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1.
Sci Rep ; 14(1): 15704, 2024 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-38977706

RESUMO

Halophiles are one of the classes of extremophilic microorganisms that can flourish in environments with very high salt concentrations. In this study, fifteen bacterial strains isolated from various crop rhizospheric soils of agricultural fields along the Southwest coastline of Saurashtra, Gujarat, and identified by 16S rRNA gene sequencing as Halomonas pacifica, H. stenophila, H. salifodinae, H. binhaiensis, Oceanobacillus oncorhynchi, and Bacillus paralicheniformis were investigated for their potentiality to produce extremozymes and compatible solute. The isolates showed the production of halophilic protease, cellulase, and chitinase enzymes ranging from 6.90 to 35.38, 0.004-0.042, and 0.097-0.550 U ml-1, respectively. The production of ectoine-compatible solute ranged from 0.01 to 3.17 mg l-1. Furthermore, the investigation of the ectoine-compatible solute production at the molecular level by PCR showed the presence of the ectoine synthase gene responsible for its biosynthesis in the isolates. Besides, it also showed the presence of glycine betaine biosynthetic gene betaine aldehyde dehydrogenase in the isolates. The compatible solute production by these isolates may be linked to their ability to produce extremozymes under saline conditions, which could protect them from salt-induced denaturation, potentially enhancing their stability and activity. This correlation warrants further investigation.


Assuntos
RNA Ribossômico 16S , Rizosfera , Microbiologia do Solo , RNA Ribossômico 16S/genética , Diamino Aminoácidos/biossíntese , Diamino Aminoácidos/metabolismo , Índia , Produtos Agrícolas/microbiologia , Celulase/metabolismo , Celulase/genética , Celulase/biossíntese , Quitinases/metabolismo , Quitinases/genética , Tolerância ao Sal/genética , Filogenia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Peptídeo Hidrolases/metabolismo , Peptídeo Hidrolases/genética , Bactérias/genética , Bactérias/metabolismo , Bactérias/isolamento & purificação , Bactérias/classificação , Bacillus/genética , Bacillus/metabolismo , Bacillus/isolamento & purificação
2.
Mem Inst Oswaldo Cruz ; 119: e240038, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38985089

RESUMO

BACKGROUND: Leishmania (Viannia) braziliensis Thor strain exhibits a heterogeneous composition comprised of subpopulations with varying levels of infectivity. Clonal subpopulations were previously obtained from the strain Thor by sorting single-parasites and proceeding cultivation. The subpopulations used in this study are named Thor03, Thor 10 and Thor22. OBJECTIVES: Phenotypic characteristics of the parasite, specially focusing on virulence factors and resistance to the antimicrobial mechanisms of macrophages, were investigate in these subpopulations. METHODS: Cellular and molecular biology, as well as biochemistry approaches were applied to obtain the data analysed in this study. FINDINGS: Relative quantification of gene expression was measured for calpain, cysteine protease B (CPB), and subtilisin proteases but no significant differences in these genes' expression among subpopulations was observed. However, subtilisin and CPB proteins were assessed as more abundant in Thor03 by fluorescence-labelled flow cytometry technique. Western Blotting assays, as semi-quantitative analysis in gel, showed higher concentrations of subtilisin (110 to 50 kDa) and CPB (40 to 18 kDa) in extract of intracellular amastigotes from subpopulations Thor03 and Thor10 and calpain (60 to 25 kDa) showed no significant differences among subpopulations. Complementary, higher trypanothione reductase activity was observed in Thor10 intracellular amastigotes and assays of susceptibility to hydrogen peroxide-inducing agents and nitric oxide donors conducted with promastigotes revealed greater resistance to in vitro oxidative stress induction for Thor10, followed by Thor03. MAIN CONCLUSIONS: The data obtained for the virulence factors explored here suggest how multiple coexisting phenotypic-distinct subpopulations may contribute in adaptability of a single L. (V.) braziliensis strain during infection in the host cells.


