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.

Biosorption Cu (II) by the yeast Saccharomyces cerevisiae.

With the industrial and population advances, the generation of effluents containing heavy metals has grown a lot. In this work, the commercial biomass of the yeast Saccharomyces cerevisiae Perlage® BB were carried out as Cu (II) ion biosorbent. The influence of some variables such as metal concentration, pH range, equilibrium time and biomass concentration were evaluated. The biosorption capacity was measured by adsorption isotherms, with the Langmuir, Freundlich, and Dubinin-Radushkevich (D-R) models. The characterization of the biomass surface were investigated by Dispersive Energy X-Ray Fluorescence Spectrometry (EDX) and Atomic Force Microscopy (AFM). The results showed that the biomass presented good biosorption efficiency. The best fit of the data was obtained with the Langmuir model, detecting the maximum biosorption capacity of 4.73 mg g-1. By the methods used in the characterization of the biomass surface, it was possible to verify the presence of the Cu (II) ion in the yeast.

Structural Studies of a Lipid-Binding Peptide from Tunicate Hemocytes with Anti-Biofilm Activity.

Clavanins is a class of peptides (23aa) histidine-rich, free of post-translational modifications. Clavanins have been studied largely for their ability to disrupt bacterial membranes. In the present study, the interaction of clavanin A with membranes was assessed by dynamic light scattering, zeta potential and permeabilization assays. We observed through those assays that clavanin A lysis bacterial cells at concentrations corresponding to its MIC. Further, the structure and function of clavanin A was investigated. To better understand how clavanin interacted with bacteria, its NMR structure was elucidated. The solution state NMR structure of clavanin A in the presence of TFE-d3 indicated an α-helical conformation. Secondary structures, based on circular dichroism measurements in anionic sodium dodecyl sulfate (SDS) and TFE (2,2,2-trifluorethanol), in silico lipid-peptide docking and molecular simulations with lipids DPPC and DOPC revealed that clavanin A can adopt a variety of folds, possibly influencing its different functions. Microcalorimetry assays revealed that clavanin A was capable of discriminating between different lipids. Finally, clavanin A was found to eradicate bacterial biofilms representing a previously unrecognized function.

Nanostructured sensor based on carbon nanotubes and clavanin A for bacterial detection.

Unusual methods for specific detection of pathogenic bacteria are becoming key points for control and identification of problems related to health and (bio)safety. In this context, this work aims to propose a new approach for the development of nanostructured biosensors based on carbon nanotubes (CNTs) and antimicrobial peptides for bacterial detection. Firstly, the antimicrobial peptide clavanin A (ClavA) was chemically immobilized on CNTs and surface-immobilized ClavA was used to detect Klebsiella pneumoniae, Enterococcus faecalis, Escherichia coli and Bacillus subtilis in a direct assay format. We used electrochemical impedance spectroscopy technique to evaluate the effectiveness and sensitivity of the ClavA-based biosensors by measuring the modifications in their electrochemical responses before and after incubation in presence of different bacteria concentrations. The biosensor was able to discriminate between bacteria concentrations in the 10(2)-10(6)CFU mL(-1) range. Atomic force microscopy analysis confirmed the biosensor functionality for bacterial recognition. This new sensor system was capable of differentiating between Gram-positive and Gram-negative bacteria, since ClavA showed different affinities toward the pathogenic bacteria species.

Elucidation of mechanisms of interaction of a multifunctional peptide Pa-MAP with lipid membranes.

This work aims to investigate the possible mechanism of action of the homologue peptide Pa-MAP based on the Antarctic fish Pleuronectes americanus, through a study by electrical impedance spectroscopy (EIS) of models of bilayer lipid membranes supported (BLM-s) on solid substrates. For comparison and validation of the data obtained by EIS, we also conducted a study evaluating the human peptide LL-37, whose mechanism of action is well described in the literature: its dielectric response was found to be similar to that of Pa-MAP. The results obtained indicate that Pa-MAP has a good potential for use as a membrane-disrupting peptide and also suggest that the corresponding mechanism of action occurs according to the carpet model followed by a detergent-like effect. The addition of either one of these peptides at different concentrations resulted in a drastic decrease in the membrane's resistance, after just 1min of exposure. Additionally, it was seen that the peptides Pa-MAP and LL-37 may act on membranes with different charges, in an indication of a possible broad spectrum antimicrobial activity. These interactions with different membrane compositions have been attributed to the peptides' structure, mainly due to the presence of many hydrophobic amino acid residues, as observed by in silico studies. Here, we describe the Pa-MAP mechanism of action for the first time. Furthermore, we report the data demonstrating that EIS can be used for studies of peptide-membranes interaction, even when small changes on the surface of the electrode can be detected.

Mechanistic aspects of peptide-membrane interactions determined by optical, dielectric and piezoelectric techniques: an overview.

Antimicrobial peptides (AMPs) have been isolated from a wide variety of organisms that include microorganisms, plants, insects, frogs and mammals. As part of the innate immune system expressed in many tissues, AMPs are able to provide protection against invasion of foreign microorganisms and exhibit a broad spectrum of activity against bacteria, fungi and/or virus. Non-AMPs cell-penetrating peptides have been used as carriers for overcoming the membrane barrier and helping in the delivery of various molecules into the cell. Physicochemical peptide-lipid interactions studies can provide us with reliable molecular information about microbe defense response, including the elucidation of the prevailing mechanisms of its action, such as the barrel-stave, toroidal pore, carpet and detergent-like models. In this paper, we present an overview of the peptide-lipid mechanisms of interaction as well as discuss alternative techniques that could help to elucidate the peptides functionality. Quartz crystal microbalance (QCM), surface plasmon resonance (SPR) spectroscopy and electrochemical impedance spectroscopy (EIS) are useful techniques to investigate in details of the peptide-membrane interaction. The techniques here discussed could also offer specific and low-cost methods that can to shed some light over the different modes of action of AMPs, contributing to the development of drugs against infectious diseases.

Evaluation of Magainin I interactions with lipid membranes: an optical and electrochemical study.

Most antimicrobial peptides (AMPs) have shown clear activity related to the disruption of lipid bilayers. In order to improve knowledge of this subject, the interaction of Magainin I (MagI) with phospholipid layers (PLs), uncoated or coated with synperonic (Synp), was studied using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and surface plasmon resonance (SPR) techniques. MagI peptide was immobilized on gold electrode via a self-assembling monolayer obtained from liposomes and liposomes covered by Synp. MagI induces pores in the supported lipid membranes, which are reflected in an increased amperometric-response and also a decreased electron-transfer resistance (R(CT)). In addition, MagI showed a significant interaction with the PL-Synp-modified gold electrode, but MagI showed a reliable contact with the PL-modified gold electrode, leading to a decrease in the relative resistance charge transfer value of -17.38%. Our results demonstrated that Synp acts as a membrane sealant after exposure of the lipid membrane to MagI. A parallel reaction model was proposed for the interaction of MagI and a hybrid layer that result in a complex bimolecular interaction. In short, the importance of triblock copolymer to stabilize liposomes for future applications as drug delivery systems for MagI was demonstrated.