Composites of sodium alginate based - Functional materials towards sustainable adsorption of benzene phenol derivatives - Bisphenol A/Triclosan.

The present study evaluates the adsorption efficiency of low-cost carbonaceous adsorbents as fly ash (FA), saw dust biochar (SDB) (untreated and alkali - treated), live/dead pulverized white rot fungus Hypocrea lixii biomass encapsulated in sodium alginate (SA) against the commercially available activated carbon (AC) and graphene oxide (GO) SA beads for removal of benzene phenol derivatives - Bisphenol A (BPA)/triclosan (TCS). Amongst bi - and tri - composites SA beads, tri-composite beads comprising of untreated flyash - dead fungal biomass - sodium alginate (UFA - DB - SA) showed at par results with commercial composite beads. The tri - composite beads with point zero charge (Ppzc) of 6.2 was characterized using FTIR, XRD, surface area BET and SEM-EDX. The batch adsorption using tri - composite beads revealed removal of 93% BPA with adsorption capacity of 16.6 mg/g (pH 6) and 83.72% TCS with adsorption capacity of 14.23 mg/g (pH 5), respectively at 50 ppm initial concentration with 6 % adsorbent dose in 5 h. Freundlich isotherm favoring multilayered adsorption provided a better fit with r2 of 0.9674 for BPA and 0.9605 for TCS respectively. Intraparticle diffusion model showed adsorption of BPA/TCS molecules to follow pseudo - second order kinetics with boundary layer diffusion governed by first step of fast adsorption and intraparticle diffusion within pores by second slow adsorption step. Thermodynamic parameters (ΔH°, ΔS°, ΔG°) revealed adsorption process as exothermic, orderly and spontaneous. Methanol showed better desorbing efficiency leading to five cycles reusability. The phytotoxicity assay revealed increased germination rate of mung bean (Vigna radiata) seeds, sprinkled with post adsorbed treated water (0 h, 5 h and 7 h) initially spiked with 50 ppm BPA/TCS. Overall, UFA - DB - SA tri - composite beads provides a cost effective and eco - friendly matrix for effective removal of hydrophobic recalcitrant compounds.

Leveraging Peptaibol Biosynthetic Promiscuity for Next-Generation Antiplasmodial Therapeutics.

Malaria remains a worldwide threat, afflicting over 200 million people each year. The emergence of drug resistance against existing therapeutics threatens to destabilize global efforts aimed at controlling Plasmodium spp. parasites, which is expected to leave vast portions of humanity unprotected against the disease. To address this need, systematic testing of a fungal natural product extract library assembled through the University of Oklahoma Citizen Science Soil Collection Program has generated an initial set of bioactive extracts that exhibit potent antiplasmodial activity (EC50 < 0.30 µg/mL) and low levels of toxicity against human cells (less than 50% reduction in HepG2 growth at 25 µg/mL). Analysis of the two top-performing extracts from Trichoderma sp. and Hypocrea sp. isolates revealed both contained chemically diverse assemblages of putative peptaibol-like compounds that were responsible for their antiplasmodial actions. Purification and structure determination efforts yielded 30 new peptaibols and lipopeptaibols (1-14 and 28-43), along with 22 known metabolites (15-27 and 44-52). While several compounds displayed promising activity profiles, one of the new metabolites, harzianin NPDG I (14), stood out from the others due to its noteworthy potency (EC50 = 0.10 µM against multi-drug-resistant P. falciparum line Dd2) and absence of gross toxicity toward HepG2 at the highest concentrations tested (HepG2 EC50 > 25 µM, selectivity index > 250). The unique chemodiversity afforded by these fungal isolates serves to unlock new opportunities for translating peptaibols into a bioactive scaffold worthy of further development.

Cadinane and carotane derivatives from the marine algicolous fungus Trichoderma virens RR-dl-6-8.

