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Cardiovascular diseases, resulting from the deposition of clots in blood vessels, are the leading cause of death worldwide. Fibrinolytic enzymatic activity can catalyze blood clot degradation. Findings show that 36 fungal isolates recovered from Caatinga soils have the potential to produce fibrinolytic protease under submerged conditions. About 58 % of the isolates displayed fibrinolytic activity above 100 U/mL, with Mucor subtilissimus UCP 1262 being the most active. The protease was biochemically and biophysically characterized, showing that the enzyme had a high affinity for SAApNA substrate and was significantly inhibited by fluoride methyl phenyl sulfonyl-C7H7FO2S, suggesting that it is a chymotrypsin-like serine protease. The highest enzyme activity was detected at pH 5.0 and 28 °C. This fibrinolytic protease's far-UV circular dichroism (CD) showed that its secondary structure was primarily α-helical. The purified fibrinolytic enzyme may represent a novel therapeutic agent for treating thrombosis. At temperatures above 65 °C, the enzyme lost all its secondary structure. Its melting temperature was 58.1 °C, the denaturation enthalpy 85.1 kcal/mol, and the denaturation entropy 0.26 kcal/K∙mol.

This study aimed to evaluate the nutritive value of three feeds ( Cecropia sp., Pterodon sp., and Inga sp.) for sloths ( Bradypus variegatus ), based on nutritional composition and in vitro gas production. After a 14-day adaptation period to these feeds, approximately 500 g of gastric contents were collected from three female sloths, processed, and incubated with the food samples to evaluate digestibility and in vitro degradation kinetics. Regarding the nutritional composition, the neutral detergent fiber (NDFcp) content was higher with 404 g kg -1 DM ( p = 0.001) in the leaves of Cecropia sp. The non-fibrous carbohydrate contents were greater with 499 g kg -1 DM in Pterodon sp. ( p = 0.002). The greatest cellulose content (211 g kg -1 DM) was found in the leaves of C. pachystachya , as well as the lowest value of 143 g kg -1 DM for hemicellulose. Significant differences in the in vitro digestibility of crude protein ( p = 0.041) were observed, with Inga sp. showing the highest value at 547 g kg -1 DM. In terms of kinetic parameters, Pterodon sp. exhibited higher total gas production (Vt) at 99 mL ( p = 0.023) and digestion rates of fibrous carbohydrates (kdFC) at 0.0223%/h ( p = 0.020) ( p < 0.05). The leaves of Pterodon sp. and Inga sp. showed potential as suitable feeds for B. variegatus , while Cecropia sp. may have negative effects on dry matter intake due to its high NDF content, because of possible repletion effects on the stomach.

This study investigated the morphology of Rhinella crucifer cutaneous glands, as well as the protein/peptide profiles and bioactivities of body gland secretions (BGS) and parotoid macrogland secretions (PS). The parotoid as well as dorsal and ventral skin fragments of male and female individuals were processed for histological analysis. The protein and peptide profiles of male and female gland secretions were evaluated. Male secretions were also assessed for proteolytic, trypsin inhibiting, hemagglutinating, hemolytic, antimicrobial, and anticoagulant activities. The R. crucifer skin structure presented protuberances that are clearly visible and formed by the integument, which has cutaneous glands throughout the body. An average of 438 and 333 glands were identified in males in females, respectively. No significant differences were observed in the distribution of glands across the body as well as for area and perimeter of glands. Differences were observed in protein composition between the PS and BGS from males and females, and secretions from animals collected from undisturbed and anthropogenically disturbed areas. Proteins with similarities to catalase and elongation factor 1-alpha were detected in the PS. Zymography revealed proteolytic activity in both male BGS and PS. Male BGS showed antibacterial activity against Enterococcus faecalis and Escherichia coli and anticoagulant activity, being able to prolong prothrombin time by 6.34-fold and activated partial thromboplastin time by 2.17-fold. Finally, male PS and BGS caused a maximum hemolysis degree of 1.4%. The data showed that the cutaneous secretions of R. crucifer are potentially promising for biotechnological prospecting., Competing Interests: Declaration of competing interest 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., (Copyright © 2024 Elsevier Inc. All rights reserved.)

