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Fungal infections are a significant global public health challenge because of their widespread occurrence, morbidity, and profound social and economic consequences. Antifungal resistance is also an increasing concern, posing a substantial risk to public health. There is a growing interest in searching for new antifungal drugs isolated from natural sources. This study aimed to evaluate the antifungal activity of novel mollusk fractions against fungal strains resistant to nystatin and amphotericin B. In addition, the role of oxidative stress in the mechanism of damage was determined. The mucus from the garden snail Cornu aspersum (MCa/1-20) and the hemolymph fraction from the marine snail Rapana venosa (HLRv/3-100) were obtained and characterized via 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometric -analyses. The results demonstrate that the spores and biomass of both mollusk fractions have a significant fungicidal effect against Penicillium griseofulvum , and Aspergillus niger . Compared to the control group, the release of intracellular proteins and reducing sugars was significantly increased in the treated groups. The data showed increased levels of oxidative stress biomarkers (lipid peroxidation and oxidatively damaged proteins) and a downregulated antioxidant enzyme defense, corresponding to increased antifungal activity. To our knowledge, this is the first study evaluating oxidative stress as a factor in mollusk fractions' antifungal activity.

Extremophiles are of significant scientific interest due to their unique adaptation to harsh environmental conditions and their potential for diverse biotechnological applications. Among these extremophiles, filamentous fungi adapted to high-salt environments represent a new and valuable source of enzymes, biomolecules, and biomaterials. While most studies on halophiles have focused on bacteria, reports on filamentous fungi remain limited. This review compiles information about salt-adapted fungi and details their distribution, adaptation mechanisms, and potential applications in various societal areas. Understanding the adaptive mechanisms of halophilic fungi not only sheds light on the biology of extremophilic fungi but also leads to promising biotechnological applications, including the development of salt-tolerant enzymes and strategies for bioremediation of saline habitats. To fully realize this potential, a comprehensive understanding of their ecology, diversity and physiology is crucial. In addition, understanding their survival mechanisms in saline environments is important for the development of astrobiology. The significant potential of applications of halophilic fungi is highlighted., Competing Interests: The authors declare no conflicts of interest.

Lactobacillus species are widely recognized for their probiotic potential, focusing on their mechanisms of health benefits and protection. Here we conducted an in vitro investigation of the probiotic potential with a role in microbiome homeostasis of four strains: Lactiplantibacillus plantarum L6 and F53, Ligilactobacillus salivarius 1, and Lactobacillus helveticus 611. A broad spectrum of antibacterial and antifungal activity was determined. The strain-specific inhibition of Staphylococcus aureus , Streptococcus mutans , Escherichia coli , Pseudomonas aeruginosa , and saprophytic/toxigenic fungi makes them promising as protective cultures. DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic) acid) measurements showed that tested samples had strain-specific capacity for scavenging of radicals. The molecular base for the antioxidant potential of two lyophilized forms of active strains was investigated by electron paramagnetic resonance spectroscopy. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, with fractions of the most active postbiotics obtained by SEC-FPLC (fast protein liquid chromatography) analysis, showed a wide variety of effects on the growth of a K562 myeloid leukemia cell line. The IC 50 (half-maximal inhibitory concentration) of L. salivarius 1 was determined to be 46.15 mg/mL. The proven in vitro functionality of the selected lactobacilli make them suitable for development of target probiotics with specific beneficial effects expected in vivo. Further investigations on produced postbiotics and safety have to be completed before they can be considered as scientifically proven probiotic strains.