Eight cadinane derivatives, trichocadinins H - N (1-7) and methylhydroheptelidate (8), and two carotane derivatives, 14-O-methyltrichocarotin G (9) and 14-O-methyl CAF-603 (10), including eight new ones (1-6, 9, and 10), were isolated from the culture of Trichoderma virens RR-dl-6-8 obtained from the organohalogen-enriched marine red alga Rhodomela confervoides. Their structures and relative configurations were established by analysis of NMR and mass spectroscopic data, and the absolute configurations were assigned on the basis of ECD curves, highlighted by the ECD diversity of carboxylic acid derivatives. Among the isolates, 1 with a halogen atom and 8, a new naturally occurring compound, are 2,3-seco-cadinane sesquiterpenes, and the epimeric 2 and 3 feature a 2-nor-cadinane skeleton. A commercially-sourced compound with the same planar structure as that of 7 has been reported in a patent, but its configuration was not given. Compounds 1-10 exhibited growth inhibition of some marine phytoplankton species.

Enantioselective Binding of Propranolol and Analogues Thereof to Cellobiohydrolase Cel7A.

At the catalytic site for the hydrolysis of cellulose the enzyme cellobiohydrolase Cel7A binds the enantiomers of the adrenergic beta-blocker propranolol with different selectivity. Methyl-to-hydroxymethyl group modifications of propranolol, which result in higher affinity and improved selectivity, were herein studied by 1 H,1 H and 1 H,13 C scalar spin-spin coupling constants as well as utilizing the nuclear Overhauser effect (NOE) in conjunction with molecular dynamics simulations of the ligands per se, which showed the presence of all-antiperiplanar conformations, except for the one containing a vicinal oxygen-oxygen arrangement governed by the gauche effect. For the ligand-protein complexes investigated by NMR spectroscopy using, inter alia, transferred NOESY and saturation-transfer difference (STD) NMR experiments the S-isomers were shown to bind with a higher affinity and a conformation similar to that preferred in solution, in contrast to the R-isomer. The fact that the S-form of the propranolol enantiomer is pre-arranged for binding to the protein is also observed for a crystal structure of dihydroxy-(S)-propranolol and Cel7A presented herein. Whereas the binding of propranolol is entropy driven, the complexation with the dihydroxy analogue is anticipated to be favored also by an enthalpic term, such as for its enantiomer, that is, dihydroxy-(R)-propranolol, because hydrogen-bond donation replaces the corresponding bonding from hydroxyl groups in glucosyl residues of the natural substrate. In addition to a favorable entropy component, albeit lesser in magnitude, this represents an effect of enthalpy-to-entropy compensation in ligand-protein interactions.

High-resolution structure of a lytic polysaccharide monooxygenase from Hypocrea jecorina reveals a predicted linker as an integral part of the catalytic domain.

For decades, the enzymes of the fungus Hypocrea jecorina have served as a model system for the breakdown of cellulose. Three-dimensional structures for almost all H. jecorina cellulose-degrading enzymes are available, except for HjLPMO9A, belonging to the AA9 family of lytic polysaccharide monooxygenases (LPMOs). These enzymes enhance the hydrolytic activity of cellulases and are essential for cost-efficient conversion of lignocellulosic biomass. Here, using structural and spectroscopic analyses, we found that native HjLPMO9A contains a catalytic domain and a family-1 carbohydrate-binding module (CBM1) connected via a linker sequence. A C terminally truncated variant of HjLPMO9A containing 21 residues of the predicted linker was expressed at levels sufficient for analysis. Here, using structural, spectroscopic, and biochemical analyses, we found that this truncated variant exhibited reduced binding to and activity on cellulose compared with the full-length enzyme. Importantly, a 0.95-Å resolution X-ray structure of truncated HjLPMO9A revealed that the linker forms an integral part of the catalytic domain structure, covering a hydrophobic patch on the catalytic AA9 module. We noted that the oxidized catalytic center contains a Cu(II) coordinated by two His ligands, one of which has a His-brace in which the His-1 terminal amine group also coordinates to a copper. The final equatorial position of the Cu(II) is occupied by a water-derived ligand. The spectroscopic characteristics of the truncated variant were not measurably different from those of full-length HjLPMO9A, indicating that the presence of the CBM1 module increases the affinity of HjLPMO9A for cellulose binding, but does not affect the active site.