A fibrinolytic enzyme from the microalga Dunaliella tertiolecta was produced under mixotrophic conditions using different corn steep liquor (CSL) concentrations ( 0 ≤ CLS ≤ 0.75%), purified using a combination of salting out and ion-exchange chromatography, and then biochemical characterized. Cultivation of this microalga using 0.5% CSL led to the highest maximum cell concentration (1.960±0.010 mg L-1) and cell productivity (0.140g L-1 day-1), besides a high fibrinolytic activity of the extract obtained by the homogenization method (102 ±1 U mL-1). The enzyme extracted from the microalgal biomass was 5-fold purified with a 20% yield and was found to have a specific activity of 670 U mg-1. The enzyme, whose molecular weight determined by fibrin zymography was 10 kDa, was shown to be stable at pH 3.0-9.0 and up to 70°C with optimal pH and temperature values of 8.0 and 50°C, respectively. When compared to other fibrinolytic enzymes, this protease stood out for its high fibrinolytic activity, which was enhanced by Fe2+, inhibited by Zn2+, Cu2+, Mg2+, and Ca2+, and strongly inhibited by phenylmethylsulfonyl fluoride, suggesting that it belongs to the serine metalloprotease family. Moreover, thanks to its thermal stability, the enzyme may be easily preserved and activated under high-temperature conditions.

Fructooligosaccharides (FOS) are prebiotics of interest to the food industry. These compounds can be produced through the transfructosylation reaction by the enzyme fructofuranosidase. This enzyme is widely produced by fungi in a medium rich in sugar. Therefore, in this work, the main objectives were production, purification, biochemical characterization of a novel fructofuranosidase enzyme by Penicillium citreonigrum URM 4459 and synthesize and evaluate the antibacterial potential of fructooligosaccharides. With respect to sucrose hydrolysis, the optimal pH was 5.5, the apparent Km for purified FFase was 3.8 mM, the molecular mass was 43.0 kDa, estimated by gel filtration on Superdex increase G75 controlled by AKTA Avant 25 and confirmed by 10% SDS-PAGE under denaturing condition. Also, the isoelectric point was 4.9. The fractions obtained with enzymatic activities, both stable at acidic pH and high temperatures, as well as being able to produce FOS. Regarding antibacterial activity, the FOS produced in this study showed better results than commercial FOS and other carbon sources. Thus, this work presents relevant data for the use of P. citreonigum to produce fructofuranosidase and consequently FOS and can be used in the food and pharmaceutical industry.

Fibrinolytic enzymes are considered promising alternative in the treatment of cardiovascular diseases by preventing fibrin clots. A protease from Mucor subtilissimus UCP 1262 was obtained by solid state fermentation and purified by ion exchange chromatography. The purified extract was administered at an acute dose of 2000 mg/mL to evaluate its toxic effects to the lungs of mice. After 14 days of treatment, a histomorphometric study was performed by the type 1 and 2 pneumocyte count and the evaluation of the lung area. As result, the experimental group showed a significant decrease of type 2 pneumocyte and although a decrease in the alveolar area was observed in relation to the control group, no significant pulmonary toxicity, emphysema, and fibrosis characteristics were detected. The in vitro tests suggest possible clinical applications for the enzyme.

This work aimed to compare the production of collagenolytic proteases produced by M. subtilissimus UCP1262 in submerged fermentation (SF) and solid-state fermentation (SSF) as well as extracting in aqueous two-phase system (ATPS). Collagenolytic protease production was performed in using MS-2 culture medium (SF) and soybean bran as substrate (SSF). Subsequently, the fermented liquid from both fermentations were used for the extraction of enzyme by ATPS, it was verified the influence of different variables from a factorial design 23. In SSF the highest protease and collagenolytic activities were achieved with 362.66 U/mL and 179.81 U/mL, respectively. When compared with SF (26.33 and 18.70 U/mL) higher values were obtained in the activities. The protease partitioning from SF and SSF in ATPS showed a similar profile showing higher affinity for the polymer rich phase. The highest value for the response variable purification factor (3.49) was obtained in the system using SSF. Thus, SSF shows promise as a bioprocess for extracellular production of collagenolytic proteases, using of soybean bran as substrate had used sustainable raw material, aiming application this possible enzyme in the treatment of burns and postoperative scarring.