Sialidases (neuraminidases) catalyze the removal of terminal sialic acid residues from glycoproteins. Novel enzymes from non-clinical isolates are of increasing interest regarding their application in the food and pharmaceutical industry. The present study aimed to evaluate the participation of carbon catabolite repression (CCR) in the regulation of cold-active sialidase biosynthesis by the psychrotolerant fungal strain Penicillium griseofulvum P29, isolated from Antarctica. The presence of glucose inhibited sialidase activity in growing and non-growing fungal mycelia in a dose- and time-dependent manner. The same response was demonstrated with maltose and sucrose. The replacement of glucose with glucose-6-phosphate also exerted CCR. The addition of cAMP resulted in the partial de-repression of sialidase synthesis. The CCR in the psychrotolerant strain P. griseofulvum P29 did not depend on temperature. Sialidase might be subject to glucose repression by both at 10 and 25 °C. The fluorescent assay using 4MU-Neu5Ac for enzyme activity determination under increasing glucose concentrations evidenced that CCR may have a regulatory role in sialidase production. The real-time RT-PCR experiments revealed that the sialidase gene was subject to glucose repression. To our knowledge, this is the first report that has studied the effect of CCR on cold-active sialidase, produced by an Antarctic strain.

The fungal strain, Penicillium griseofulvum P29, isolated from a soil sample taken from Terra Nova Bay, Antarctica, was found to be a good producer of sialidase (P29). The present study was focused on the purification and structural characterization of the enzyme. P29 enzyme was purified using a Q-Sepharose column and fast performance liquid chromatography separation on a Mono Q column. The determined molecular mass of the purified enzyme of 40 kDa by SDS-PAGE and 39924.40 Da by matrix desorption/ionization mass spectrometry (MALDI-TOF/MS) analysis correlated well with the calculated mass (39903.75 kDa) from the amino acid sequence of the enzyme. P29 sialidase shows a temperature optimum of 37 °C and low-temperature stability, confirming its cold-active nature. The enzyme is more active towards α(2 → 3) sialyl linkages than those containing α(2 → 6) linkages. Based on the determined amino acid sequence and 3D structural modeling, a 3D model of P29 sialidase was presented, and the properties of the enzyme were explained. The conformational stability of the enzyme was followed by fluorescence spectroscopy, and the new enzyme was found to be conformationally stable in the neutral pH range of pH 6 to pH 9. In addition, the enzyme was more stable in an alkaline environment than in an acidic environment. The purified cold-active enzyme is the only sialidase produced and characterized from Antarctic fungi to date., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Extremely cold habitats are a serious challenge for the existing there organisms. Inhabitants of these conditions are mostly microorganisms and lower mycetae. The mechanisms of microbial adaptation to extreme conditions are still unclear. Low temperatures cause significant physiological and biochemical changes in cells. Recently, there has been increasing interest in the relationship between low-temperature exposure and oxidative stress events, as well as the importance of antioxidant enzymes for survival in such conditions. The catalase is involved in the first line of the cells' antioxidant defense. Published information supports the concept of a key role for catalase in antioxidant defense against cold stress in a wide range of organisms isolated from the Antarctic. Data on representatives of microscopic fungi, however, are rarely found. There is scarce information on the characterization of catalase synthesized by adapted to cold stress organisms. Overall, this study aimed to observe the role of catalase in the survival strategy of filamentous fungi in extremely cold habitats and to identify the gene encoded catalase enzyme. Our results clearly showed that catalase is the main part of antioxidant enzyme defense in fungal cells against oxidative stress caused by low temperature exposure., (© Association of Microbiologists of India 2023. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Sialidase preparations are applied in structural and functional studies on sialoglycans, in the production of sialylated therapeutic proteins and synthetic substrates for use in biochemical research, etc. They are obtained mainly from pathogenic microorganisms; therefore, the demand for apathogenic producers of sialidase is of exceptional importance for the safe production of this enzyme. Here, we report for the first time the presence of a sialidase gene and enzyme in the saprophytic actinomycete Oerskovia paurometabola strain O129. An electrophoretically pure, glycosylated enzyme with a molecular weight of 70 kDa was obtained after a two-step chromatographic procedure using DEAE cellulose and Q-sepharose. The biochemical characterization showed that the enzyme is extracellular, inductive, and able to cleave α(2→3,6,8) linked sialic acids with preference for α(2→3) bonds. The enzyme production was strongly induced by glycomacropeptide (GMP) from milk whey, as well as by sialic acid. Investigation of the deduced amino acid sequence revealed that the protein molecule has the typical six-bladed β-propeller structure and contains all features of bacterial sialidases, i.e., an YRIP motif, five Asp-boxes, and the conserved amino acids in the active site. The presence of an unusual signal peptide of 40 amino acids was predicted. The sialidase-producing O. paurometabola O129 showed high and constant enzyme production. Together with its saprophytic nature, this makes it a reliable producer with high potential for industrial application.