Assuntos
Leishmania braziliensis , Leishmania braziliensis/enzimologia , Leishmania braziliensis/genética , Leishmania braziliensis/efeitos dos fármacos , Animais , Macrófagos/parasitologia , Western Blotting , Citometria de Fluxo , Fatores de Virulência , Peptídeo Hidrolases/metabolismo , Fenótipo , NADH NADPH Oxirredutases
3.
Appl Microbiol Biotechnol ; 108(1): 413, 2024 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-38985324

RESUMO

Environmental concerns arising from the increasing use of polluting plastics highlight polylactic acid (PLA) as a promising eco-friendly alternative. PLA is a biodegradable polyester that can be produced through the fermentation of renewable resources. Together with its excellent properties, suitable for a wide range of applications, the use of PLA has increased significantly over the years and is expected to further grow. However, insufficient degradability under natural conditions emphasizes the need for the exploration of biodegradation mechanisms, intending to develop more efficient techniques for waste disposal and recycling or upcycling. Biodegradation occurs through the secretion of depolymerizing enzymes, mainly proteases, lipases, cutinases, and esterases, by various microorganisms. This review focuses on the enzymatic degradation of PLA and presents different enzymes that were isolated and purified from natural PLA-degrading microorganisms, or recombinantly expressed. The review depicts the main characteristics of the enzymes, including recent advances and analytical methods used to evaluate enantiopurity and depolymerizing activity. While complete degradation of solid PLA particles is still difficult to achieve, future research and improvement of enzyme properties may provide an avenue for the development of advanced procedures for PLA degradation and upcycling, utilizing its building blocks for further applications as envisaged by circular economy principles. KEY POINTS: • Enzymes can be promisingly utilized for PLA upcycling. • Natural and recombinant PLA depolymerases and methods for activity evaluation are summarized. • Approaches to improve enzymatic degradation of PLA are discussed.


Assuntos
Biodegradação Ambiental , Poliésteres , Poliésteres/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Hidrolases de Éster Carboxílico/genética , Lipase/metabolismo , Esterases/metabolismo , Bactérias/enzimologia , Bactérias/metabolismo , Peptídeo Hidrolases/metabolismo
4.
Food Res Int ; 188: 114463, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38823831

RESUMO

To investigate the prevalence of Pseudomonas in the pasteurized milk production process and its effect on milk quality, 106 strains of Pseudomonas were isolated from the pasteurized milk production process of a milk production plant in Shaanxi Province, China. The protease, lipase and biofilm-producing capacities of the 106 Pseudomonas strains were evaluated, and the spoilage enzyme activities of their metabolites were assessed by simulating temperature incubation in the refrigerated (7 °C) and transport environment (25 °C) segments and thermal treatments of pasteurization (75 °C, 5 min) and ultra-high temperature sterilization (121 °C, 15 s). A phylogenetic tree was drawn based on 16S rDNA gene sequencing and the top 5 strains were selected as representative strains to identify their in situ spoilage potential by examining their growth potential and ability to hydrolyze proteins and lipids in milk using growth curves, pH, whiteness, Zeta-potential, lipid oxidation, SDS-PAGE and volatile flavor compounds. The results showed that half and more of the isolated Pseudomonas had spoilage enzyme production and biofilm capacity, and the spoilage enzyme activity of metabolites was affected by the culture temperature and sterilization method, but ultra-high temperature sterilization could not completely eliminate the enzyme activity. The growth of Pseudomonas lundensis and Pseudomonas qingdaonensis was less affected by temperature and time, and the hydrolytic capacity of extracellular protease and lipase secreted by Pseudomonas lurida was the strongest, which had the greatest effect on milk quality. Therefore, it is crucial to identify the key contamination links of Pseudomonas, the main bacteria responsible for milk spoilage, and the influence of environmental factors on its deterioration.


Assuntos
Biofilmes , Microbiologia de Alimentos , Lipase , Leite , Pasteurização , Pseudomonas , Pseudomonas/metabolismo , Pseudomonas/genética , Pseudomonas/isolamento & purificação , Pseudomonas/crescimento & desenvolvimento , Leite/microbiologia , Animais , Biofilmes/crescimento & desenvolvimento , Lipase/metabolismo , China , Filogenia , Peptídeo Hidrolases/metabolismo , RNA Ribossômico 16S/genética , Contaminação de Alimentos/análise , Temperatura
5.
Food Res Int ; 188: 114513, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38823886

RESUMO

This study reports the effect of thermal pretreatment and the use of different commercial proteolytic enzymes (Protamex, Flavourzyme, Protana prime, and Alcalase) on the free amino acid content (FAA), peptide profile, and antioxidant, antidiabetic, antihypertensive, and anti-inflammatory potential (DPPH, FRAP, and ABTS assay, DPP-IV, ACE-I, and NEP inhibitory activities) of dry-cured ham bone hydrolyzates. The effect of in vitro digestion was also determined. Thermal pretreatment significantly increased the degree of hydrolysis, the FAA, and the DPP-IV and ACE-I inhibitory activities. The type of peptidase used was the most significant factor influencing antioxidant activity and neprilysin inhibitory activity. Protana prime hydrolyzates failed to inhibit DPP-IV and neprilysin enzymes and had low values of ACE-I inhibitory activity. After in vitro digestion, bioactivities kept constant in most cases or even increased in ACE-I inhibitory activity. Therefore, hydrolyzates from dry-cured ham bones could serve as a potential source of functional food ingredients for health benefits.


