Luteapyrone,a Novel ƴ-Pyrone Isolated from the Filamentous Fungus Metapochonia lutea

In the process of screening for new bioactive microbial metabolites we found a novel ƴ-pyrone derivative for which we propose the trivial name luteapyrone, in a recently described microscopic filamentous fungus, Metapochonia lutea BiMM-F96/DF4. The compound was isolated from the culture extract of the fungus grown on modified yeast extract sucrose medium by means of flash chromatography followed by preparative HPLC. The chemical structure was elucidated by NMR and LC-MS. The new compound was found to be non-cytotoxic against three mammalian cell lines (HEK 263, KB-3.1 and Caco-2). Similarly, no antimicrobial activity was observed in tested microorganisms (gram positive and negative bacteria, yeast and fungi).     

An Indigenous Hyperproductive Species of Aureobasidium pullulans RYLF-10: Influence of Fermentation Conditions on Exopolysaccharide (EPS) Production

In recent years, a significant interest has been generated in discovering and developing exopolysaccharides (EPS) produced by microorganisms, especially fungi due to their multifaceted industrial and pharmacological applications. A number of filamentous and cellular fungi have been explored for this; however, according to the existing literature, the work on exopolysaccharide production by indigenous culture on this aspect is still very less and requires a serious attention. The present work is an attempt in this regard and aims to optimize the submerged culture conditions to produce the exopolysaccharides from an indigenous yeast Aureobasidium pullulans RYLF-10 with respect to several operating parameters in shake flask fermentation. The yeast A. pullulans RYLF-10 was identified by 18s RNA sequencing and detailed study on its nutritional requirements, and environmental conditions for submerged culture have been optimized. The optimal temperature and pH for both the vegetative growth and EPS production were found to be 28?±?1 °C and 5.0, respectively, while the agitation speed and inoculum size were reported to be 150 rpm and 1 % (v/v), respectively. Sucrose (50 g/l) and yeast extract (1 g/l) were found to be the most suitable carbon and nitrogen sources which worked best in the ratio of 60:1 and resulted in the maximum EPS yield. Similarly, the other variables like growth regulator (riboflavin) and minerals (NaCl?+?K₂HPO₄?+?MgSO₄) altogether resulted in a noteworthy EPS yield of 45.24 g/l which is the maximum yield from this indigenous isolate of A. pullulans RYLF-10.     

A new pentanorlanostane derivative, cladosporide A, as a characteristic antifungal agent against Aspergillus fumigatus, isolated from Cladosporium sp

In the course of searching for new antifungal agents, a new pentanorlanostane derivative, cladosporide A (1), was isolated along with ergosterol, ergosterol peroxide and 23,24,25,26,27-pentanorlanost-8-ene-3beta,22-diol (2) from Cladosporium sp. as a characteristic antifungal agent against the human pathogenic filamentous fungus Aspergillus fumigatus. The structure of 1 was established as 3beta,22-dihydroxy-23,24,25,26,27-pentanorlanostane-29-al by spectroscopic and chemical investigation and X-ray crystallographic analysis. Inhibitory activity against A. fumigatus (IC80 0.5-4.0 microg/ml) was observed for cladosporide A (1), but no activity was observed against pathogenic yeasts, Candida albicans and Cryptococcus neoformans, and other pathogenic filamentous fungi, Aspergillus niger and A. flavus. The 4beta-aldehyde residue in 1 might be essential for the antifungal activity, since 23,24,25,26,27-pentanorlanost-8-ene-3beta,22-diol (2) showed no inhibition against the above four fungi.     

Epipyrone A,a Broad-Spectrum Antifungal Compound Produced by Epicoccum nigrum ICMP 19927

We have isolated a filamentous fungus that actively secretes a pigmented exudate when growing on agar plates. The fungus was identified as being a strain of Epicoccum nigrum. The fungal exudate presented strong antifungal activity against both yeasts and filamentous fungi, and inhibited the germination of fungal spores. The chemical characterization of the exudate showed that the pigmented molecule presenting antifungal activity is the disalt of epipyrone A—a water-soluble polyene metabolite with a molecular mass of 612.29 and maximal UV–Vis absorbance at 428 nm. This antifungal compound showed excellent stability to different temperatures and neutral to alkaline pH.     

