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Purpose of this study is to explore the extraction of potentially valuable cosmetic ingredients from rice crop residues, aiming to mitigate their environmental impact. Methods: We employed AOAC methods to analyze the fat, protein, ash, fiber, soluble, and insoluble carbohydrate content in these residues. To identify sugars rich in galactose and acidic sugars, a total soluble carbohydrate extraction was performed. Cellulose, as part of the insoluble carbohydrates, was isolated through alkaline and acid hydrolysis, while sodium silicate was derived from the ash. Characterization of insoluble cellulose and silicate involved techniques like FTIR, DSC, PXRD, microphotography, porosity assessments, and water absorption studies. For proteins, alkaline solubilization and precipitation at the isoelectric point were utilized, with quantification via BCA and amino acid profiling through gas chromatography. Evaluation of radical scavenging capacity using DPPH led to the calculation of apparent molecular weight via SDS-PAGE. Results: The results revealed low levels of gum, mucilage, and pectin in both residues, contrasting with a high concentration of insoluble polysaccharides. Among these, Iß cellulose displayed potential attributes for cosmetic applications due to its oil and water adsorption characteristics. However, silicates obtained from the ashes did not exhibit direct use potential. In terms of protein extraction, we observed antioxidant properties, with enhanced performance through enzymatic hydrolysis, achieving a hydrolysis degree of 30.41% and a DPPH radical absorption rate exceeding 70%. Conclusion: Rice residues, particularly husk and straw, shown valuable substances suitable for potential cosmetic applications, encompassing cellulose, hydrolyzed proteins, and ash as a silicate precursor.

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Spider venoms are composed, among other substances, of peptide toxins whose selectivity for certain physiological targets has made them powerful tools for applications such as bioinsecticides, analgesics, antiarrhythmics, antibacterials, antifungals and antimalarials, among others. Bioinsecticides are an environmentally friendly alternative to conventional agrochemicals. In this paper, the primary structure of an insecticidal peptide was obtained from the venom gland transcriptome of the ctenid spider Phoneutria depilata (Transcript ID PhdNtxNav24). The peptide contains 53 amino acids, including 10 Cys residues that form 5 disulfide bonds. Using the amino acid sequence of such peptide, a synthetic gene was constructed de novo by overlapping PCRs and cloned into an expression vector. A recombinant peptide, named delta-ctenitoxin (rCtx-4), was obtained. It was expressed, folded, purified and validated using mass spectrometry (7994.61 Da). The insecticidal activity of rCtx-4 was demonstrated through intrathoracic injection in crickets (LD50 1.2 µg/g insect) and it was not toxic to mice. rCtx-4 is a potential bioinsecticide that could have a broad spectrum of applications in agriculture.

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The methylotrophic yeast Pichia pastoris has been one of the most widely used organisms in recent years as an expression system for a wide variety of recombinant proteins with therapeutic potential. Its popularity as an alternative system to Escherichia coli is mainly due to the easy genetic manipulation and the ability to produce high levels of heterologous proteins, either intracellularly or extracellularly. Being a eukaryotic organism, P. pastoris carries out post-translational modifications that allow it to produce soluble and correctly folded recombinant proteins. This work, evaluated the expression capacity in P. pastoris of two single-chain variable fragments (scFvs) of human origin, 10FG2 and LR. These scFvs were previously obtained by directed evolution against scorpion venom toxins and are able to neutralize different toxins and venoms of Mexican species. The yield obtained in P. pastoris was higher than that obtained in bacterial periplasm (E. coli), and most importantly, biochemical and functional properties were not modified. These results confirm that P. pastoris yeast can be a good expression system for the production of antibody fragments of a new recombinant antivenom.