Fibrinolytic proteases are a promising alternative in the pharmaceutical industry, they are used in the treatment of cardiovascular diseases, especially thrombosis. Microorganisms are the most interesting source of fibrinolytic proteases. The aim of this study was the production of fibrinolytic protease from Streptomyces parvulus DPUA 1573, the recovery of the protease by aqueous two-phase system and partial biochemical characterization of the enzyme. The aqueous two-phase system was performed according to a 24-full factorial design using polyethylene glycol molar mass, polyethylene glycol concentration, citrate concentration and pH as independent variables. It was analyzed the effect of different ions, surfactants, inhibitors, pH and temperature on enzyme activity. The best conditions for purifying the enzyme were 17.5% polyethylene glycol 8,000, 15% Phosphate and pH 8.0, it was obtained a partition coefficient of 7.33, a yield of 57.49% and a purification factor of 2.10-fold. There was an increase in enzyme activity in the presence of Fe2+ and a decrease in the presence of $\beta$-Mercaptoethanol, phenylmethylsulfonyl fluoride and Iodoacetic acid. The optimum pH was 7.0 and the optimum temperature was 40 ºC. The purified protease exhibited a molecular mass of 41 kDa. The fibrinolytic protease from Streptomyces parvulus proved to be a viable option for the development of a possible drug with fibrinolytic action.

Precipitation of blood products from plasma fractionation has played a fundamental role in the industrial purification of important therapeutic products. Only a few studies have been reported by using tannins as proteins precipitant agent from whole plasma while, several conditions have been analyzed. Here, we decided to verify the effect of the temperature on the precipitation process of plasma proteins using tannic acid (TA). Plasma proteins were precipitated with tannic acid by using different temperature incubations. Subsequently, the protein-TA complex was analyzed by SDS-PAGE and quantified. In addition, the protein activity of the complex was measured after heating, as well as the structural changes of the complexes were accompanied by thermogravimetric analysis, differential scanning calorimetry and circular dichroism. In all conditions tested, tannic acid was able to precipitate without selectively separating the proteins in the mixture by using different temperatures during the precipitation process. Furthermore, the protein concentration from the plasma precipitate was not affected by different temperatures and the plasma precipitate was able to dissolve fibrin clots in vitro., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Copaiba oil is a natural product used by Amazonian populations and recognized for its medicinal properties because it has significant antimicrobial activity for several pathogenic microorganisms. The present work aimed to evaluate and characterize the effect of natural oil produced by copaiba - Copaifera multijuga against multiresistant isolates of bubaline mastitis. The nitrocefin test was performed with isolates of Staphylococcus aureus from bubaline mastitis, which were 100% positive for beta-lactamase enzyme detection. Minimum Bactericidal Concentration (MBC) of 25% to 3.12% was obtained for Enterococcus faecalis and Escherichia coli and 50% and 25% for S. aureus, but Klebsiella pneumoniae and Bacillus subtilis were resistant. MBC with 12.5% and 6.25% oil were obtained for most multiresistant bubaline mastitis isolates from the states of Pernambuco, Ceará, Bahia and Alagoas. The results demonstrated the great potential of using copaiba natural oil in the treatment of buffalo mastitis.

The industrial demand for proteolytic enzymes is stimulating the search for new enzyme sources. Fungal enzymes are preferred over bacterial enzymes, and more effective and easier to extract. The aim of this work was to evaluate the potential of protease production by solid state fermentation (SSF) of Mucor subtilissimus UCP 1262, evaluate different specific activities, purify and partially characterize the enzyme in terms of biochemical as to the optimal pH and temperature. Initially, the enzyme crude extract was screened for 3 different proteolytic activities, collagenolytic (161.4 U/mL), keratinolytic (39.6 U/mL) and fibrinolytic (26.1 U/mL) in addition to conventional proteinase activity. After ammonium sulfate precipitation, the active fractions with fibrinolytic activity were dialyzed in 15 mM Tris-HCl buffer, pH 8, loaded onto DEAE-Sephadex A50 ion-exchange column and gel filtrated through Superdex 75 HR10/300. The enzyme showed a fibrinolytic maximum activity at 40 C and pH 9,0. The purified enzyme showed activity against a chromogenic chymotrypsin substrate, SDS-PAGE showing a molecular mass of approximately 70 kDa and, the specific activity of 25.93 U/mg. These characteristics suggest that the enzyme could be and efficiently produced in a simple and low-cost way using Mucor subtilissimus UCP 1262 in SSF.