Different Covalent Immobilizations Modulate Lipase Activities of Hypocrea pseudokoningii.

Enzyme immobilization can promote several advantages for their industrial application. In this work, a lipase from Hypocrea pseudokoningii was efficiently linked to four chemical supports: agarose activated with cyanogen bromide (CNBr), glyoxyl-agarose (GX), MANAE-agarose activated with glutaraldehyde (GA) and GA-crosslinked with glutaraldehyde. Results showed a more stable lipase with both the GA-crosslinked and GA derivatives, compared to the control (CNBr), at 50 °C, 60 °C and 70 °C. Moreover, all derivatives were stabilized when incubated with organic solvents at 50%, such as ethanol, methanol, n-propanol and cyclohexane. Furthermore, lipase was highly activated (4-fold) in the presence of cyclohexane. GA-crosslinked and GA derivatives were more stable than the CNBr one in the presence of organic solvents. All derivatives were able to hydrolyze sardine, açaí (Euterpe oleracea), cotton seed and grape seed oils. However, during the hydrolysis of sardine oil, GX derivative showed to be 2.3-fold more selectivity (eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) ratio) than the control. Additionally, the types of immobilization interfered with the lipase enantiomeric preference. Unlike the control, the other three derivatives preferably hydrolyzed the R-isomer of 2-hydroxy-4-phenylbutanoic acid ethyl ester and the S-isomer of 1-phenylethanol acetate racemic mixtures. On the other hand, GX and CNBr derivatives preferably hydrolyzed the S-isomer of butyryl-2-phenylacetic acid racemic mixture while the GA and GA-crosslink derivatives preferably hydrolyzed the R-isomer. However, all derivatives, including the control, preferably hydrolyzed the methyl mandelate S-isomer. Moreover, the derivatives could be used for eight consecutive cycles retaining more than 50% of their residual activity. This work shows the importance of immobilization as a tool to increase the lipase stability to temperature and organic solvents, thus enabling the possibility of their application at large scale processes.

Bioreducible Hydrophobin-Stabilized Supraparticles for Selective Intracellular Release.

One of the main hurdles in nanomedicine is the low stability of drug-nanocarrier complexes as well as the drug delivery efficiency in the region-of-interest. Here, we describe the use of the film-forming protein hydrophobin HFBII to organize dodecanethiol-protected gold nanoparticles (NPs) into well-defined supraparticles (SPs). The obtained SPs are exceptionally stable in vivo and efficiently encapsulate hydrophobic drug molecules. The HFBII film prevents massive release of the encapsulated drug, which, instead, is activated by selective SP disassembly triggered intracellularly by glutathione reduction of the protein film. As a consequence, the therapeutic efficiency of an encapsulated anticancer drug is highly enhanced (2 orders of magnitude decrease in IC50). Biodistribution and pharmacokinetics studies demonstrate the high stability of the loaded SPs in the bloodstream and the selective release of the payloads once taken up in the tissues. Overall, our results provide a rationale for the development of bioreducible and multifunctional nanomedicines.

Inactivation kinetics and conformation change of Hypocrea orientalis ß-glucosidase with guanidine hydrochloride.

The relationship between unfolding and inactivation of Hypocrea orientalis ß-glucosidase has been investigated for the first time. The secretion of ß-glucosidase from H. orientalis is induced by raw cassava residues. The enzyme was 75 kD without glycosylation. Guanidine hydrochloride (GuHCl) could reversibly inactivate the enzyme with an estimated IC50 value of 0.4 M. The inactivation kinetics model by GuHCl has been established and the microscopic inactivation rate constants are determined. The values of forward inactivation rate constants of free enzyme are found to be larger than that of substrate-enzyme complex suggesting the enzyme could be protected by substrate during denaturation. Conformational change of the enzyme during denaturation is observed as the intrinsic fluorescence emission peaks appeared red-shift (334-354 nm) with intensity decreased following increase of GuHCl concentrations. Inactivation extent is found to be greater than conformation change of the whole enzyme, indicating that the active site of H. orientalis ß-glucosidase might be a more flexible region than the whole enzyme.