Asymmetric microbial conversion of (E)-2-benzylideneindan-1-one by the filamentous fungi Botrytis cinerea,Trichoderma viride,and Eutypa lata

The transformation of (E)-2-benzylideneindan-1-one 1 by the filamentous fungi Botrytis cinerea, Trichoderma viride, and Eutypa lata as biocatalysts was studied. The results showed the catalytic potential of these fungi in affording several hydroxylation and reduction products, three of them reported here for the first time. The absolute configuration of enantiomerically pure 2-benzylindane derivatives was determined.     

The Isolation and Characterization of Antagonist Trichoderma spp. from the Soil of Abha,Saudi Arabia

Background: The genus Trichoderma is widely spread in the environment, mainly in soils. Trichoderma are filamentous fungi and are used in a wide range of fields to manage plant patho-genic fungi. They have proven to be effective biocontrol agents due to their high reproducibility, adaptability, efficient nutrient mobilization, ability to colonize the rhizosphere, significant inhibitory effects against phytopathogenic fungi, and efficacy in promoting plant growth. In the present study, the antagonist Trichoderma isolates were characterized from the soil of Abha region, Saudi Arabia. Methodology: Soil samples were collected from six locations of Abha, Saudi Arabia to isolate Trichoderma having the antagonistic potential against plant pathogenic fungi. The soil dilution plate method was used to isolate Trichoderma (Trichoderma Specific Medium (TSM)). Isolated Trichoderma were evaluated for their antagonistic potential against Fusarium oxysporum, Alternaria alternata and Helminthosporium rostratum. The antagonist activity was assessed by dual culture assay, and the effect of volatile metabolites and culture filtrate of Trichoderma. In addition, the effect of different temperature and salt concentrations on the growth of Trichoderma isolates were also evaluated. Results: The most potent Trichoderma species were identified by using ITS4 and ITS 5 primers. Total 48 Trichoderma isolates were isolated on (TSM) from the soil samples out of those six isolates were found to have antagonist potential against the tested plant pathogenic fungi. In general, Trichoderma strains A (1) 2.1 T, A (3) 3.1 T and A (6) 2.2 T were found to be highly effective in reducing the growth of tested plant pathogenic fungi. Trichoderma A (1) 2.1 T was highly effective against F. oxysporum (82%), whereas Trichoderma A (6) 2.2 T prevented the maximal growth of H. rostratum (77%) according to the dual culture data. Furthermore, Trichoderma A (1) 2.1 T volatile metabolites hindered F. oxysporum growth. The volatile metabolite of Trichoderma A (6) 2.2 T, on the other hand, had the strongest activity against A. alternata (45%). The Trichoderma A (1) 2.1 T culture filtrate was proven to be effective in suppressing the growth of H. rostratum (47%). The temperature range of 26 °C to 30 °C was observed to be optimum for Trichoderma growth. Trichoderma isolates grew well at salt concentrations (NaCl) of 2%, and with the increasing salt concentration the growth of isolates decreased. The molecular analysis of potent fungi by ITS4 and ITS5 primers confirmed that the Trichoderma isolates A (1) 2.1 T, A (3) 3.1 and A (6) 2.2 T were T. harzianum, T. brevicompactum, and T. velutinum, respectively. Conclusions: The study concludes that the soil of the Abha region contains a large population of diverse fungi including Trichoderma, which can be explored further to be used as biocontrol agents.     

Methods for isolation of chitin-glucan complexes from higher fungi native biomass

Chitin-containing materials have been isolated from the biomass of Armillariella mellea natural higher fungi (Mari El, Russia) and have been identified with the use of a new simplified procedure based on a domestic detergent and soft reagents and two standard methods. The properties of the materials are compared with those of pure chitin isolated from crustaceans. Variations in the semiproduct composition (the contents of water, mineral and organic substances, total nitrogen, and D-glucosamine) during the isolation process are monitored. The advantages of the new procedure are demonstrated; it enables preservation of the native properties of the material in the course of chitin isolation. It has been shown that Armillariella mellea fungus may be used as a potential raw material for chitin production.     