Artrhospira (Spirulina) platensis produced fibrinolytic enzyme under mixotrophic conditions using corn steep liquor (CSL). The enzyme was extracted, purified by combination of two chromatographic techniques and biochemically characterized. Maximum fibrinolytic production (268.14 U mg -1 ) was obtained using liquid medium culture composed by 0.2% CLS after 10th day of cultivation. Fibrinolytic activity was higher when extracted by homogenization methods and was purified 32.72-fold with specific activity of 7988 U mg -1 . Fibrin zymography showed an active band, indicated acts as a plasmin-like protein with molecular weight of 72 kDa. Fibrinolytic enzyme have optimum pH of 6.0, stable in the range of 6.0 to 10.0 during 24 h and optimum temperature at 40 °C with a stability below 50 °C. Fibrinolytic enzyme is a serine metalloprotease by to be enhanced by Fe 2+ and inhibited by PMSF. The enzyme has higher enzymatic activity than most other fibrinolytic enzymes and is stable at temperature and pH human physiological. Overall, the fibrinolytic enzyme from A. platensis has attractive biochemical properties to potential applications in the treatment of thrombosis., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Microalgae are considered one of the most promising raw materials for the development of high value products for pharmaceuticals, nutraceuticals, and cosmetic industries, as well as being potential sources of protein, vitamins, and minerals for human consumption. Hence, the present research focuses extraction of antioxidant and antimicrobial compounds from Scenedesmus subspicatus using solvents of different polarities. Different solvents such as ethanol, methanol, butanol, acetone, dimethyl sulfoxide, and water were used to extract compounds from the green microalgae S. subspicatus and then they were examined for phytochemical screening, antioxidant activity, and antimicrobial properties. In vitro free radical quenching and total antioxidant activity of extracts were investigated with 1,1-diphenyl-2-picryl hydrazyl and compared with catequin and gallic acid as positive controls. The antimicrobial activity was evaluated in gram-negative and gram-positive bacteria. Aqueous extracts and dimethyl sulfoxide presented better performance in phytochemical analysis. This result showed consistency in the sequential tests. The antioxidant activity was also better using the two solvents cited above. The extracts acetone, water, and dimethyl sulfoxide showed ability to inhibit the growth of Bacillus subtilis. However, only dimethyl sulfoxide inhibited the growth of Klebsiella pneumoniae and Escherichia coli. Use of the aqueous extract, proven its effectiveness, is an economic protocol and avoids the use of toxic substances.

The fibrinolytic enzyme produced by Mucor subtilissimus UCP 1262 was obtained by solid fermentation and purified by ion exchange chromatography using DEAE-Sephadex A50. The enzyme toxicity was evaluated using mammalian cell lineages: HEK-293, J774.A1, Sarcoma-180 and PBMCs which appeared to be viable at a level of 80%. The biochemical parameters of the mice treated with an acute dose of enzyme (2000 mg/mL) identified alterations of AST and ALT and the histomorphometric analysis of the liver showed a loss of endothelial cells (P < 0.001). However, these changes are considered minimal to affirm that there was a significant degree of hepatotoxicity. The comet assay and the micronucleus test did not identify damage in the DNA of the erythrocytes of the animals treated. The protease did not degrade the Aα and Bβ chains of human and bovine fibrinogens, thus indicating that it does not act as anticoagulant, but rather as a fibrinolytic agent. The assay performed to assess blood biocompatibility shows that at dose of 0.3-5 mg/mL the hemolytic grade is considered insignificant. Moreover, the enzyme did not prolong bleeding time in mice when dosed with 1 mg/kg. These results indicate that this enzyme produced is a potential competitor for developing novel antithrombotic drugs., (Copyright © 2019. Published by Elsevier Inc.)

