Comparative bioinformatic analysis and biochemical characterization of digestive proteinases from Pacific whiting (Merluccius productus).

The hake fishery plays a crucial role due to its significant economic impact. The genus Merluccius includes 12 extant species found along the coasts of the Americas, Europe, and Africa. However, research on their digestive physiology and the enzymes involved in digestion, including proteases, remains limited. Proteases play a key role in protein digestion, a vital process for all living organisms. This study focused on screening the genomes of eight Merluccius spp. for eight specific proteases previously identified in Merluccius polli. Additionally, the study conducted biochemical analyses of proteases found in the stomach and intestine of Pacific whiting fish (Merluccius productus), comparing the results with the genomic findings. The analysis revealed that proteases across Merluccius spp. are conserved, although with slight variations, particularly in chymotrypsin and aspartic proteases. Biochemical characterization of M. productus identified at least three main proteases in the stomach, active at acidic pH, and at least seven proteases in the intestine, active at alkaline pH, as determined by electrophoresis. Further investigation, including specific inhibition studies, determination of molecular mass, and assessment of pH and temperature preferences for catalysis, revealed that one of the stomach proteases functioning at acidic pH likely belongs to the acid peptidase class, likely pepsin. Similarly, analysis of proteases active at alkaline pH indicated the presence of a chymotrypsin and a trypsin, consistent with genomic findings in M. productus. These results are important as they provide insights into the digestive physiology of Merluccius spp., contributing to a better understanding of their nutritional needs.

Evaluation of Cytotoxicity and Acute Oral Toxicity of Saline Extract and Protein-Rich Fraction from Moringa oleifera Lam. Leaves.

Moringa oleifera Lam. (horseradish tree) leaves demonstrate high nutritional value, are rich in proteins, and are widely used in folk medicine and food. This study investigated the presence of secondary metabolites and antinutritional proteins in leaf extract (LE) and the protein-rich fraction (PRF) derived from M. oleifera leaves, as well as the cytotoxicity to human cells, hemolytic activity, and in vivo acute toxicity and genotoxicity in mice. The flavonoids rutin and vitexin as well as trypsin inhibitors and lectins were detected in LE and PRF. Neither sample demonstrated toxicity against human peripheral blood mononuclear cells and both showed low hemolytic action. In vivo, LE and PRF did not show antinutritional effects and caused no death. The hematological parameters of the animals in the treated group were similar to those of the control. A significant increase in the serum levels of alanine aminotransferase and a discrete leukocyte infiltration with cytoplasmic vacuolization of the hepatocytes in the liver were detected in LE-treated animals. The preparations were not genotoxic or mutagenic. This study shows that LE and PRF are not antinutritional agents and presented low acute toxicity and no genotoxicity or mutagenicity. The present study contributes to the determination of the safety of using M. oleifera leaf proteins.

The monodomain Kunitz protein EgKU-7 from the dog tapeworm Echinococcus granulosus is a high-affinity trypsin inhibitor with two interaction sites.

Typical Kunitz proteins (I2 family of the MEROPS database, Kunitz-A family) are metazoan competitive inhibitors of serine peptidases that form tight complexes of 1:1 stoichiometry, mimicking substrates. The cestode Echinococcus granulosus, the dog tapeworm causing cystic echinococcosis in humans and livestock, encodes an expanded family of monodomain Kunitz proteins, some of which are secreted to the dog host interface. The Kunitz protein EgKU-7 contains, in addition to the Kunitz domain with the anti-peptidase loop comprising a critical arginine, a C-terminal extension of ∼20 amino acids. Kinetic, electrophoretic, and mass spectrometry studies using EgKU-7, a C-terminally truncated variant, and a mutant in which the critical arginine was substituted by alanine, show that EgKU-7 is a tight inhibitor of bovine and canine trypsins with the unusual property of possessing two instead of one site of interaction with the peptidases. One site resides in the anti-peptidase loop and is partially hydrolyzed by bovine but not canine trypsins, suggesting specificity for the target enzymes. The other site is located in the C-terminal extension. This extension can be hydrolyzed in a particular arginine by cationic bovine and canine trypsins but not by anionic canine trypsin. This is the first time to our knowledge that a monodomain Kunitz-A protein is reported to have two interaction sites with its target. Considering that putative orthologs of EgKU-7 are present in other cestodes, our finding unveils a novel piece in the repertoire of peptidase-inhibitor interactions and adds new notes to the evolutionary host-parasite concerto.

Purification and partial physical-chemical characterization of a new bovine trypsin proteoform (zeta-trypsin).