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Background: Scorpion neurotoxins such as those that modify the mammalian voltage-gated sodium ion channels (Nav) are the main responsible for scorpion envenomation. Their neutralization is crucial in the production of antivenoms against scorpion stings. Methods: In the present study, two in silico designed genes - one that codes for a native neurotoxin from the venom of the Anatolian scorpion Androctonus crassicauda, named Acra 4 - and another non-native toxin - named consensus scorpion toxin (SccTx) obtained from the alignment of the primary structures of the most toxic neurotoxins from the Middle Eastern and North African scorpions - were recombinantly expressed in E. coli Origami. Results: Following bacterial expression, the two expressed neurotoxins, hereafter named HisrAcra4 and HisrSccTx, were obtained from inclusion bodies. Both recombinant neurotoxins were obtained in multiple Cys-Cys isoforms. After refolding, the active protein fractions were identified with molecular masses of 8,947.6 and 9,989.1 Da for HisrAcra4 and HisrSccTx, respectively, which agreed with their expected theoretical masses. HisrAcra4 and HisrSccTx were used as antigens to immunize two groups of rabbits, to produce either anti-HisrAcra4 or anti-HisrSccTx serum antibodies, which in turn could recognize and neutralize neurotoxins from venoms of scorpion species from the Middle East and North Africa. The antibodies obtained from rabbits neutralized the 3LD50 of Androctonus australis, Leiurus quinquestriatus hebraeus and Buthus occitanus venoms, but they did not neutralize A. crassicauda and A. mauritanicus venoms. In addition, the anti-HisrAcra4 antibodies did not neutralize any of the five scorpion venoms tested. However, an antibody blend of anti-HisrAcra4 and anti-HisrSccTx was able to neutralize A. crassicauda and A. mauritanicus venoms. Conclusions: Two recombinant Nav neurotoxins, from different peptide families, were used as antigens to generate IgGs for neutralizing scorpion venoms of species from the Middle East and North Africa.

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Given consumer trends propelling a movement toward using plant protein in the food industry and searching for alternative protein ingredients by the industry, this study aimed to assess the influence of factors such as protein concentration, medium pH, and the presence of a divalent ion (Ca2+) upon the rheological properties such as viscosity change and gel formation of dispersion proteins extracted from quinoa, black beans, and lentils. A solution of each protein was prepared by varying its concentration (2.5%, 5.0%, and 10%), the pH (5.0, 7.0, and 9.0), and the incorporation of calcium chloride (0.0% and 1.0%). Each obtained solution was subjected to rheological tests to determine the parameters: consistency index (K), flow behavior (n), the storage (G') and loss (G'') modules, and the phase shift angle (δ). The results demonstrate that the incorporation of Ca2+, the shift in protein levels, and the decrease in pH modified the rheological behaviors of proteins, which were also influenced by the structural characteristics of each protein studied. However, thermal treatment and protein concentrations caused the most significant impact on proteins' rheological behavior, forming gels independently of other conditions. It was possible to study and interpret the studied proteins' rheological variations according to the environment's conditions.

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Crotoxin complex CA/CB and crotamine are the main toxins associated with Crotalus envenomation besides the enzymatic activities of phospholipases (PLA2) and proteases. The neutralization at least of the crotoxin complex by neutralizing the subunit B could be a key in the production process of antivenoms against crotalids. Therefore, in this work, a Crotoxin B was recombinantly expressed to evaluate its capacity as an immunogen and its ability to produce neutralizing antibodies against crotalid venoms. A Crotoxin B transcript from Crotalus tzabcan was cloned into a pCR®2.1-TOPO vector (Invitrogen, Waltham, MA, USA) and subsequently expressed heterologously in bacteria. HisrCrotoxin B was extracted from inclusion bodies and refolded in vitro. The secondary structure of HisrCrotoxin B was comparable to the secondary structure of the native Crotoxin B, and it has PLA2 activity as the native Crotoxin B. HisrCrotoxin B was used to immunize rabbits, and the obtained antibodies partially inhibited the activity of PLA2 from C. tzabcan. The anti-HisrCrotoxin B antibodies neutralized the native Crotoxin B and the whole venoms from C. tzabcan, C. s. salvini, and C. mictlantecuhtli. Additionally, anti-HisrCrotoxin B antibodies recognized native Crotoxin B from different Crotalus species, and they could discriminate venom in species with high or low levels of or absence of Crotoxin B.

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Sonicated protein isolates were recovered from Chenopodium quinua, Phaseoulus vulgaris and Lens culinaris to develop a functional matrix by assessing the physicochemical and functional properties. The plant protein isolates were prepared from powdered materials followed by sonication in alkaline medium using a Box-Behnken design. pH (6-10), a buffer-to-material ratio (5:1 to 15:1) and sonication time (0-20 min) were taken as independent variables, whereas protein yield was taken as the dependent variable. A pH of 9, 20 min treatment, and a buffer-to-material ratio of 5:1 were the optimal extraction conditions for quinoa and black beans, whereas a 1:10 ratio was suitable for lentils. Sonication in alkaline medium caused partial protein unfolding and these isolates; in turn, the molecular weight affected the emulsifying activity and stability. Moreover, sonication had a strong effect on the gelation temperature, emulsifying activity, the water, and oil sorption. Sonication improved protein yield and exposed amino acids such as glutamic acid, aspartic acid, leucine and glycine. In turn, thiol groups were responsible for the increased in gelation temperature. The better gelling property coupled with high emulsifying property of these proteins show potential application as protein emulsifiers in the production of gels, sausages, and pet foods.

