenoids (1-9), together with 13 known congeners (10-22). The structures of the new compounds were elucidated by 1D and 2D NMR analysis in combination with HRESIMS data. The absolute configuration of the new natural products 1, 3, and 7 was determined using the TDDFT-ECD approach and confirmed for 1 by single-crystal X-ray determination through anomalous dispersion. The isolated compounds were tested for cytotoxicity against L5178Y, A2780, J82, and HEK-293 cell lines. Compound 1 was the most active metabolite toward L5178Y cells, with an IC50 value of 3.6 µM, and an IC50 against A2780 cells of 8.7 µM. Interestingly, 1 features a new type of indole diterpenoid scaffold with a rare 6/5/6/6/6/6/5 heterocyclic system bearing an aromatic ring C, which is suggested to be important for the cytotoxic activity of this natural product against L5278Y and A2780 cells.

Evaluation of diclofenac biodegradation by the ascomycete fungus Penicillium oxalicum at flask and bench bioreactor scales.

Diclofenac (DFC) is a common anti-inflammatory drug, and has attracted the significant attention due to its massive use around the world and its environmental impact. In this work, we describe for the first time the use of Penicillium oxalicum, an ascomycetes fungus, for the biotransformation of DFC at flask and bench bioreactor scales. We present a complete study of the role of enzymes, metabolic pathway, acute toxicity assays and comparison between free and immobilised biomass. Pellets of P. oxalicum degraded 100 µM of DFC within 24 h, and the activity of CYP450 enzymes was key for the elimination of the drug. The scaling-up to bench bioreactor was optimised by the reduction of nutrients, and characterising the actions of free pellets, polyurethane foam- and plastic K1-immobilised biomass revealed free pellets to be the most efficient DFC removal system (total elimination occurred in 36 h). Hydroxylated metabolites were detected during the process, suggesting that a mixture of biological and physical processes were involved in the elimination of DFC. The use of P. oxalicum reduced the acute toxicity of the medium supplemented with diclofenac and represents a novel and attractive alternative for the elimination of pharmaceutical compounds.