Structural and functional studies of the glycoside hydrolase family 3 ß-glucosidase Cel3A from the moderately thermophilic fungus Rasamsonia emersonii.

The filamentous fungus Hypocrea jecorina produces a number of cellulases and hemicellulases that act in a concerted fashion on biomass and degrade it into monomeric or oligomeric sugars. ß-Glucosidases are involved in the last step of the degradation of cellulosic biomass and hydrolyse the ß-glycosidic linkage between two adjacent molecules in dimers and oligomers of glucose. In this study, it is shown that substituting the ß-glucosidase from H. jecorina (HjCel3A) with the ß-glucosidase Cel3A from the thermophilic fungus Rasamsonia emersonii (ReCel3A) in enzyme mixtures results in increased efficiency in the saccharification of lignocellulosic materials. Biochemical characterization of ReCel3A, heterologously produced in H. jecorina, reveals a preference for disaccharide substrates over longer gluco-oligosaccharides. Crystallographic studies of ReCel3A revealed a highly N-glycosylated three-domain dimeric protein, as has been observed previously for glycoside hydrolase family 3 ß-glucosidases. The increased thermal stability and saccharification yield and the superior biochemical characteristics of ReCel3A compared with HjCel3A and mixtures containing HjCel3A make ReCel3A an excellent candidate for addition to enzyme mixtures designed to operate at higher temperatures.

Hypocriols A-F, Heterodimeric Botryane Ethers from Hypocrea sp., an Insect-Associated Fungus.

The new heterodimeric botryane ethers hypocriols A-F (1-6) and the known compounds 4ß-acetoxy-9ß,10ß,15α-trihydroxyprobotrydial (7), dihydrobotrydial (8), 10-oxodehydrodihydrobotrydial (9), and dehydrobotrydienol (10) were isolated from the solid cultures of an insect-associated fungus Hypocrea sp. The structures of 1-6 were elucidated primarily by NMR experiments. The absolute configuration of 1 was assigned using the modified Mosher method and electronic circular dichroism (ECD) calculations, whereas those for 3-5, and 2 and 6 were deduced via ECD calculations and circular dichroism data, respectively. Compounds 1-6 appear to be the first heterodimeric botryane ethers and showed antiproliferative effects against a small panel of four human tumor cell lines.

Hypocrol A, a new tyrosol derivative from a sponge-derived strain of the fungus Hypocrea koningii.

In continuation of our search for new antibacterial and antioxidant metabolites from sponge-derived fungi, one new tyrosol derivative, hypocrol A (1), together with four known congeners, trichodenol B (2), 4-hydroxyphenethyl acetate (3), 4-hydroxyphenethyl tetradecanoate (4) and 1-oleyltyrosol (5), was isolated from the strain Hypocrea koningii PF04. Their planar structures were unequivocally elucidated by spectroscopic methods and comparison with the literature data. All the compounds displayed weak antibacterial activities against Staphylococcus aureus, methicillin-resistant S. aureus and Escherichia coli, whereas compounds 1 and 2 exhibited a moderate antioxidant efficacy in the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay with IC50 values of 48.5 and 97.4 µg/mL, respectively.

Informatic search strategies to discover analogues and variants of natural product archetypes.

Natural products are a crucial source of antimicrobial agents, but reliance on low-resolution bioactivity-guided approaches has led to diminishing interest in discovery programmes. Here, we demonstrate that two in-house automated informatic platforms can be used to target classes of biologically active natural products, specifically, peptaibols. We demonstrate that mass spectrometry-based informatic approaches can be used to detect natural products with high sensitivity, identifying desired agents present in complex microbial extracts. Using our specialised software packages, we could elaborate specific branches of chemical space, uncovering new variants of trichopolyn and demonstrating a way forward in mining natural products as a valuable source of potential pharmaceutical agents.

Characterisation of branched gluco-oligosaccharides to study the mode-of-action of a glucoamylase from Hypocrea jecorina.