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The antimicrobial and immunomodulatory capacities of the peptide Css54 and the chemokine MCP-1 were tested. The first, a peptide isolated from the venom of the scorpion Centruroides suffusus suffusus was synthesized chemically. In contrast, the second is a monocyte chemoattractant expressed as a recombinant protein in our lab. It was observed in vitro that Css54 inhibited the growth of Salmonella enterica serovar Typhimurium (6.2 µg/mL). At high concentrations, it was toxic to macrophages (25 µg/mL), activated macrophage phagocytosis (1.5 µg/mL), and bound Salmonella LPS (3 µg/mL). On the other hand, the recombinant MCP-1 neither inhibited the growth of Salmonella Typhimurium nor was it toxic to macrophages (up to 25 µg/mL), nor activated macrophage phagocytosis or bound Salmonella LPS (up to 3 µg/mL). Although it was observed in vivo in mice Balb/C that both Css54 and MCP-1 did not resolve the intraperitoneal infection by S. Typhimurium, Css54 decreased the expression of IL-6 and increased IL-10, IL-12p70, and TNF-α levels; meanwhile, MCP-1 decreased the expression of IFN-γ and increased IL-12p70 and TNF-α. It was also observed that the combination of both molecules Css54 and MCP-1 increased the expression of IL-10 and TNF-α.

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The transcriptome of the venom glands of the Phoneutria depilata spider was analyzed using RNA-seq with an Illumina protocol, which yielded 86,424 assembled transcripts. A total of 682 transcripts were identified as potentially coding for venom components. Most of the transcripts found were neurotoxins (156) that commonly act on sodium and calcium channels. Nevertheless, transcripts coding for some enzymes (239), growth factors (48), clotting factors (6), and a diuretic hormone (1) were found, which have not been described in this spider genus. Furthermore, an enzymatic characterization of the venom of P. depilata was performed, and the proteomic analysis showed a correlation between active protein bands and protein sequences found in the transcriptome. The transcriptomic analysis of P. depilata venom glands show a deeper description of its protein components, allowing the identification of novel molecules that could lead to the treatment of human diseases, or could be models for developing bioinsecticides.

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Background: Scorpion neurotoxins such as those that modify the mammalian voltagegated sodium ion channels (Nav) are the main responsible for scorpion envenomation. Their neutralization is crucial in the production of antivenoms against scorpion stings. Methods: In the present study, two in silico designed genes ­ one that codes for a native neurotoxin from the venom of the Anatolian scorpion Androctonus crassicauda, named Acra 4 ­ and another non-native toxin ­ named consensus scorpion toxin (SccTx) obtained from the alignment of the primary structures of the most toxic neurotoxins from the Middle Eastern and North African scorpions ­ were recombinantly expressed in E. coli Origami. Results: Following bacterial expression, the two expressed neurotoxins, hereafter named HisrAcra4 and HisrSccTx, were obtained from inclusion bodies. Both recombinant neurotoxins were obtained in multiple Cys-Cys isoforms. After refolding, the active protein fractions were identified with molecular masses of 8,947.6 and 9,989.1 Da for HisrAcra4 and HisrSccTx, respectively, which agreed with their expected theoretical masses. HisrAcra4 and HisrSccTx were used as antigens to immunize two groups of rabbits, to produce either anti-HisrAcra4 or anti-HisrSccTx serum antibodies, which in turn could recognize and neutralize neurotoxins from venoms of scorpion species from the Middle East and North Africa. The antibodies obtained from rabbits neutralized the 3LD50 of Androctonus australis, Leiurus quinquestriatus hebraeus and Buthus occitanus venoms, but they did not neutralize A. crassicauda and A. mauritanicus venoms. In addition, the anti-HisrAcra4 antibodies did not neutralize any of the five scorpion venoms tested. However, an antibody blend of anti-HisrAcra4 and anti-HisrSccTx was able to neutralize A. crassicauda and A. mauritanicus venoms. Conclusions: Two recombinant Nav neurotoxins, from different peptide families, were used as antigens to generate IgGs for neutralizing scorpion venoms of species from the Middle East and North Africa.(AU)