Transformation of Cinchona alkaloids into 1-N-oxide derivatives by endophytic Xylaria sp isolated from Cinchona pubescens

The microbial transformation of four Cinchona alkaloids (quinine, quinidine, cinchonidine, and cinchonine) by endophytic fungi isolated from Cinchona pubescens was investigated. The endophytic filamentous fungus Xylaria sp. was found to transform the Cinchona alkaloids into their 1-N-oxide derivatives.     

Total Aseptization of Boar Semen,to Increase the Biosecurity of Reproduction in Swine

The aim of the study was to establish the complete microbiological profile of boar semen (Sus scrofa domesticus) and to choose the most effective antiseptic measures in order to control and optimize AI reproduction in pig farms. One hundred and one semen samples were collected and analyzed from several pig farms. The microbiological profile of ejaculates was determined by evaluating the degree of contamination of fresh semen and after dilution with specific extenders. The bacterial and fungal load of fresh boar semen recorded an average value of 82.41/0.149 × 10³ CFU/mL, while after diluting the ejaculates the contamination value was 0.354/0.140 × 10³ CFU/mL. Twenty-four germs (15 bacterial and 9 fungal species) were isolated, the most common being Candida parapsilosis/sake (92%) and Escherichia coli (81.2%). Modification of the sperm collection protocol (HPBC) reduced contamination in raw sperm by 49.85% in bacteria (significant (p < 0.00001) and by 9.67% in fungi (non-significant (p < 0.111491). The load in bacteria and filamentous fungi can be controllable, but not in levuras fungi. Some fluconazole-added extenders (12.5 mg%), ensure fungal aseptization, and even an increase in sperm progressivity (8.39%) for at least a 12 h shelf life after dilution. Validation of sperm aseptization was done by maintaining sow fecundity unchanged after AI (insignificant p > 0.05).     

Structural insights of a cellobiose dehydrogenase enzyme from the basidiomycetes fungus Termitomyces clypeatus

Filamentous fungi secrete various oxidative enzymes to degrade the glycosidic bonds of polysaccharides. Cellobiose dehydrogenase (CDH) (E.C.1.1.99.18) is one of the important lignocellulose degrading enzymes produced by various filamentous fungi. It contains two stereo specific ligand binding domains, cytochrome and dehydrogenase - one for heme and the other for flavin adenine dinucleotide (FAD) respectively. The enzyme is of commercial importance for its use in amperometric biosensor, biofuel production, lactose determination in food, bioremediation etc. Termitomyces clypeatus, an edible fungus belonging to the basidiomycetes group, is a good producer of CDH. In this paper we have analyzed the structural properties of this enzyme from T. clypeatus and identified a distinct carbohydrate binding module (CBM) which is not present in most fungi belonging to the basidiomycetes group. In addition, the dehydrogenase domain of T. clypeatus CDH exhibited the absence of cellulose binding residues which is in contrast to the dehydrogenase domains of CDH of other basidiomycetes. Sequence analysis of cytochrome domain showed that the important residues of this domain were conserved like in other fungal CDHs. Phylogenetic tree, constructed using basidiomycetes and ascomycetes CDH sequences, has shown that very surprisingly the CDH from T. clypeatus, which is classified as a basidiomycetes fungus, is clustered with the ascomycetes group. A homology model of this protein has been constructed using the CDH enzyme of ascomycetes fungus Myricoccum thermophilum as a template since it has been found to be the best match sequence with T. clypeatus CDH. We also have modelled the protein with its substrate, cellobiose, which has helped us to identify the substrate interacting residues (L354, P606, T629, R631, Y649, N732, H733 and N781) localized within its dehydrogenase domain. Our computational investigation revealed for the first time the presence of all three domains - cytochrome, dehydrogenase and CBM - in the CDH of T. clypeatus, a basidiomycetes fungus. In addition to discovering the unique structural attributes of this enzyme from T. clypeatus, our study also discusses the possible phylogenetic status of this fungus.     