Cold-active catalase (CAT) elicits great interest because of its vast prospective at the medical, commercial, and biotechnological levels. The study paper reports the production of cold-active CAT by the strain Penicillium griseofulvum P29 isolated from Antarctic soil. Improved enzyme production was achieved by optimization of medium and culture conditions. Maximum CAT was demonstrated under low glucose content (2%), 10% inoculum size, temperature 20°C, and dissolved oxygen concentration (DO) 40%. An effective laboratory technology based on changing the oxidative stress level through an increase of DO in the bioreactor was developed. The used strategy resulted in a 1.7- and 1.4-fold enhanced total enzyme activity and maximum enzyme productivity. The enzyme was purified and characterized. P. griseofulvum P29 CAT was most active at approximately 20°C and pH 6.0. Its thermostability was in the range between 5°C and 40°C., (© 2021 Wiley-VCH GmbH.)

Sialidases (neuraminidases, EC 3.2.1.18) are widely distributed in biological systems but there are only scarce data on its production by filamentous fungi. The aim of this study was to obtain information about sialidase distribution in filamentous fungi from non-clinical isolates, to determine availability of sialidase gene, and to select a perspective producer. A total of 113 fungal strains belonging to Ascomycota and Zygomycota compassing 21 genera and 51 species were screened. Among them, 77 strains (11 orders, 14 families and 16 genera) were able to synthesize sialidase. Present data showed a habitat-dependent variation of sialidase activity between species and within species, depending on location. Sialidase gene was identified in sialidase-positive and sialidase-negative strains. . Among three perspective strains, the best producer was chosen based on their sialidase production depending on type of cultivation, medium composition, and growth temperature. The selected P. griseofulvum Р29 was cultivated in 3L bioreactor at 20 °C on medium supplemented with 0.5% milk whey. The results demonstrated better growth and 2.3-fold higher maximum enzyme activity compared to the shaken flask cultures. Moreover, the early occurring maximum (48 h) is an important prerequisite for future up scaling of the process., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Because of the crucial role of ligninolytic enzymes in a variety of industrial processes, the demand for a new effective producer has been constantly increasing. Furthermore, information on enzyme synthesis by autochthonous fungal strains is very seldom found. Two fungal strains producing ligninolytic enzymes were isolated from Bulgarian forest soil. They were identified as being Trametes trogii and T. hirsuta . These two strains were assessed for their enzyme activities, laccase (Lac), lignin peroxidase (LiP) and Mn-dependent peroxidase (MnP) in culture filtrate depending on the temperature and the type of nutrient medium. T. trogii was selected as the better producer of ligninolytic enzymes. The production process was further improved by optimizing a number of parameters such as incubation time, type of cultivation, volume ratio of medium/air, inoculum size and the addition of inducers. The maximum activities of enzymes synthesized by T. trogii was detected as 11100 U/L for Lac, 2.5 U/L for LiP and 4.5 U/L for MnP after 14 days of incubation at 25°C under static conditions, volume ratio of medium/air 1:6, and 3 plugs as inoculum. Among the supplements tested, 5% glycerol increased Lac activity to a significant extent. The addition of 1% veratryl alcohol had a positive effect on MnP., (© 2018 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.)