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This study aimed to assess the thermal stability of the bioactive compounds from annatto seed extract, encapsulated by ionic gelation using quinoa proteins, lentil proteins, soy proteins, and sodium caseinate as carrying materials. The 10.0% aqueous dispersions of the different proteins (carriers) were prepared and mixed with the annatto seed extract. The dispersions were then extruded into a calcium chloride solution to induce the extract encapsulation. The capsules were characterized by encapsulation efficiency, particle size, infrared transmission spectroscopy, confocal microscopy, and scanning electron microscopy (SEM). The antioxidant and antimicrobial activities, the polyphenol compounds, and bixin content from the free and encapsulated extract were assessed once stored for 12 d at different temperatures (4 °C, 25 °C, and 65 °C). The results demonstrated the ability of the proteins to encapsulate the annatto extract with encapsulation efficiencies ranging from 58% to 80%, where the protein structure and amino acid content were the relevant factors to obtain high encapsulation efficiencies. The free extracts stored at 65 °C for 12 d experienced a degradation of bixin and polyphenol compounds, respectively. Conversely, the encapsulated extract had degradations from ~34.00% to ~4.05% for polyphenol compounds and ~20.0% for bixin, respectively. These proteins have a potential encapsulation capacity of annatto extract by ionic gelation.

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The three-fingered toxin family and more precisely short-chain α-neurotoxins (also known as Type I α-neurotoxins) are crucial in defining the elapid envenomation process, but paradoxically, they are barely neutralized by current elapid snake antivenoms. This work has been focused on the primary structural identity among Type I neurotoxins in order to create a consensus short-chain α-neurotoxin with conserved characteristics. A multiple sequence alignment considering the twelve most toxic short-chain α-neurotoxins reported from the venoms of the elapid genera Acanthophis, Oxyuranus, Walterinnesia, Naja, Dendroaspis and Micrurus led us to propose a short-chain consensus α-neurotoxin, here named ScNtx. The synthetic ScNtx gene was de novo constructed and cloned into the expression vector pQE30 containing a 6His-Tag and an FXa proteolytic cleavage region. Escherichia coli Origami cells transfected with the pQE30/ScNtx vector expressed the recombinant consensus neurotoxin in a soluble form with a yield of 1.5 mg/L of culture medium. The 60-amino acid residue ScNtx contains canonical structural motifs similar to α-neurotoxins from African elapids and its LD50 of 3.8 µg/mice is similar to the most toxic short-chain α-neurotoxins reported from elapid venoms. Furthermore, ScNtx was also able to antagonize muscular, but not neuronal, nicotinic acetylcholine receptors (nAChR). Rabbits immunized with ScNtx were able to immune-recognize short-chain α-neurotoxins within whole elapid venoms. Type I neurotoxins are difficult to isolate and purify from natural sources; therefore, the heterologous expression of molecules such ScNtx, bearing crucial motifs and key amino acids, is a step forward to create common immunogens for developing cost-effective antivenoms with a wider spectrum of efficacy, quality and strong therapeutic value.

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The scorpionism in Panama is notorious for the confluence and coexistence of buthid scorpions from the genera Centruroides and Tityus. This communication describes an overview of the larger representative toxic venom fractions from eight dangerous buthid scorpion species of Panama: Centruroides (C. granosus, C. bicolor, C. limbatus and C. panamensis) and Tityus (T. (A.) asthenes, T. (A.) festae, T. (T.) cerroazul and T. (A.) pachyurus). Their venoms were separated by HPLC and the corresponding sub-fractions were tested for lethality effects on mice and insects. Many fractions toxic to either mice or insects, or both, were found and have had their molecular masses determined by mass spectrometry analysis. The great majority of the lethal components had a molecular mass close to 7000 Da, assumed to be peptides that recognize Na+-channels, responsible for the toxicity symptoms observed in other buthids scorpion venoms. A toxic peptide isolated from the venom of T. pachyurus was sequenced by Edman degradation, allowing the synthesis of nucleotide probe for cloning the correspondent gene. The mature toxin based on the cDNA sequencing has the C-terminal residue amidated, contains 62 amino acid packed by 4 disulfide linkages, with molecular mass of 7099.1 Da. This same toxic peptide seems to be present in scorpions of the species T. pachyurus collected in 5 different regions of Panama, although the overall HPLC profile is quite different. The most diverse neurotoxic venom components from the genus Centruroides were found in the species C. panamensis, whereas T. cerroazul was the one from the genus Tityus. The most common neurotoxins were observed in the venoms of T. festae, T. asthenes and T. pachyurus with closely related molecular masses of 7099.1 and 7332 Da. The information reported here is considered very important for future generation of a neutralizing antivenom against scorpions from Panama. Furthermore, it will contribute to the growing interest in using bioactive toxins from scorpions for drug discovery purposes.