In the conversion of starch to fermentable glucose for bioethanol production, hydrolysis of amylopectin by α-amylases and glucoamylases is the slowest step. In this process, α-1,6-branched gluco-oligosaccharides accumulate and are slowly degraded. Glucoamylases that are able to degrade such branched oligosaccharides faster are economically beneficial. This research aimed at the isolation and characterisation of branched gluco-oligosaccharides produced from amylopectin digestion by α-amylase, to be used as substrates for comparing their degradation by glucoamylases. Branched gluco-oligosaccharides with a DP between five and twelve were purified using size exclusion chromatography. These structures were characterised after labelling with 2-aminobenzamide using UHPLC-MS(n) analysis. Further, the purified oligosaccharides were used to evaluate the mode-of-action of a glucoamylase from Hypocrea jecorina. The enzyme cleaves the α-1,4-linkage adjacent to the α-1,6-linkage at a lower rate than that of α-1,4-linkages in linear oligosaccharides. Hence, the branched gluco-oligosaccharides are a suitable substrate to evaluate glucoamylase activity on branched structures.

Crystallization and preliminary X-ray diffraction analysis of Hypocrea jecorina Cel7A in two new crystal forms.

Cel7A (previously known as cellobiohydrolase I) from Hypocrea jecorina was crystallized in two crystalline forms, neither of which have been previously reported. Both forms co-crystallize under the same crystallization conditions. The first crystal form belonged to space group C2, with unit-cell parameters a=152.5, b=44.9, c=57.6 Å, ß=101.2°, and diffracted X-rays to 1.5 Šresolution. The second crystal form belonged to space group P6322, with unit-cell parameters a=b≃155, c≃138 Å, and diffracted X-rays to 2.5 Šresolution. The crystals were obtained using full-length Cel7A, which consists of a large 434-residue N-terminal catalytic domain capable of cleaving cellulose, a 27-residue flexible linker and a small 36-residue C-terminal carbohydrate-binding module (CBM). However, a preliminary analysis of the electron-density maps suggests that the linker and CBM are disordered in both crystal forms. Complete refinement and structure analysis are currently in progress.

Production and optimization of L-glutaminase enzyme from Hypocrea jecorina pure culture.

L-Glutaminase (L-glutamine amidohydrolase, EC 3.5.1.2) is the important enzyme that catalyzes the deamination of L-glutamine to L-glutamic acid and ammonium ions. Recently, L-glutaminase has received much attention with respect to its therapeutic and industrial applications. It acts as a potent antileukemic agent and shows flavor-enhancing capacity in the production of fermented foods. Glutaminase activity is widely distributed in plants, animal tissues, and microorganisms, including bacteria, yeasts, and fungi. This study presents microbial production of glutaminase enzyme from Hypocrea jecorina pure culture and determination of optimum conditions and calculation of kinetic parameters of the produced enzyme. The optimum values were determined by using sa Nesslerization reaction for our produced glutaminase enzyme. The optimum pH value was determined as 8.0 and optimum temperature as 50°C for the glutaminase enzyme. The Km and Vmax values, the kinetic parameters, of enzyme produced from Hypocrea jecorina, pure culture were determined as 0.491 mM for Km and 13.86 U/L for Vmax by plotted Lineweaver-Burk graphing, respectively. The glutaminase enzyme from H. jecorina microorganism has very high thermal and storage stability.

Anti-Trichomonas vaginalis activity of marine-associated fungi from the South Brazilian Coast.