Recent advancements in enzyme research have unveiled a new proteoform of bovine trypsin, expanding our understanding of this well-characterized enzyme. While generally similar to other trypsins, this novel proteoform comprises three polypeptide chains, marking a significant difference in activity, kinetic properties, and conformational stability. Compared with the already known bovine trypsin proteoforms, the results showed a lower: activity, kcat and kcat.KM-1 and protein 'foldedness' ratio for the new proteoform. Molecular autolysis, a common feature in trypsin and chymotrypsin, has been explored through comparative physical chemistry properties with other proteoforms. This new proteoform of trypsin not only enriches the existing enzyme repertoire but also promises to shed light on the intricate physiological pathway for enzyme inactivation. Our results suggest that the new trypsin proteoform is one of the likely final pathways for enzyme inactivation in a physiological environment. This discovery opens up new avenues for further research into the functional implications of this new trypsin proteoform.

A Novel Trypsin Kunitz-Type Inhibitor from Cajanus cajan Leaves and Its Inhibitory Activity on New Cancer Serine Proteases and Its Effect on Tumor Cell Growth.

A novel trypsin inhibitor from Cajanus cajan (TIC) fresh leaves was partially purified by affinity chromatography. SDS-PAGE revealed one band with about 15 kDa with expressive trypsin inhibitor activity by zymography. TIC showed high affinity for trypsin (Ki = 1.617 µM) and was a competitive inhibitor for this serine protease. TIC activity was maintained after 24 h of treatment at 70 °C, after 1 h treatments with different pH values, and ß-mercaptoethanol increasing concentrations, and demonstrated expressive structural stability. However, the activity of TIC was affected in the presence of oxidizing agents. In order to study the effect of TIC on secreted serine proteases, as well as on the cell culture growth curve, SK-MEL-28 metastatic human melanoma cell line and CaCo-2 colon adenocarcinoma was grown in supplemented DMEM, and the extracellular fractions were submitted salting out and affinity chromatography to obtain new secreted serine proteases. TIC inhibited almost completely, 96 to 89%, the activity of these serine proteases and reduced the melanoma and colon adenocarcinoma cells growth of 48 and 77% respectively. Besides, it is the first time that a trypsin inhibitor was isolated and characterized from C. cajan leaves and cancer serine proteases were isolated and partial characterized from SK-MEL-28 and CaCo-2 cancer cell lines. Furthermore, TIC shown to be potent inhibitor of tumor protease affecting cell growth, and can be one potential drug candidate to be employed in chemotherapy of melanoma and colon adenocarcinoma.

Íleo meconial y falsos negativos del resultado de tripsina inmunorreactiva en una niña portadora de fibrosis quística / Meconium Ileus and false negative immunoreactive trypsine results in a cystic fibrosis carrier infant / Íleo meconial e resultados falso-negativos de tripsina imunorreativa em uma menina com fibrose cística

Introducción: la fibrosis quística (FQ) es una enfermedad genética, multisistémica, caracterizada por la disfunción de las glándulas de secreción exocrina secundaria a la mutación de una proteína transmembrana que actúa como canal de cloro. La inclusión de su búsqueda en el Sistema Nacional de Pesquisa Neonatal (SNPN) determinó un gran impacto sobre la morbimortalidad de esta enfermedad, permitiendo un diagnóstico y tratamiento precoz. El íleo meconial (IM) es la manifestación más precoz de la enfermedad, ocurriendo en un 10% a 18% de los pacientes. Objetivo: describir una forma de presentación precoz de FQ con resultado de pesquisa neonatal normal. Caso clínico: 18 meses. Sexo femenino; 33 semanas de edad gestacional. Retraso en la expulsión del meconio. Al tercer día de vida íleo meconial con oclusión intestinal, vólvulo de intestino medio y necrosis intestinal. Se realiza resección quirúrgica. Tripsina inmunorreactiva (TIR) normal a los 7 y 23 días de vida. No se solicita proteína asociada a la pancreatitis (PAP). Desnutrición crónica, bronquiolitis grave a los 3 meses, neumonías virales a los 7 y 11 meses. Ingreso con diagnóstico de neumonía aguda comunitaria con insuficiencia respiratoria. Test del sudor alterado en dos oportunidades. Elastasa pancreática insuficiente. Se aísla Staphylococcus aureus y Pseudomonas aeruginosa en exudado nasal y de secreciones respiratorias. Estudio genético: mutación Delta F508. Con diagnóstico de FQ se inició abordaje multidisciplinario, tratamiento y derivación al centro especializado. Conclusiones: la existencia de IM puede dar falsos negativos en el valor de la TIR, siendo necesaria la dosificación de la PAP. El pronóstico de estos pacientes va a depender de un diagnóstico precoz y el manejo terapéutico oportuno en centros especializados.