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Background: The choice between heterologous expression versus chemical synthesis for synthesizing short cysteine-rich insecticidal peptides from arthropods may impact the obtainment of yields and well-folded bioactive molecules for scientific research. Therefore, two recombinant expression systems were compared to that of chemical synthesis for producing Ba1, a cysteine-rich spider neurotoxin. Methods: The transcription of the insecticidal neurotoxin Ba1 was obtained from a cDNA library of venom glands of the spider Brachypelma albiceps.It was cloned into the pCR®2.1-TOPO® cloning vector and then introduced in two different expression vectors, pQE40 and pET28a+. Each vector was transfected into E. coli M15 and BL21 cells, respectively, and expressed under induction with isopropyl thiogalactoside (IPTG). The chemical synthesis of Ba1 was performed in an Applied Biosystems 433A peptide synthesizer. Results: Both expression systems pQE40 and pET28a+ expressed the His-tagged recombinant protein products, HisrDFHRBa1 and HisrBa1, respectively, as inclusion bodies. The recombinant proteins HisrDFHRBa1 and HisrBa1 presented respective molecular masses of 28,289 and 8274.6 Da, and were not biologically active. These results suggested that both HisrDFHRBa1 and HisrBa1 were oxidized after cell extraction, and that their insecticidal activities were affected by their N-terminal pro-peptides and different disulfide bridge arrangements. The respective protein expression yields for HisrDFHRBa1 and HisrBa1 were 100 μg/L and 900 μg/L of culture medium. HisrBa1 was reduced and folded under in vitroconditions. The in vitro folding of HisrBa1 produced several isoforms, one of which, after removing its N-terminal pro-peptide by enzymatic cleavage, presented elevated insecticidal activities compared to the native Ba1. Furthermore, the His-tagged protein HisrDFHRBa1 underwent enzymatic cleavage to obtain recombinant Ba1 (rBa1). As expected, the molecular mass of rBa1 was 4406.4 Da. On the other hand, Ba1 was chemically synthesized (sBa1) with a yield of 11 mg per 0.1 mmol of amino acid assembly. Conclusions: The two recombinant insecticidal peptides and the one synthesized chemically were as active as the native Ba1; however, toxin yields differed drastically.(AU)

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BACKGROUND: The choice between heterologous expression versus chemical synthesis for synthesizing short cysteine-rich insecticidal peptides from arthropods may impact the obtainment of yields and well-folded bioactive molecules for scientific research. Therefore, two recombinant expression systems were compared to that of chemical synthesis for producing Ba1, a cysteine-rich spider neurotoxin. METHODS: The transcription of the insecticidal neurotoxin Ba1 was obtained from a cDNA library of venom glands of the spider Brachypelma albiceps. It was cloned into the pCR®2.1-TOPO® cloning vector and then introduced in two different expression vectors, pQE40 and pET28a(+). Each vector was transfected into E. coli M15 and BL21 cells, respectively, and expressed under induction with isopropyl thiogalactoside (IPTG). The chemical synthesis of Ba1 was performed in an Applied Biosystems 433A peptide synthesizer. RESULTS: Both expression systems pQE40 and pET28a(+) expressed the His-tagged recombinant protein products, HisrDFHRBa1 and HisrBa1, respectively, as inclusion bodies. The recombinant proteins HisrDFHRBa1 and HisrBa1 presented respective molecular masses of 28,289 and 8274.6 Da, and were not biologically active. These results suggested that both HisrDFHRBa1 and HisrBa1 were oxidized after cell extraction, and that their insecticidal activities were affected by their N-terminal pro-peptides and different disulfide bridge arrangements. The respective protein expression yields for HisrDFHRBa1 and HisrBa1 were 100 µg/L and 900 µg/L of culture medium. HisrBa1 was reduced and folded under in vitro conditions. The in vitro folding of HisrBa1 produced several isoforms, one of which, after removing its N-terminal pro-peptide by enzymatic cleavage, presented elevated insecticidal activities compared to the native Ba1. Furthermore, the His-tagged protein HisrDFHRBa1 underwent enzymatic cleavage to obtain recombinant Ba1 (rBa1). As expected, the molecular mass of rBa1 was 4406.4 Da. On the other hand, Ba1 was chemically synthesized (sBa1) with a yield of 11 mg per 0.1 mmol of amino acid assembly. CONCLUSIONS: The two recombinant insecticidal peptides and the one synthesized chemically were as active as the native Ba1; however, toxin yields differed drastically.