The Antarctic fungal strain Aspergillus glaucus 363 produces cold-active (CA) Cu/Zn-superoxide dismutase (SOD). The strain contains at least one gene encoding Cu/Zn-SOD that exhibited high homology with the corresponding gene of other Aspergillus species. To our knowledge, this is the first nucleotide sequence of a CA Cu/Zn-SOD gene in fungi. An effective laboratory technology for A. glaucus SOD production in 3 L bioreactors was developed on the basis of transient cold-shock treatment. The temperature downshift to 10 °C caused 1.4-fold increase of specific SOD activity compared to unstressed culture. Maximum enzyme productivity was 64 × 10(3) U kg(-1) h(-1). Two SOD isoenzymes (Cu/Zn-SODI and Cu/Zn-SODII) were purified to electrophoretic homogeneity. The specific activity of the major isoenzyme, Cu/Zn-SODII, after Q-Sepharose chromatography was 4000 U mg(-1). The molecular mass of SODI (38 159 Da) and of SODII (15 835 Da) was determined by electrospray quadropole time-of-flight (ESI-Q-TOF) mass spectrometry and dynamic light scattering (DLS). The presence of Cu and Zn were confirmed by inductively coupled plasma mass spectrometry (ICP-MS). The N-terminal amino acid sequence of Cu/Zn-SODII revealed a high degree of structural homology with Cu/Zn-SOD from other fungi, including Aspergillus species., (Copyright © 2016 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

The study ob]ective was assessment of pathogenetic and prognostic significance of gynecologic and obstetrical pathology and the concentrations of sex steroids in adult women with acute coronaty syndrome (ACS). The study group included 120 postmenopausal women with ACS treated in the Clinic of Cardiology, University Hospital "Alexandrovska" between 2011 and 2013. Sex hormones were measured in 57 patients. Enzyme, electrochemiluminescent, enzyme-linked immunologic and immunoturbodimeric methods were used for the examined indices assessment. The history for gynecologic disorders and pregnancy complications was associated with coronaiy atherosclerotic burden (SYNTAX score - 4,6+/-8,8 vs 8,5+/-9,3, p=0,003), gynecologic history only - with lower 17Beta-estradiol levels (139,01+/-167,66 vs 113,51+/-304,1, p=0,004) and coronaly atherosclerosis severity (5,5+/-9,3 vs 8,0+/-10,3, p=0,058). Abnormally high endogenous concentrations of androgens were found among the patients with ACS with ST elevation, STEMI (27,5% vs 77,8%, p=0,004), with significantly more intense acute infiammatoty response (8,7+/-3,21 vs 11,07+/-2,85, p=0,044 3a WBC) and more extensive acute myocardial damage (57,8+/-12,6 vs 45,3 ml, p=O,OO8 for e]ection fraction 33,7+/-37,4 vs 117+/-144,22 U/L, p=0,031 for CPK-MB; 0,89+/-8 18 vs 1,87+/-0,4 ng/ml, p=0,009 for hsTnT). The gynecologic and obstetrical history and hyperandrogenism are related to the extent and severity of coronary atherosclerosis, occurrence of STEMI, more intense acute inflammatory response and myocardial injury among postmenopausal women with ACS.

Vibrio cholerae neuraminidase (VCNA) is widely used in biochemical and medical research, in processes for preparing homogenous sialoconjugates, and in the pharmaceutical industry. Its production by non-toxigenic strains is quite desirable, in order to avoid the expensive safety measures. Here, we report the first method for highly effective production of a novel, purified V. cholerae extracellular neuraminidase from a non-toxigenic strain. The enzyme is highly active, and its properties, as well as the responsible gene nanH, are practically identical with those of the toxigenic strains. It cleaves α,2 → 3 and α,2 → 6 glycosidic bonds with highest affinity (K M 1.7 × 10(-5) μM) for human transferrin. The deduced amino acid sequence of the enzyme reveals three binding sites for Ca(2+) and one for sialic acid. The sequence analysis of the nanH gene, being the first for a V. cholerae non-O1 strain, shows 99% identity with a new nanH allele of an O1 Vibrio strain. The simple laboratory technology for efficient production of the new VCNA is based on the use of common and cheap nutrient media and easily available inducer--glycomacropeptide. The rapid purification consists of salting-out and diethylaminoethanol (DEAE) and Q-Sepharose chromatography steps. Purified preparation contains no aldolase and protease, which gives the production scheme a great potential for industrial application.