Trichomonas vaginalis is the causative agent of trichomonosis, the most common non-viral sexually transmitted disease. Infection with this protozoan may have serious consequences, especially for women. Currently, 5-nitroimidazole drugs are the treatment of choice for trichomonosis, but the emergence of resistance has limited the effectiveness of this therapy. In this context, this study aimed to evaluate the anti-T. vaginalis activity of marine-associated fungi found in the South Brazilian Coast. A total of 42 marine-associated fungal species (126 filtrate samples) isolated from 39 different marine organisms, mainly sponges, were selected to be screened against T. vaginalis. Of these, two filtrate samples from Hypocrea lixii F02 and Penicillium citrinum F40 showed significant growth-inhibitory activity (up to 100%) against ATCC 30236 and fresh clinical isolates, including a metronidazole-resistant isolate. Minimum inhibitory concentration (MIC) values of H. lixii F02 and P. citrinum F40 samples for all isolates tested, including the metronidazole-resistant isolate, were 2.5 mg/mL. The kinetic growth curve showed that the filtrate samples were able to reduce the density of parasites to zero within 24 h of incubation, which was confirmed by microscopy. Both fungal filtrate samples exhibited no hemolytic activity, and the P. citrinum F40 filtrate sample showed low cytotoxicity against Vero cells. These data suggest that marine-associated fungi from the South Brazilian Coast may produce potential candidates for further investigation and possible use in the treatment of metronidazole-resistant trichomonosis.

A botryane metabolite with a new hexacyclic skeleton from an entomogenous fungus Hypocrea sp.

Hypocrolide A (1), a botryane metabolite with a new hexacyclic skeleton, was isolated from cultures of the entomogenous fungus Hypocrea sp. The proposed structure was confirmed by X-ray crystallography using Cu Kα radiation. The mixed-biogenetic skeleton could be derived from the hypothetical precursors related to coumarin and dihydrobotrydiol, and the latter may be derived from the coisolated 10-oxodehydrodihydrobotrydial (2) or a similar analogue.

A cellulolytic Hypocrea strain isolated from South American brave straw produces a modular xylanase.

Cellulase-producing fungi from the Andean regions in Bolivia, an ecosystem characterized as an extreme arid highland, were studied. Thirty-two isolates were screened for presence of cellulase activity using carboxymethyl cellulose (CMC) as carbon source, and activity was confirmed using a filter paper assay. One isolate, denoted as BLT1C was selected from this screening, and sequence analysis of the internal transcribed spacer (ITS) classified the strain as Hypocrea lixii. The secretome of BLT1C showed high xylanase activity (compared to that of two reference Trichoderma reesei strains) when cultivated using brave straw, an abundant native grass from the area, as carbon source. SDS-PAGE analysis revealed three main protein-bands (18, 32 and 65 kDa) and in-gel digestion and mass spectrometry combined with activity analysis showed that these proteins were active xylanases with molecular masses corresponding to (I) a single glycoside hydrolase family 11 catalytic module (18 kDa), and (II, III) modular enzymes, with the GH11 catalytic domain connected to a module of unknown function (32 kDa) or putatively connected to a GH7 catalytic module (65 kDa). The N-terminal sequence of the 65 kDa xylanase did not show significant sequence similarities to deposited sequences. The collected data on xylanase activity, molecular mass, GH11-sequence conservation, combined with lack of sequence similarities in the N-terminus show that the 65 kDa band corresponds to a novel modular xylanase.

A new alkaloid from the marine-derived fungus Hypocrea virens.

A new alkaloid, 2-methylimidazo[1,5-b]isoquinoline-1,3,5(2H)-trione, was isolated from the EtOAc extract of the marine-derived fungus Hypocrea virens. Its structure was confirmed by spectroscopic methods (1D and 2D NMR, MS).

Novel hydrophobins from Trichoderma define a new hydrophobin subclass: protein properties, evolution, regulation and processing.

Hydrophobins are small proteins, characterised by the presence of eight positionally conserved cysteine residues, and are present in all filamentous asco- and basidiomycetes. They are found on the outer surfaces of cell walls of hyphae and conidia, where they mediate interactions between the fungus and the environment. Hydrophobins are conventionally grouped into two classes (class I and II) according to their solubility in solvents, hydropathy profiles and spacing between the conserved cysteines. Here we describe a novel set of hydrophobins from Trichoderma spp. that deviate from this classification in their hydropathy, cysteine spacing and protein surface pattern. Phylogenetic analysis shows that they form separate clades within ascomycete class I hydrophobins. Using T. atroviride as a model, the novel hydrophobins were found to be expressed under conditions of glucose limitation and to be regulated by differential splicing.