Introduction: cystic fibrosis (CF) is a genetic, multisystemic disease, characterized by dysfunction of the exocrine secretion glands secondary to the mutation of a transmembrane protein that acts as a chloride channel. Including its research by the National Neonatal Screening System (SNPN) caused a great impact on the morbidity and mortality of this disease, enabling early diagnosis and treatment. Meconium ileus (MI) is the earliest manifestation of the disease, occurring in 10% to 18% of patients. Objective: to describe a form of early presentation of CF with normal neonatal screening results. Clinical case: 18 months. Female, 33 weeks gestational age. Delay in the expulsion of meconium. On the third day of life, meconium ileus with intestinal obstruction, midgut volvulus and intestinal necrosis. Surgical resection is performed. Trypsin immunoreactive (TIR) normal at 7 and 23 days of age. Pancreatitis-associated protein (PAP) is not requested. Chronic malnutrition, severe bronchiolitis at 3 months, viral pneumonia at 7 and 11 months. Admission with a diagnosis of acute community pneumonia with respiratory failure. Sweat test altered on two occasions. Insufficient pancreatic elastase. Staphylococcus aureus and Pseudomonas aeruginosa were isolated in nasal exudate and respiratory secretions. Genetic study: Delta F508 mutation. With the CF diagnosis, a multidisciplinary approach, treatment and referral to a specialized center began. Conclusions: the existence of MI can provide false negatives in the IRR value, making PAP dosage necessary. The prognosis of these patients will depend on early diagnosis and timely therapeutic management at specialized centers.

Introdução: a fibrose cística (FC) é uma doença genética, multissistêmica, caracterizada pela disfunção das glândulas secretoras exócrinas secundária à mutação de uma proteína transmembrana que age como canal de cloreto. A inclusão da sua pesquisa no Sistema Nacional de Triagem Neonatal (SNPN) determinou grande impacto na morbimortalidade desta doença, permitindo diagnóstico e tratamento precoces. O íleo meconial (IM) é a manifestação mais precoce da doença, ocorrendo em 10 a 18% dos pacientes. Objetivo: descrever uma forma de apresentação precoce da FC com resultados normais de triagem neonatal. Caso clínico: 18 meses. Sexo feminino. 33 semanas de idade gestacional. Atraso na expulsão do mecônio. No terceiro dia de vida, íleo meconial com obstrução intestinal, vólvulo de intestino médio e necrose intestinal. A ressecção cirúrgica é realizada. Imunorreativo à tripsina (TIR) normal aos 7 e 23 dias de vida. A proteína associada à pancreatite (PAP) não é solicitada. Desnutrição crônica, bronquiolite grave aos 3 meses, pneumonia viral aos 7 e11 meses. Internação com diagnóstico de pneumonia comunitária aguda com insuficiência respiratória. Teste do suor alterado em duas ocasiões. Elastase pancreática insuficiente. Staphylococcus aureus e Pseudomonas aeruginosa foram isolados em exsudato nasal e secreções respiratórias. Estudo genético: mutação Delta F508. Com o diagnóstico de FC iniciou-se abordagem multidisciplinar, tratamento e encaminhamento para centro especializado. Conclusões: a existência de IM pode dar falsos negativos no valor da TIR, tornando necessária a dosagem de PAP. O prognóstico destes pacientes dependerá do diagnóstico precoce e do manejo terapêutico oportuno em centros especializados.

A soybean trypsin inhibitor reduces the resistance to transgenic maize in a population of Spodoptera frugiperda (Lepidoptera: Noctuidae).

Lepidopteran pests have been successfully managed by the adoption of insect resistant transgenic plants expressing Cry and/or Vip insecticidal proteins derived from Bacillus thuringiensis (Bt plants). Among such pests, Spodoptera frugiperda (Smith, 1797) (Lepidoptera: Noctuidae) is highlighted for its destructive potential in maize crops and for cases of field-evolved resistance to Bt plants. Cry insecticidal proteins expressed in Bt plants are known for their interaction with insect midgut receptors and subsequent midgut cell disruption that leads to target pest death. In the midgut of lepidopteran larval pests such as S. frugiperda, serine proteases are important in dietary protein digestion and activation or degradation of insecticidal proteins. This work was conducted to evaluate if the use of a soybean trypsin inhibitor (SBTI) could disrupt the development of a Bt-susceptible and a Bt-resistant population of S. frugiperda ingesting Bt (expressing Cry1F, Cry1A.105, and Cry2Ab2 Cry proteins) and non-Bt maize plants. The SBTI was produced and purified using recombinant expression in E. coli followed by purification in Ni-Sepharose. Bioassays using non-Bt maize leaves indicated that the development of susceptible and resistant populations of S. frugiperda was not influenced by the ingestion of SBTI. However, when the resistant population consumed Bt maize plants amended with SBTI, high mortality along with a reduction in larval weight and reduced activity of digestive trypsins were observed. Although the mode of action was not elucidated, it is possible that the consumption of SBTI increased susceptibility to Bt maize in the resistant population of S. frugiperda.