Gastrodin Production from p-2-Hydroxybenzyl Alcohol Through Biotransformation by Cultured Cells of Aspergillus foetidus and Penicillium cyclopium

The objective of this work was to take advantage of the resting cells of suitable fungus as an in vitro model to prepare gastrodin from p-2-hydroxybenzyl alcohol (HBA), which mainly exists in the metabolites of the plant Gastrodia elata Blume. The one-step biotransformation of HBA into gastrodin was examined with the filamentous fungi cells of Aspergillus foetidus and Penicillium cyclopium AS 3.4513 in this study. The fundamental conditions of biotransformation were screened and compared for both fungi. P. cyclopium AS 3.4513 had better gastrodin-producing capability than A. foetidus through one-step bioconversion. The highest yield of gastrodin was 36 mg/L for A. foetidus ZU-G1 and 65 mg/L for P. cyclopium AS 3.4513 under the respective development condition during 6 days of biotransformation. The comparative results show that P. cyclopium AS 3.4513 reveals great potential to form gastrodin using HBA as the precursor. The products catalyzed by the resting cells of P. cyclopium AS 3.4513 were identified through NMR and ESI-MS. Current results can be applied not only to the chemical synthesis processes that may involve the hydroxylation reaction but also to the industrial production. The selected fungus is the potential biocatalyst for HBA glucosylation.     

Polyamines Upregulate Cephalosporin C Production and Expression of β-Lactam Biosynthetic Genes in High-Yielding Acremonium chrysogenum Strain

The high-yielding production of pharmaceutically significant secondary metabolites in filamentous fungi is obtained by random mutagenesis; such changes may be associated with shifts in the metabolism of polyamines. We have previously shown that, in the Acremonium chrysogenum cephalosporin C high-yielding strain (HY), the content of endogenous polyamines increased by four- to five-fold. Other studies have shown that the addition of exogenous polyamines can increase the production of target secondary metabolites in highly active fungal producers, in particular, increase the biosynthesis of β-lactams in the Penicillium chrysogenum Wis 54–1255 strain, an improved producer of penicillin G. In the current study, we demonstrate that the introduction of exogenous polyamines, such as spermidine or 1,3-diaminopropane, to A. chrysogenum wild-type (WT) and HY strains, leads to an increase in colony germination and morphological changes in a complete agar medium. The addition of 5 mM polyamines during fermentation increases the production of cephalosporin C in the A. chrysogenum HY strain by 15–20% and upregulates genes belonging to the beta-lactam biosynthetic cluster. The data obtained indicate the intersection of the metabolisms of polyamines and beta-lactams in A. chrysogenum and are important for the construction of improved producers of secondary metabolites in filamentous fungi.     

Separation and Immobilization of Lipase from Penicillium simplicissimum by Selective Adsorption on Hydrophobic Supports

Lipases are an enzyme class of a great importance as biocatalysts applied to organic chemistry. However, it is still necessary to search for new enzymes with special characteristics such as good stability towards high temperatures, organic solvents, and high stereoselectivity presence. The present work’s aim was to immobilize the lipases pool produced by Penicillium simplissicimum, a filamentous fungi strain isolated from Brazilian babassu cake residue. P. simplissicimum lipases were separated into three different fractions using selective adsorption method on different hydrophobic supports (butyl-, phenyl-, and octyl-agarose) at low ionic strength. After immobilization, it was observed that these fractions’ hyperactivation is in the range of 131% to 1133%. This phenomenon probably occurs due to enzyme open form stabilization when immobilized onto hydrophobic supports. Those fractions showed different thermal stability, specificity, and enantioselectivity towards some substrates. Enantiomeric ratio for the hydrolysis of (R,S) 2-O-butyryl-2-phenylacetic acid ranged from 1 to 7.9 for different immobilized P. simplissicimum lipase fractions. Asymmetry factor for diethyl 2-phenylmalonate hydrolysis ranged from 11.8 to 16.4 according to the immobilized P. simplissicimum lipase fractions. Those results showed that sequential adsorption methodology was an efficient strategy to obtain new biocatalysts with different enantioselectivity degrees, thermostability, and specificity prepared with a crude extract produced by a simple and low-cost technology.     