Fermented milks are a source of bioactive peptides and may be considered as functional foods. Among these, sheep's milk fermented with kefir has not been widely studied and its most relevant properties need to be more thoroughly characterized. This research study is set out to investigate and evaluate the antioxidant and antimicrobial properties of peptides from fermented sheep's milk in Brazil when produced by using kefir. For this, the chemical and microbiological composition of the sheep's milk before and after the fermentation was evaluated. The changes in the fermented milk and the peptides extracted before the fermentation and in the fermented milk during its shelf life were verified. The antimicrobial and antioxidant activities of the peptides from the fermented milk were evaluated and identified according to the literature. The physicochemical properties and mineral profile of the fermented milk were like those of fresh milk. The peptide extract presented antimicrobial activity and it was detected that 13 of the 46 peptides were able to inhibit the growth of pathogenic microorganisms. A high antioxidant activity was observed in the peptides extracted from fermented milk (3.125 mg/mL) on the 28th day of storage. Two fractions displayed efficient radical scavenging properties by DPPH and ABTS methods. At least 11 peptides distributed in the different fractions were identified by tandem mass spectrometry. This sheep's milk fermented by Brazilian kefir grains, which has antioxidant and antimicrobial activities and probiotic microorganisms, is a good candidate for further investigation as a source for bioactive peptides. The fermentation process was thus a means by which to produce potential bioactive peptides.

Blood coagulation and platelet-dependent primary homeostasis are important defense mechanisms against bleeding and novel inhibitors have been researched to obtain pharmacological and clinical applications. In this work, the PpyLL, a lectin obtained from Phthirusa pyrifolia, was characterized in terms of its molecular structure and biological functions (anticoagulant, antiplatelet agreggation and hemagglutinating activities) in presence or absence of Gamma radiation exposure. Results revealed a lectin with secondary-structure content by approximately 49% of β-sheet, 20% of β-turn and 31% of disordered structure. Irradiation effect demonstrated possible different sites of function by lectin on anticoagulant and hemagglutinating activities, once a decrease about 80% was observed when compared the activities under 0.5kGy of exposition to gamma radiation. An emphatic discussion about the use of gamma radiation as a possible modulator of the lectin activity was made, and once the ionizing radiation affected differently the anticoagulation and hemagglutinating activities, we speculated that the results are determined by selective molecular damages in different binding sites. PpyLL biological activities and gamma radiation modulation could be considered for future researches in biomedical field aiming possible medical applications., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In this work, chitosan-based films containing gelatin and chondroitin-4-sulfate (C4S) with and without ZnO particles were produced and tested in vitro to investigate their potential wound healing properties. Chitosans were produced from shrimp-head processing waste by alkaline deacetylation of chitin to obtain chitosans differing in molecular weight and degree of deacetylation (80 ± 0.5%). The film-forming solutions (chitosan, C4S and gelatin) and ZnO suspension showed no toxicity towards fibroblasts or keratinocytes. Chitosan was able to agglutinate red blood cells, and film-forming solutions induced no hemolysis. Film components were released into solution when incubated in PBS as demonstrated by protein and sugar determination. These data suggest that a stable, chitosan-based film with low toxicity and an ability to release components would be able to establish a biocompatible microenvironment for cell growth. Chitosan-based films significantly increased the percentage of wound healing (wound contraction from 65 to 86%) in skin with full-thickness excision when compared with control (51%), after 6 days. Moreover, histological analysis showed increased granulation tissue in chitosan and chitosan/gelatin/C4S/ZnO films. Chitosan-based biopolymer composites could be used for improved biomedical applications such as wound dressings, giving them enhanced properties.

Fibrinolytic proteases are enzymes that degrade fibrin. They provide a promising alternative to existing drugs for thrombolytic therapy. A protease isolated from the filamentous fungus Mucor subtilissimus UCP 1262 was purified in three steps by ammonium sulfate fractionation, ion exchange, and molecular exclusion chromatographies, and characterized biochemically and structurally. The purified protease exhibited a molecular mass of 20 kDa, an apparent isoelectric point of 4.94 and a secondary structure composed mainly of α-helices. Selectivity for N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as substrate suggests that this enzyme is a chymotrypsin-like serine protease, whose activity was enhanced by the addition of Cu 2+ , Mg 2+ , and Fe 2+ . The enzyme showed a fibrinolytic activity of 22.53 U/mL at 40 °C and its contact with polyethylene glycol did not lead to any significant alteration of its secondary structure. This protein represents an important example of a novel fibrinolytic enzyme with potential use in the treatment of thromboembolic disorders such as strokes, pulmonary emboli, and deep vein thrombosis.