A Multifunctional Trypsin Protease Inhibitor from Yellow Bell Pepper Seeds: Uncovering Its Dual Antifungal and Hypoglycemic Properties.

Fungal infections are a growing public health concern worldwide and the emergence of antifungal resistance has limited the number of therapeutic options. Therefore, developing novel strategies for identifying and developing new antifungal compounds is an active area of research in the pharmaceutical industry. In this study, we purified and characterized a trypsin protease inhibitor obtained from Yellow Bell Pepper (Capsicum annuum L.) seeds. The inhibitor not only showed potent and specific activity against the pathogenic fungus Candida albicans, but was also found to be non-toxic against human cells. Furthermore, this inhibitor is unique in that it also inhibits α-1,4-glucosidase, positioning it as one of the first plant-derived protease inhibitors with dual biological activity. This exciting discovery opens new avenues for the development of this inhibitor as a promising antifungal agent and highlights the potential of plant-derived protease inhibitors as a rich source for the discovery of novel multifunctional bioactive molecules.

Molecular Docking and In Vitro Studies of Ochratoxin A (OTA) Biodetoxification Testing Three Endopeptidases.

Ochratoxin A (OTA) is considered one of the main mycotoxins responsible for health problems and considerable economic losses in the feed industry. The aim was to study OTA's detoxifying potential of commercial protease enzymes: (i) Ananas comosus bromelain cysteine-protease, (ii) bovine trypsin serine-protease and (iii) Bacillus subtilis neutral metalloendopeptidase. In silico studies were performed with reference ligands and T-2 toxin as control, and in vitro experiments. In silico study results showed that tested toxins interacted near the catalytic triad, similar to how the reference ligands behave in all tested proteases. Likewise, based on the proximity of the amino acids in the most stable poses, the chemical reaction mechanisms for the transformation of OTA were proposed. In vitro experiments showed that while bromelain reduced OTA's concentration in 7.64% at pH 4.6; trypsin at 10.69% and the neutral metalloendopeptidase in 8.2%, 14.44%, 45.26% at pH 4.6, 5 and 7, respectively (p < 0.05). The less harmful α-ochratoxin was confirmed with trypsin and the metalloendopeptidase. This study is the first attempt to demonstrate that: (i) bromelain and trypsin can hydrolyse OTA in acidic pH conditions with low efficiency and (ii) the metalloendopeptidase was an effective OTA bio-detoxifier. This study confirmed α-ochratoxin as a final product of the enzymatic reactions in real-time practical information on OTA degradation rate, since in vitro experiments simulated the time that food spends in poultry intestines, as well as their natural pH and temperature conditions.

An innovative insecticidal approach based on plant protease inhibitor and Bt protoxins inhibits trypsin-like activity in zebrafish.

The Leucaena leucocephala trypsin inhibitor (LTI) + Bacillus thuringiensis (Bt) protoxins mix has been proposed as a novel larvicide agent in order to control the vector mosquito of dengue virus, Aedes aegypti, in their aquatic breeding sites. However, use of this insecticide formulation has raised concerns about its impacts on aquatic biota. In this context, this work aimed to assess the effects of LTI and Bt protoxins, separately or in combination, in zebrafish, in regard to the evaluation of toxicity at early life stages and to the presence of LTI inhibitory effects on intestinal proteases of this fish. Results showed that LTI and Bt concentrations (250 mg/L, and 0.13 mg/L, respectively), and LTI + Bt mix (250 mg/L + 0.13 mg/L) - 10 times superior to those with insecticidal action - did not cause death nor did it induce morphological changes during embryonic and larval development (3 to 144 h post-fertilization) of zebrafish. Molecular docking analyses highlighted a possible interaction between LTI and zebrafish trypsin, especially through hydrophobic interactions. In concentrations near to those with larvicidal action, LTI (0.1 mg/mL) was able to inhibit in vitro intestinal extracts of trypsin in female and male fish by 83 % and 85 %, respectively, while LTI + Bt mix promoted trypsin inhibition of 69 % in female and 65 % in male ones. These data show that the larvicidal mix can potentially promote deleterious effects to nutrition and survival in non-target aquatic organisms, especially those with trypsin-like dependent protein digestion.