New phomalone derivatives from the endolichenic fungus Cochliobolus kusanoi in Ny‐Alesund Arctic

Natural products from endolichenic fungi, isolated from different lichen species, have been attracting much attention because of their potential to produce metabolites with activities and/or new structures. The filamentous fungus M227‐2 isolated from lichen in Ny‐Alesund Arctic showed significant anti‐microbial activity and was identified as Cochliobolus kusanoi based on 18S rDNA. Secondary metabolites of M227‐2 were investigated in order to exploit them as lead compounds for new antibiotics. Chemical investigation on M227‐2 led to the isolation of three phomalone derivatives (products 1 – 3) . Their structures were elucidated on the basis of their NMR and MS spectral data. Product 2 was new, whereas products 1 and 3 were isolated for the first time from the species. The biosynthetic pathway is also proposed.     

Statistical Analysis and Modeling of Pelletized Cultivation of Mucor circinelloides for Microbial Lipid Accumulation

Microbial oil accumulation via oleaginous fungi has some potential benefits because filamentous fungi can form pellets during cell growth and these pellets are easier to harvest from the culture broth than individual cells. This research studied the effect of various culture conditions on the pelletized cell growth of Mucor circinelloides and its lipid accumulation. The results showed that cell pelletization was positively correlated to biomass accumulation; however, pellet size was negatively correlated to the oil content of the fungal biomass, possibly due to the mass transfer barriers generated by the pellet structure. How to control the size of the pellet is the key to the success of the pelletized microbial oil accumulation process.     

Screening and Production of Ligninolytic Enzyme by a Marine-Derived Fungal Pestalotiopsis sp. J63

Marine-derived fungi are prone to produce structurally unique secondary metabolites, a considerable number of which display the promising biological properties and/or industrial applications. Among those, ligninolytic enzymes have attracted great interest in recent years. In this work, about 20 strains were isolated from sea mud samples collected in the East China Sea and then screened for their capacity to produce lignin-degrading enzymes. The results showed that a strain, named J63, had a great potential to secrete a considerable amount of laccase. Using molecular method, it was identified as an endophytic fungus, Pestalotiopsis sp. which was rarely reported as ligninolytic enzyme producer in the literature. The production of laccase by Pestalotiopsis sp. J63 was investigated under submerged fermentation (SF) and solid state fermentation (SSF) with various lignocellulosic by-products as substrates. The SSF of rice straw powder accumulated the highest level of laccase activity (10,700 IU/g substrate), whereas the SF of untreated sugarcane bagasse provided the maximum amount of laccase activity (2,000 IU/ml). The value was far higher than those reported by other reports. In addition, it produced 0.11 U/ml cellulase when alkaline-pretreated sugarcane bagasse was used as growth substrate under SF. Meanwhile, the growth of fungi and laccase production under different salinity conditions were also studied. It appeared to be a moderately halo-tolerant organism.     

Immunomodulatory Properties of Filamentous Fungi Cultivated through Solid-State Fermentation on Rapeseed Meal