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Background:The choice between heterologous expression versus chemical synthesis for synthesizing short cysteine-rich insecticidal peptides from arthropods may impact the obtainment of yields and well-folded bioactive molecules for scientific research. Therefore, two recombinant expression systems were compared to that of chemical synthesis for producing Ba1, a cysteine-rich spider neurotoxin.Methods:The transcription of the insecticidal neurotoxin Ba1 was obtained from a cDNA library of venom glands of the spider Brachypelma albiceps.It was cloned into the pCR®2.1-TOPO® cloning vector and then introduced in two different expression vectors, pQE40 and pET28a+. Each vector was transfected into E. coli M15 and BL21 cells, respectively, and expressed under induction with isopropyl thiogalactoside (IPTG). The chemical synthesis of Ba1 was performed in an Applied Biosystems 433A peptide synthesizer.Results:Both expression systems pQE40 and pET28a+ expressed the His-tagged recombinant protein products, HisrDFHRBa1 and HisrBa1, respectively, as inclusion bodies. The recombinant proteins HisrDFHRBa1 and HisrBa1 presented respective molecular masses of 28,289 and 8274.6 Da, and were not biologically active. These results suggested that both HisrDFHRBa1 and HisrBa1 were oxidized after cell extraction, and that their insecticidal activities were affected by their N-terminal pro-peptides and different disulfide bridge arrangements. The respective protein expression yields for HisrDFHRBa1 and HisrBa1 were 100 μg/L and 900 μg/L of culture medium. HisrBa1 was reduced and folded under in vitroconditions. The in vitro folding of HisrBa1 produced several isoforms, one of which, after removing its N-terminal pro-peptide by enzymatic cleavage, presented elevated insecticidal activities compared to the native Ba1. Furthermore, the His-tagged protein HisrDFHRBa1 underwent enzymatic cleavage to obtain recombinant Ba1 (rBa1). As expected, the molecular mass of rBa1 was 4406.4 Da. On the other hand, Ba1 was chemically synthesized (sBa1) with a yield of 11 mg per 0.1 mmol of amino acid assembly.Conclusions:The two recombinant insecticidal peptides and the one synthesized chemically were as active as the native Ba1; however, toxin yields differed drastically.(AU)

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Background: The choice between heterologous expression versus chemical synthesis for synthesizing short cysteine-rich insecticidal peptides from arthropods may impact the obtainment of yields and well-folded bioactive molecules for scientific research. Therefore, two recombinant expression systems were compared to that of chemical synthesis for producing Ba1, a cysteine-rich spider neurotoxin. Methods: The transcription of the insecticidal neurotoxin Ba1 was obtained from a cDNA library of venom glands of the spider Brachypelma albiceps.It was cloned into the pCR®2.1-TOPO® cloning vector and then introduced in two different expression vectors, pQE40 and pET28a+. Each vector was transfected into E. coli M15 and BL21 cells, respectively, and expressed under induction with isopropyl thiogalactoside (IPTG). The chemical synthesis of Ba1 was performed in an Applied Biosystems 433A peptide synthesizer. Results: Both expression systems pQE40 and pET28a+ expressed the His-tagged recombinant protein products, HisrDFHRBa1 and HisrBa1, respectively, as inclusion bodies. The recombinant proteins HisrDFHRBa1 and HisrBa1 presented respective molecular masses of 28,289 and 8274.6 Da, and were not biologically active. These results suggested that both HisrDFHRBa1 and HisrBa1 were oxidized after cell extraction, and that their insecticidal activities were affected by their N-terminal pro-peptides and different disulfide bridge arrangements. The respective protein expression yields for HisrDFHRBa1 and HisrBa1 were 100 μg/L and 900 μg/L of culture medium. HisrBa1 was reduced and folded under in vitroconditions. The in vitro folding of HisrBa1 produced several isoforms, one of which, after removing its N-terminal pro-peptide by enzymatic cleavage, presented elevated insecticidal activities compared to the native Ba1. Furthermore, the His-tagged protein HisrDFHRBa1 underwent enzymatic cleavage to obtain recombinant Ba1 (rBa1). As expected, the molecular mass of rBa1 was 4406.4 Da. On the other hand, Ba1 was chemically synthesized (sBa1) with a yield of 11 mg per 0.1 mmol of amino acid assembly. Conclusions: The two recombinant insecticidal peptides and the one synthesized chemically were as active as the native Ba1; however, toxin yields differed drastically.