Clinical implementation of comprehending dentin on a microscopic level

SUMMARY: The aim of the present in vitro study is to visualize dentin to get an in-depth knowledge of the nature of dentin that could provide useful information regarding conditioning dentinal substrate when treating dentinal lesions. Forty-nine extracted human third molars were obtained and prepared to produce artificial dentinal lesions through demineralizing with acetic acid for 7 and 14 days, or lactic acid for 7 days. The teeth were divided into groups and treated with either NaOCl, pepsin, trypsin, or phosphoric acid. To obtain information on the morphology of the treated dentinal surfaces, all samples were visualized under high resolution field emission scanning electron microscope. With high magnification reaching x50000 dentin was clearly visualized together with its constitutes. The effect of various demineralization approaches and various treatment protocols were demonstrated clearly. The relationship between the conditioning procedure steps and the subsequent bond strength was discussed. To our best knowledge, there is no previous clear highly magnified scanning electron microscope images for dentin, and dentinal components and constitutes with and without various treatments. The current in vitro study suggests the complexity nature of dentin as a substrate that should be treated carefully especially with technique sensitive procedures such as adhesive restorations.

El objetivo del presente estudio in vitro fue visualizar la dentina para obtener un conocimiento completo de la naturaleza de ella lo que podría proporcionar información útil sobre el acondicionamiento del sustrato dentinario en el tratamiento de lesiones dentinarias. Se obtuvieron 49 terceros molares humanos extraídos y se prepararon para producir lesiones dentinales artificiales mediante desmineralización con ácido acético por 7 y 14 días, o ácido láctico por 7 días. Los dientes se dividieron en grupos y se trataron con NaOCl, pepsina, tripsina o ácido fosfórico. Para obtener información sobre la morfología de las superficies dentinarias tratadas, todas las muestras se visualizaron bajo un microscopio electrónico de barrido de emisión de campo de alta resolución. Con un gran aumento que alcanzó x50000, la dentina se visualizó claramente junto con sus componentes. Se demostró el efecto de varios enfoques de desmineralización y varios protocolos de tratamiento. Se discutió la relación entre los pasos del procedimiento de acondicionamiento y la subsiguiente fuerza de unión. Hasta donde sabemos, no hay imágenes claras previas de microscopio electrónico de barrido altamente ampliadas para la dentina y los componentes y constituyentes de la dentina con y sin diferentes tratamientos. El estudio in vitro actual sugiere la naturaleza compleja de la dentina como sustrato que debe tratarse con cuidado, especialmente en los procedimientos sensibles a la técnica, tal como las restauraciones adhesivas.

Nanoparticles Containing Tamarind Isolate Protein Potentiate the Satiety without Promoting the Anti-Inflammatory Effect in a Preclinical Model of Diet-Induced Obesity.

The study aimed to evaluate the nanoparticles (ECW) containing tamarind trypsin inhibitor (TTI) concerning the storage effect under different conditions on antitrypsin activity and the bioactive potential in a preclinical model. ECW was exposed to different pH and temperatures to evaluate the interaction between TTI and its encapsulating agents, monitored by antitrypsin activity. Wistar rats (n = 25) with obesity induced by diet were divided into groups: untreated; treatment with nutritionally adequate diet; treatment with nutritionally adequate diet and ECW/12.5 mg/kg; treatment with ECW/12.5 mg/kg; and treatment with TTI/25 mg/kg. The groups were evaluated over ten days with regards to satiety, zoometric, biochemical, and inflammatory parameters, using ten times less TTI (2.5 mg/kg) contained in ECW. TTI was protected and encapsulated in ECW without showing residual inhibitory activity. Only at gastric pH did ECW show antitrypsin activity. At different temperatures, it showed high antitrypsin activity, similar to TTI. The animals treated with ECW had significantly reduced body weight variation (p < 0.05), and only TTI treatment reduced the inflammatory parameters significantly (p < 0.05). The study showed that by using lower concentrations of TTI in ECW it was possible to perceive promising effects with perspectives of use in functional products for managing obesity and its complications.

Physicochemical, Functional, and In Vitro Digestibility of Protein Isolates from Thai and Peru Sacha Inchi (Plukenetia volubilis L.) Oil Press-Cakes.