Water extracts from solid-state fermentation (SSF) on rapeseed meal using filamentous fungi exhibit interesting immunomodulatory activities in vitro. Immunomodulation was determined by the capacity of the compounds to activate blood neutrophils and to influence cytokine production in human peripheral blood mononuclear cells (PBMC) and mouse bone marrow-derived macrophages (BMDM). Among the strains tested, Aspergillus sojae mycelium and SSF extracts were the most promising in terms of enhancing the immune response. The filamentous fungus was also successfully cultivated in a pre-pilot bioreactor with forced aeration. The results indicated that the extracts not only activated blood neutrophils but also significantly modulated IL-1β cytokine levels with lipopolysaccharide (LPS)-stimulated PBMC and BMDM without any cytotoxicity in immune cells. IL-1β was down-regulated in a dose-dependent manner in the presence of A. sojae crude mycelium and SSF extract with PBMC, which indicated that there was an anti-inflammatory activity, whereas IL-1β secretion was up-regulated in the presence of stimulated BMDM with the highest concentration that was tested (100 μg/mL). The non-fermented rapeseed had no effect at the same concentration. SSF culture, as a natural product, may be a good source for the development of functional feed with an immunostimulating effect or could potentially be used in medicinal applications.     

Gymnemagenin-producing Endophytic Fungus Isolated from a Medicinal Plant Gymnema sylvestre R.Br

Gymnema sylvestre is a plant containing the triterpenoid gymnemagenin, which is used in the pharmaceutical industry as an antidiabetic agent. The objective of this study was to determine whether endophytic fungi, isolated from G. sylvestre, produce gymnemagenin. We isolated an endophytic fungal strain from the leaves of G. sylvestre which produces gymnemagenin in the medium. The fungus was identified as Penicillium oxalicum based on morphological and molecular methods. The strain had a component with the same TLC Rf value and HPLC retention time as authentic gymnemagenin. The presence of gymnemagenin was further confirmed by FTIR, UV, and ¹H NMR analyses.     

Thermoanalytical investigation and biological properties of zinc(II) 4-chloro- and 5-chlorosalicylates with N-donor ligands

New zinc(II) 4- and 5-chlorosalicylate complexes of general formula [Zn(X-sal)₂(L) _n (H₂O) _x ] (where X-sal?=?4-Clsalicylate, 5-Clsalicylate; L?=?N,N-diethylnicotinamide, isonicotinamide, theophylline; n?=?1, 2; x?=?0, 1, 2, 4) were prepared. The complexes were determined by elemental analysis and characterised by infrared spectroscopy. The thermal behaviour of the complexes was studied by simultaneous TG, DTG and DTA methods under dynamic air conditions. The thermal decomposition is a multi-step process. In the first step of the thermal decomposition, water is released in hydrated compounds. The anhydrous compounds start to decompose by the release of organic ligand, followed by chlorosalicylic acid, chlorophenol and carbon monoxide. The final solid product of the thermal decomposition is zinc oxide. The volatile products of the thermal decomposition were determined by mass spectrometry. The antimicrobial activities of the complexes were evaluated against selected pathogen and probiotic bacteria, yeasts and fungi strains. Bioactivities of the tested compounds are different against bacteria, yeasts and filamentous fungi. It was found that bacteria were more sensitive to the studied zinc(II) complex compounds than yeasts or filamentous fungi.     

Acetylcholinesterase Inhibitory Potential and Insecticidal Activity of an Endophytic Alternaria sp. from Ricinus communis

Keeping in view the vast potential of endophytic fungi to produce bioactive molecules, this study aimed at isolating and screening endophytes for the production of acetylcholinesterase inhibitors. Fifty-four endophytic fungi were isolated from Ricinus communis and screened for their AChE inhibitory activity using Ellman??s colorimetric assay method. Six isolates were found to possess AChE inhibitory activity with maximum inhibition of 78?% being evinced by culture Cas1 which was identified to be Alternaria sp. on the basis of molecular as well as microscopic methods. Optimization of inhibitor production was carried out using one factor at a time approach. Maximum production of inhibitor was obtained on potato dextrose broth after 10?days incubation. The IC₅₀ of the chloroform extract was observed to be 40???g/ml. The extract was purified on silica gel and eluted stepwise with a gradient of chloroform/methanol. The insecticidal potential of the extract was evaluated by feeding the larvae of Spodoptera litura on diet containing varying concentrations of the extract. It was observed that with increase in the concentration of the extract, mortality of the larvae increased. The culture has the potential of being exploited in medicine as well as a biocontrol agent.