The study focuses on the morphological and physiological cell responses to oxidative stress induced by high temperature treatment in the industrially relevant fungus Aspergillus niger 26. Temperatures above 30 °C lead to growth suppression and changes in morphological characteristics: decrease in the size of hyphal elements and increase in "active length" by switching from slightly branched long filaments to a multitude of branched forms containing active cytoplasm. Transmission electron microscopy of fungal cultures heated at 40 °C demonstrated abnormal wavy septation with reduced amount of chitin (as shown by WGA-gold labelling), intrahyphal hyphae development, disintegration of mitochondria and extensive autolysis. Temperature-dependent decrease in the total intracellular protein content and a sharp increase (six to tenfold) in oxidatively damaged proteins were also demonstrated. Elevated temperatures caused a two and threefold increase in catalase and superoxide dismutase activities, respectively.

The members of Verbascum L. (Scrophulariaceae) are known to be rich in phenylethnoid glycosides, and among them Verbascum xanthophoeniceum is an endemic plant species for the Balkan region, Northwestern, and Southern Turkey. A scheme was developed for the isolation of the main active constituents that accumulate in plant aerial parts. The antioxidant activities of total methanol extracts, collected phenylethanoid glycosides fractions and specific active constituents (forsythoside B, verbascoside and leucosceptoside B) were then evaluated in 2,2'-diphenyl-1-picrylhydrazyl (DPPH·), oxygen radical absorbance capacity (ORACFL), hydroxyl radical averting capacity (HORACFL), ferric-reducing antioxidant power (FRAP), and superoxide anion (O2(-)) radical scavenging assays. In vitro acetylcholinesterase (AChE) and butyrylcholinesterase (BChe) inhibitory activities of abovementioned extracts, fractions and isolated pure compounds were also examined. Depending on the method used, forsythoside B, verbascoside and leucosceptoside B proved to be effective radical scavengers and cholinesterases inhibitors. On the basis of these findings it can be proposed that in addition to providing a potent source of antimicrobial and anti-inflammatory compounds, Verbascum plants could serve as attractive mines of powerful antioxidants for various purposes., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Neuraminidase is a key factor in the infectious process of many viruses and pathogenic bacteria. The neuraminidase enzyme secreted by the etiological agent of cholera - Vibrio cholerae О1 - is well studied in contrast with the one produced by non-O1/non-O139 V. cholerae. Environmental non-O1/non-O139 V. cholerae isolates from Bulgaria were screened for production of neuraminidase. The presence of the neuraminidase gene nanH was detected in 18.5% of the strains. Тhe strain showing highest activity (30 U/mL), V. cholerae non-O1/13, was used to investigate the enzyme production in several media and at different aeration conditions. The highest production of extracellular neuraminidase was observed under microaerophilic conditions, which is possibly related to its role in the infection of intestine epithelium, where the oxygen content is low. On the other hand, this is another advantage of the microbe in such microaerophilic environments as sediments and lake mud. The highest production of intracellular neuraminidase was observed at anaerobic conditions. The ratio of extracellular to intracellular neuraminidase production in V. cholerae was investigated. The temperature optimum of the enzyme was determined to be 50 °C and the pH optimum to be 5.6-5.8.