Proteins from Sacha inchi (SI) have been widely known for their health-benefiting properties. This study aimed to investigate the different protein isolates obtained from oil press-cakes of Thai and Peru SI. The protein content and protein recovery of Thai and Peru SI were estimated to be 93.27, 90.67%, and 49.15, 59.32%, respectively. The protein patterns of the Thai and Peru SI samples analyzed by SDS-PAGE showed glycoprotein as a major protein, with a molecular weight of 35 kDa. Both protein isolates (PI) showed water and oil holding capacities in the range of 2.97−3.09 g/g sample and 2.75−2.88 g/g sample, respectively. The emulsifying properties of the PI from Thai SI were higher than those of Peru (p < 0.05), while the foaming properties were not analogous to the emulsion properties. The Thai SI sample showed lower digestibility up to 120 min of in vitro digestion time than that of the Peru SI sample (p < 0.05). However, simulated in vitro pepsin digestion of Thai and Peru Si samples displayed hydrolyzed protein bands compared to trypsin digestion, which showed no protein patterns in both SI samples on a 4−20% gradient gel. These results suggest that the protein isolates from Thai and Peru SI exhibit marked variations in physical and techno-functional properties and have a high potential to be employed as plant-based protein additives for future non-animal-based protein-rich foods.

An Insulin Receptor-Binding Multifunctional Protein from Tamarindus indica L. Presents a Hypoglycemic Effect in a Diet-Induced Type 2 Diabetes-Preclinical Study.

The objectives of this study were to evaluate the hypoglycemic effect of the trypsin inhibitor isolated from tamarind seeds (TTI) in an experimental model of T2DM and the in silico interaction between the conformational models of TTI 56/287 and the insulin receptor (IR). After inducing T2DM, 15 male Wistar rats were randomly allocated in three groups (n = 5): 1-T2DM group without treatment; 2-T2DM group treated with adequate diet; and 3-T2DM treated with TTI (25 mg/kg), for 10 days. Insulinemia and fasting glucose were analyzed, and the HOMA-IR and HOMA-ß were calculated. The group of animals treated with TTI presented both lower fasting glucose concentrations (p = 0.0031) and lower HOMA-IR indexes (p = 0.0432), along with higher HOMA-ß indexes (p = 0.0052), than the animals in the other groups. The in silico analyses showed that there was an interaction between TTIp 56/287 and IR with interaction potential energy (IPE) of -1591.54 kJ mol-1 (±234.90), being lower than that presented by insulin and IR: -894.98 kJ mol-1 (±32.16). In addition, the presence of amino acids, type of binding and place of interaction other than insulin were identified. This study revealed the hypoglycemic effect of a bioactive molecule of protein origin from Tamarind seeds in a preclinical model of T2DM. Furthermore, the in silico analysis allowed the prediction of its binding in the IR, raising a new perspective for explaining TTI's action on the glycemic response.

Thermostable trypsin-like protease by Penicillium roqueforti secreted in cocoa shell fermentation: Production optimization, characterization, and application in milk clotting.

The increased demand for cheese and the limited availability of calf rennet justifies the search for milk-clotting enzymes from alternative sources. Trypsin-like protease by Penicillium roqueforti was produced by solid-state fermentation using cocoa shell waste as substrate. The production of a crude enzyme extract that is rich in this enzyme was optimized using a Doehlert-type multivariate experimental design. The biochemical characterization showed that the enzyme has excellent activity and stability at alkaline pH (10-12) and an optimum temperature of 80°C, being stable at temperatures above 60°C. Enzymatic activity was maximized in the presence of Na+ (192%), Co2+ (187%), methanol (153%), ethanol (141%), and hexane (128%). Considering the biochemical characteristics obtained and the milk coagulation activity, trypsin-like protease can be applied in the food industry, such as in milk clotting and in the fabrication of cheeses.

The enzymatic disaggregation by trypsin does not alter cell quality and genomic stability of adipose-derived stem cells cultivated for human cell therapy.

There is no consensus between the protocols used for the isolation, maintenance and cultivation of Adipose-derived stem cells (ADSCs) for therapeutic purposes. Thus, was evaluated the maintenance method of ADSCs submitted to enzymatic disaggregation by trypsin. Was made (1st until 10th passage) immunophenotyping, cell differentiation assays, comet assay, differential cell death, apoptosis, cell viability and membrane integrity by flow cytometry.The results showded that trypsinization,did not induce genomic instability, also did not alter the tail moment. The cell death assay, showed that only on the 10th passage there was a significant reduction and was cofirmed by flow cytometry that is apoptosis. The viability showded significant reduction only in 10th passage, this was related to the loss of integrity of membrane, proven by flow cytometry. The quantities varied along the passages (11 × 105 to 2 × 105). Qualitatively, it can be observed that as the number of cells decreases, there is also a reduction in the juxtaposition of ADSCs and increased of the cell size, it is started in 6th passage. In view of the results, it is suggested for more safety, that ADSCs cultured until the 5th passage being used in human transplantation procedures.

