Production and biochemical and biophysical characterization of fibrinolytic protease of a Mucor subtilissimus strain isolated from the caatinga biome.

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.

Recovery and partial purification of fibrinolytic protease from Pleurotus ostreatus and P. eryngii and cytotoxic and antioxidant activity of their extracts.

Mushrooms are a source of primary and secondary metabolites. Little is known about the most suitable conditions for production of mushrooms by submerged fermentation. This article reports antioxidant and cytotoxic assays, in addition to quantitatively evaluating the content of proteases with fibrinolytic action in the crude extracts of two species of edible mushrooms produced in different formulations, as well as evaluating the recovery of these enzymes by aqueous two-phase systems (ATPS). The mushrooms Pleurotus ostreatus and Pleurotus eryngii, at concentration of 100 µg/mL, displayed inhibition of DPPH and ABTS radicals below 50%. In the cytotoxicity test, the cells human fibroblast cell lines (MRC-5) showed cell viability greater than 80%. Concerning fibrinolytic activity, P. eryngii presented 226.47 ± 7.26 U/mL, therefore being more efficient than P. ostreatus (71.5 ± 0.56 U/mL). In the recovery of the P. eryngii extract by ATPS, the fibrinolytic protease was partitioned in the salt phase (30.25 U/mL). The molecular mass of the proteases was between 75 and 100 kDa. These results prove the low cytotoxicity of the extracts produced and that fermentation in supplemented malt broth favored the excretion of fibrinolytic proteases compared to the other evaluated media.

Cost-effective fibrinolytic enzyme production by microalga Dunaliella tertiolecta using medium supplemented with corn steep liquor.

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.

Histomorphometric analysis of the lung of Swiss mice treated with a fibrinolytic protease.

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.

A novel collagenolytic protease from Mucor subtilissimus UCP 1262: Comparative analysis of production and extraction in submerged and stated-solid fermentation.

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.

Purification and characterization of fibrinolytic protease from Streptomyces parvulus by polyethylene glycol-phosphate aqueous two-phase system.

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.

Purification and characterization of a protease from Aspergillus sydowii URM5774: Coffee ground residue for protease production by solid state fermentation.

Solid state fermentation is a promising technology largely used in biotechnology process and is a suitable strategy for producing low-cost enzymatic products. At the present study, a novel enzyme obtained through solid state fermentation using Aspergillus sydowii was herein purified and characterized. The fermentations used coffee ground residue as substrate and the crude enzyme was submitted through further purification steps of: acetonic precipitation, DEAE-Sephadex and Superdex G-75 column. Both crude and purified enzymes were submitted to biochemical characterization of their thermostability, optimal temperature and pH, effects of inhibitors and metal ions. A purified protease was obtained with yield of 5.9-fold and 53% recovery, with maximal proteolytic activity of 352.0 U/mL. SDS-PAGE revealed a band of protein at 47.0 kDa. The enzyme activity was abolished in the presence of phenyl-methyl sulfonyl fluoride and partially inhibited against Triton X-100 (78.0%). The optimal activity was found in pH 8.0 at 45°C of temperature. Besides, the enzyme showed stability between 35°C and 50°C. It was possible to determine appropriate conditions to the obtainment of thermostable proteases with biotechnological interest associated with a method that concomitantly shows excellent production levels and recovery waste raw material in a very profitable process.

Silver nanoprisms as plasmonic enhancers applied in the photodynamic inactivation of Staphylococcus aureus isolated from bubaline mastitis.

Mastitis is a bacterial infection that affects all lactating mammals, and in dairy cattle, it leads to a reduction in their milk production and, in worse cases, it may lead to animal death. One viable therapeutic modality for overcoming bacterial resistance can be photodynamic inactivation (PDI), a therapeutic modality for bacterial infection treatment. One of the main factors that can lead to an efficient PDI process is the association of metallic nanoparticles in the close vicinity of photosensitizers, which has shown promising results due to localized surface plasmon resonance phenomena. In this work, methylene blue (MB) molecules were associated with Ag prismatic nanoplatelets (AgNPrs) to use as PDI photosensitizer against Staphylococcus aureus isolated from bubaline mastitis. The optical plasmonic activity of AgNPrs was tuned to the MB absorption region (600-700 nm) by inducing their growth into prismatic shapes by a seed-mediated procedure, using poly (sodium 4-styrene sulfonate) as the surfactant. A simulation on the plasmonic properties of the nanoprisms, applying particle size within the dimensions determined by TEM image analysis (d = 32 ± 6 nm), showed a 30 % increase of the incident field on the prismatic tips. Photodynamic results showed that the electrostatic AgNPr-MB conjugates promoted enhancement (ca. 15 %) of the reactive oxygen species production. Besides, PDI mediated by AgNPrs-MB led to the complete inactivation of the mastitis S. aureus strain after 6 min inactivation, in contrast to PDI mediated by MB, which reduced less than a 0.5 bacterial log. Thus, the results show this plasmonic enhanced photodynamic tool's potential to be applied in the inactivation of multi-resistant bacterial strains.

Antimicrobial potential of Copaiba Oil (Copaifera multijuga Hayne-Leguminosae) against bubaline mastitis multiresistant isolates.

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.

Protease from Mucor subtilissimus UCP 1262: Evaluation of several specific protease activities and purification of a fibrinolytic enzyme.

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.