The growth and intracellular protein content of lavender (Lavandula vera MM) cell suspension culture was followed along with some antioxidant defense system members-non-enzymatic (rosmarinic acid) and enzymatic [superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6)]. It was found that the media content and the cultivation mode strongly influenced the production of plant defense compounds as well as the ratio between non-enzymatic and enzymatic ones. The bioreactor culture contains about two times more rosmarinic acid, superoxide dismutase, and catalase compared to the shake-flask cultivation. These findings are discussed with respect to the relative stress levels and plant antioxidant orchestra system. It was concluded that investigated defense system components (enzymatic and non-enzymatic) were closely associated in a complex balance. The three isoenzyme forms of SOD (Cu/ZnSOD, FeSOD, and MnSOD) in the cells of Lavandula vera were revealed by polyacrylamide gel electrophoresis analysis, and the FeSOD isoform exhibited highest activity.

The fungal strain Aspergillus niger produces two superoxide dismutases, Cu/Zn-SOD and Mn-SOD. The primary structure of the Cu/Zn-SOD has been determined by Edman degradation of peptide fragments derived from proteolytic digests. A single chain of the protein, consisting of 153 amino acid residues, reveals a very high degree of structural homology with the amino acid sequences of other Aspergillus Cu/Zn-SODs. The molecular mass of ANSOD, measured by MALDI-MS and ESI-MS, and calculated by its amino acid sequence, was determined to be 15821 Da. Only one Trp residue, at position 32, and one disulfide bridge were identified. However, neither a Tyr residue nor a carbohydrate chain occupying an N-linkage site (-Asn-Ile-Thr-) were found. Studies on the temperature and pH dependence of fluorescence, and on the temperature dependence of CD spectroscopic properties, confirmed that the enzyme is very stable, which can be explained by the stabilising effect of the disulfide bridge. The enzyme retains about 53% of its activity after incubation for a period of 30 min at 60 degrees C, and 15% at 85 degrees C.

Oxidative stress events have been shown to be associated with reduced consumption of nutrients in yeasts, but there are very few studies in filamentous fungi. In the present study we investigated the impact of oxidative stress on glucose and ammonia utilization in batch cultures of Aspergillus niger B1-D. The addition of 1mM H(2)O(2) significantly reduced both glucose and ammonia uptake rates in these cultures. Associated with the decreased nutrient uptake, the activity of glyceraldehyde-3-phosphate dehydrogenase was greatly reduced; conversely, the activity of glucose-6-phosphate dehydrogenase remained unchanged. During the period of reduced nutrient uptake, the intracellular ATP and NADPH levels decreased while the amount of trehalose increased. The activities of glutamine synthetase and glutamate dehydrogenase, two key enzymes of ammonia assimilation, remained unchanged in response to H(2)O(2) up to 1mM, suggesting the decreased ammonia uptake rate noted under such conditions is not due to enzyme inactivation caused by oxidative stress, but may be due to an insufficient supply of ATP and NADPH, which are required for ammonia assimilation.

Keyhole limpet hemocyanin is a respiratory glycoprotein of high molecular weight from the gastropod mollusc Megathura crenulata. Two subunits, KLH1 and KLH2, were isolated using ion exchange chromatography and their physical properties are compared with the parent molecule. The various proteins are characterized by fluorescence spectroscopy, combined with fluorescence quenching studies, using acrylamide, cesium chloride and potassium iodide as tryptophan quenchers. The conformational stability of the native aggregate and its isolated structural subunits are also studied by circular dichroism and fluorescence spectroscopy as a function of temperature, as well as in the presence of guanidinium hydrochloride and urea. The associated subunits in the hemocyanin aggregates increase considerably the melting temperature to 67 degrees C and the free energy of stabilization in water, DeltaG(H(2)O)(D), towards guanidinium hydrochloride is higher for the decamer as compared to the isolated subunits; this difference can be accounted for by the stabilizing effects of intra-subunit interactions exerted within the oligomer. The copper-dioxygen complex at the active site additionally stabilizes the molecule, and removing of the copper ions increases the tryptophan emission and the quantum yield of the fluorescence.