Prospection of putative genes for digestive enzymes based on functional genome of the hepatopancreas of Amazon river prawn

Over recent years, Macrobrachium amazonicum has become a popular species for shrimp farming due to their fast growth, high survival rates, and marketability. Several studies have focused on the development of new technology for the culture of this species, but many aspects of their nutrition and physiology remain unknown. Thus, the goal of the present study was to obtain transcripts of putative genes encoding digestive enzymes, based on a library of the cDNA from the hepatopancreas of M. amazonicum, sequenced in the Ion TorrentTM platform. We identified fragments of nine genes related to digestive enzymes, acting over proteins, carbohydrates and lipids. Endo and exoproteases were also recorded in the hepatopancreas, indicating adaptation to the digestion of protein-richfoods. Nonetheless, the enzymes involved in the carbohydrate metabolism formed the largest functional group in M. amazonicum, including enzymes related to the digestion of starch, chitin, and cellulose. These findings indicate that the species has a genetic apparatus of a well-adapted omnivorous animal. This information may provide important insights for the selection of ingredients for the formulation of a more appropriate diet to the enzymatic repertoire of M. amazonicum.(AU)

Avaliação preditiva estrutural das serino peptidases de aranhas: as relações evolutivas e de estrutura/função

Serine peptidases (SP) are hydrolases represented in many living organisms, with chymotrypsins and trypsins being the main digestive SPs. Studies of genomes, transcriptomes and proteomes of spiders allow identifying these enzymes in different tissues and/or secretions of these animals and suggest that SPs are involved in poisoning and digestion, two very important processes for the evolutionary success of the group. In order to compare the SPs involved in these processes, in silico analyses of the databases of a total of 38 distinct species of spiders were performed, totaling 1,200 sequences of SPs. Proteomic analyses of Nephilingis cruentata, Stegodyphus mimosarum and Acanthoscurria geniculata show the presence of SPs in poisons (SPV) and digestive process (SPD). In general, the SPVs present a molecular mass in the range between 30 and 37 kDa and pI in the range of 4.65 and 9.79, presenting, in most sequences, only the catalytic domain. Fluid and digestive system SPDs mandatorily present the catalytic domain associated with CUB-LDLa, indicating that CUB-LDL SPcatalytic combination is a signature of the digestive SPs of spiders. These enzymes have a molecular mass of 36 to 140 kDa and pI between 4.4 and 5.6. Other differences found among SPV and SPD are: predicted patterns of glycosilation, cell addressing and structural prediction. Among the different digestive SPs there is a distinction between hydrophobicity, electrostatic potential, and substrate accessibility. Molecular docking also allowed predicting the interaction behavior of SPs with inhibitors that mimic substrates, as Kunitz type inhibitors. Analyses indicate that poison enzymes have greater accessibility of the catalytic triad to the substrate. Phylogenetic analyses show that the different groups of digestive SPs are represented in all studied spider species, keeping CUB-LDLa as a signature. The conservation of the CUB-LDLa domain was also observed in SPs of other Groups of Arachnida such as Scorpionidae, Acari and Opiliones, being even present in Limulus representative of the Chelicerata group. The association of these domains is possibly related to different functionalities of SPs in arachnids maintained throughout the evolutionary process.

As serino peptidases (SP) são hidrolases representadas em muitos organismos vivos, sendo as quimotripsinas e tripsinas as principais SPs digestivas. Estudos de genomas, transcriptomas e proteomas de aranhas permitem identificar essas enzimas em diferentes processos fisiológicos desses animais e sugerem que as SPs estão envolvidas no envenenamento e no processo digestivo, dois processos muito importantes para o sucesso evolutivo do grupo. Com o objetivo de comparar as SPs envolvidas nestes processos foram realizadas análises in silico dos bancos de dados de um total de 38 espécies distintas de aranhas, totalizando 1.200 sequências de SPs. Análises proteômicas de Nephilingis cruentata, Stegodyphus mimosarum e Acanthoscurria geniculata mostram a presença de SPs nos venenos (SPV) e no processo digestivo (SPD). Em geral, as SPVs apresentam uma massa molecular na faixa entre 30 e 37 kDa e pI na faixa de 4,65 e 9,79, apresentando, na maioria das sequências, apenas o domínio catalítico. As SPDs de fluido e sistema digestório apresentam obrigatoriamente o domínio catalítico associado a CUB-LDLa, indicando que a combinação CUB-LDLa-SP catalítico é uma assinatura das SPs digestivas de aranhas. Essas enzimas apresentam uma massa molecular de 36 a 140 kDa e o pI entre 4,4 e 5,6. Outras diferenças encontradas são: os padrões preditos de glicosilação, endereçamento celular e predição estrutural. Entre as diferentes SPs digestivas há distinção entre a hidrofobicidade, o potencial eletrostático e a acessibilidade ao substrato. Docking molecular permitiu ainda prever o comportamento de interação das SPs com inibidores que mimetizam substratos como inibidores do tipo Kunitz. As análises indicam que as enzimas de veneno têm maior acessibilidade da tríade catalítica ao substrato. Análises filogenéticas mostram que os diferentes grupos de SPs digestivas estão representados em todos as espécies de aranhas estudadas mantendo CUB-LDLa como assinatura. A conservação do domínio CUB-LDLa também foi observada em SPs de outros grupos de Arachnida como Scorpionidae, Acari e Opiliones, estando inclusive presente em Limulus polyphemus representante do grupo Chelicerata. A associação destes domínios possivelmente está relacionada a diferentes funcionalidades de SPs em aracnídeos mantidas ao longo do processo evolutivo.