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Five variants of human ß-defensins (HBDs) were expressed in Escherichia coli using two vector systems (pET28a(+) and pQE30) with inducible expression by IPTG. The last vector has not been previously reported as an expression system for HBDs. The recombinant peptides were different in their lengths and overall charge. The HBDs were expressed as soluble or insoluble proteins depending on the expression system used, and the final protein yields ranged from 0.5 to 1.6 mg of peptide/g of wet weight cells, with purities higher than 90%. The recombinant HBDs demonstrated a direct correlation between antimicrobial activity and the number of basic charged residues; that is, their antimicrobial activity was as follows: HBD3-M-HBD2 > HBD3 = HBD3-M = HB2-KLK > HBD2 when assayed against E. coli, Staphylococcus aureus and Pseudomonas aeruginosa. Interestingly, HBD2 had the best antimicrobial activity against the Mycobacterium tuberculosis strain H37Rv (1.5 µM) and the heterologous tandem peptide, HBD3-M-HBD2, had the best minimal inhibitory concentration (MIC) value (2.7 µM) against a multidrug resistance strain (MDR) of M. tuberculosis, demonstrating the feasibility of the use of HBDs against pathogenic M. tuberculosis reported to be resistant to commercial antibiotics.

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Al igual que las proteínas, los péptidos antimicrobianos (PAM) son moléculas versátiles sintetizadas por microorganismos siguiendo rutas enzimáticas, y con interesantes propiedades alimentarias y farmacéuticas, entre ellas la capacidad antibiótica sobre patógenos. La búsqueda convencional de biomoléculas provenientes de microorganismos consiste en analizar químicamente su extracto crudo, lo cual es engorroso y prolongado. El método de aislamiento usado en esta investigación permitió localizar microorganismos con capacidad para producir PAM por interacción de cargas entre moléculas generadas en el metabolismo microbiano y un colorante cargado presente en el medio de cultivo. Se aislaron 20 muestras de suelos de varios pisos térmicos en medios selectivos. Se purificaron 35 cepas con base en la interacción con un colorante básico y se identificaron microorganismos del género Streptomyces sp. y Bacillus sp. Se obtuvieron proteínas de los extractos crudos de fermentaciones, identificando péptidos y aminoácidos por cromatografía de capa fina y electroforesis. Aquellos extractos con alto contenido proteico se evaluaron por bioautografía, y se encontraron 2 extractos de 35 con actividad inhibitoria sobre E. coli ATCC 8739 (halos de 8 mm). Se demuestra la efectividad del método para aislar y purificar microorganismos productores de péptidos cargados y que poseen actividad biológica e industrial de gran interés.

Like proteins, antimicrobial peptides (AMP) are versatile molecules synthesized by microorganisms using enzymatic pathways with no genetic code instruction. AMP have interesting properties in the food and pharmaceutical industries, like their antimicrobial ability against pathogens. Looking for biomolecules from microorganisms requires hard and time consuming chemical analysis of each microorganism extract. The microorganism isolation method proposed in this research allowed us to find antimicrobial peptides produced by bacteria, through interaction between a charged dye mixed with selective agar and metabolites produced by microorganisms. Twenty soil samples from different zones were isolated in selective media; thirty five strains were purified based on interaction between basic dye and charged molecules from bacteria. Streptomyces sp. y Bacillus sp. both genera were identified. Protein extracts were obtained from the isolated microorganisms cultivated in liquid media; peptides and amino acids were identified by thin layer chromatography and electrophoresis. Those extracts with high protein level were used to evaluate bioautography. Two extracts from 35 showed inhibitory activity against E. coli ATCC 8739 (8 mm halo). Method effectiveness for the isolation and the purifying of microorganisms able to produce charged molecules, of industrial interest is demonstrated.

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