Trypsin Genes Are Regulated through the miRNA Bantam and Associated with Drug Sensitivity in the Sea Louse Caligus rogercresseyi.

The role of trypsin genes in pharmacological sensitivity has been described in numerous arthropod species, including the sea louse Caligus rogercresseyi. This ectoparasite species is mainly controlled by xenobiotic drugs in Atlantic salmon farming. However, the post-transcriptional regulation of trypsin genes and the molecular components involved in drug response remain unclear. In particular, the miRNA bantam family has previously been associated with drug response in arthropods and is also found in C. rogercresseyi, showing a high diversity of isomiRs. This study aimed to uncover molecular interactions among trypsin genes and bantam miRNAs in the sea louse C. rogercresseyi in response to delousing drugs. Herein, putative mRNA/miRNA sequences were identified and localized in the C. rogercresseyi genome through genome mapping and blast analyses. Expression analyses were obtained from the mRNA transcriptome and small-RNA libraries from groups with differential sensitivity to three drugs used as anti-sea lice agents: azamethiphos, deltamethrin, and cypermethrin. The validation was conducted by qPCR analyses and luciferase assay of selected bantam and trypsin genes identified from in silico transcript prediction. A total of 60 trypsin genes were identified in the C. rogercresseyi genome, and 39 bantam miRNAs were differentially expressed in response to drug exposure. Notably, expression analyses and correlation among values obtained from trypsin and bantam revealed an opposite trend and potential binding sites with significant ΔG values. The luciferase assay showed a reduction of around 50% in the expression levels of the trypsin 2-like gene, which could imply that this gene is a potential target for bantam. The role of trypsin genes and bantam miRNAs in the pharmacological sensitivity of sea lice and the use of miRNAs as potential markers in these parasites are discussed in this study.

Multiple conformations in solution of the maize C4-phosphoenolpyruvate carboxylase isozyme.

The photosynthetic phosphoenolpyruvate carboxylase isozyme from C4 plants (PEPC-C4) has a complex allosteric regulation, involving positive cooperativity in binding the substrate phosphoenolpyruvate as well as positive and negative allosteric effectors. Besides the proposed R- and T-states, previous kinetic results suggested functionally relevant different R-states of the maize enzyme (ZmPEPC-C4) elicited by PEP or its two kinds of activators, glucose 6-phosphate or glycine. To detect these different R-state conformations, we used as conformational probes the fluorescence of 8-anilino-1-naphthalene sulfonate (ANS), near-UV circular dichroism (CD) spectroscopy, and limited proteolysis by trypsin. Phosphoenolpyruvate and malate binding caused distinct concentration-dependent fluorescence changes of ZmPEPC-C4/ANS, suggesting that they elicited conformational states different from that of the free enzyme, while glucose 6-phosphate or glycine binding did not produce fluorescence changes. Differences were also observed in the near UV CD spectra of the enzyme, free or complexed with its substrate or allosteric effectors. Additionally, differences in the trypsin-digestion fragmentation patterns, as well as in the susceptibility of the free and complexed enzyme to digestion and digestion-provoked loss of activity, provided evidence of several ZmPEPC-C4 conformations in solution elicited by the substrate and the allosteric effectors. Using the already reported ZmPEPC-C4 crystal structures and bioinformatics methods, we predicted that the most probable trypsin-cleavage sites are located in superficial flexible regions, which seems relevant for the protein dynamics underlying the function and allosteric regulation of this enzyme. Together, our findings agree with previous kinetic results, shed light on this enzyme's complex allosteric regulation, and place ZmPEPC-C4 in the growing list of allosteric enzymes possessing an ensemble of closely related R-state conformations.