Assuntos
Antioxidantes , Digestão , Animais , Hidrólise , Antioxidantes/metabolismo , Antioxidantes/análise , Osso e Ossos/metabolismo , Suínos , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/metabolismo , Manipulação de Alimentos/métodos , Temperatura Alta , Aminoácidos/metabolismo , Aminoácidos/análise , Produtos da Carne/análise , Hipoglicemiantes/farmacologia , Anti-Hipertensivos/farmacologia , Anti-Inflamatórios/farmacologia , Peptídeo Hidrolases/metabolismo , Inibidores da Dipeptidil Peptidase IV , Neprilisina/metabolismo , Neprilisina/antagonistas & inibidores , Endopeptidases
6.
J Oleo Sci ; 73(7): 963-976, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38945925

RESUMO

The objective of this research was to evaluate the efficiency of aqueous enzymatic extraction (AEE) to obtain oil from hemp seeds (Cannabis sativa L.) grown in northern Morocco. Optimisation of AEE extraction parameters, including pH, enzyme concentration (hemicellulase, protease and pectinase), temperature and incubation time, to maximize oil yield was achieved using response surface methodology with a central composite design. For comparison, the solvent extraction (Soxhlet) (SE) method was also used. Optimized hydrolysis conditions involved incubation for 4 hours at 60°C with a pH of 6.5, using a multi-enzyme preparation comprising protease, hemicellulase and pectinase at concentrations of 55, 202.5 and 234 U/mg, respectively. Referring to the conventional Soxhlet extraction (SE), Aqueous Enzymatic Extraction (AEE) achieved a 30.65% oil recovery rate under the optimized parameters mentioned above. The use of enzymes produced an oil that was more stable against oxidation than the solvent-extracted oil, with a peroxide value (PV) of 19.54 and 47.87 meq O 2 /kg, respectively. Furthermore, HPLC-DAD analysis of tocopherol content indicated a higher total tocopherol content (547.2 mg/kg) in Aqueous Enzymatic Extraction (AEE) compared to Soxhlet Extraction (SE) (513.51 mg/kg), with γ-tocopherol being the predominant form. No significant differences in fatty acid composition were observed between the two extraction methods with linoleic acid and alpha-linolenic acid being the predominant constituents.


Assuntos
Cannabis , Glicosídeo Hidrolases , Peptídeo Hidrolases , Óleos de Plantas , Poligalacturonase , Sementes , Cannabis/química , Poligalacturonase/metabolismo , Óleos de Plantas/química , Óleos de Plantas/isolamento & purificação , Glicosídeo Hidrolases/metabolismo , Sementes/química , Peptídeo Hidrolases/metabolismo , Hidrólise , Extração Líquido-Líquido/métodos , Qualidade dos Alimentos , Água , Tocoferóis/análise , Tocoferóis/isolamento & purificação , Concentração de Íons de Hidrogênio , Temperatura , Solventes/química , Química Verde/métodos
7.
Mol Biol (Mosk) ; 58(1): 171-177, 2024.
Artigo em Russo | MEDLINE | ID: mdl-38943589

RESUMO

Many viruses, including SARS-CoV-2, the coronavirus responsible for the COVID-19 pandemic, enter host cells through a process of cell-viral membrane fusion that is activated by proteolytic enzymes. Typically, these enzymes are host cell proteases. Identifying the proteases that activate the virus is not a simple task but is important for the development of new antiviral drugs. In this study, we developed a bioinformatics method for identifying proteases that can cleave viral envelope glycoproteins. The proposed approach involves the use of predictive models for the substrate specificity of human proteases and the application of a structural analysis method for predicting the vulnerability of protein regions to proteolysis based on their 3D structures. Specificity models were constructed for 169 human proteases using information on their known substrates. A previously developed method for structural analysis of potential proteolysis sites was applied in parallel with specificity models. Validation of the proposed approach was performed on the SARS-CoV-2 spike protein, whose proteolysis sites have been well studied.


Assuntos
Biologia Computacional , Peptídeo Hidrolases , Proteólise , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/química , Humanos , SARS-CoV-2/enzimologia , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Biologia Computacional/métodos , Especificidade por Substrato , Peptídeo Hidrolases/metabolismo , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , COVID-19/virologia , COVID-19/metabolismo , Pandemias , Modelos Moleculares , Betacoronavirus/enzimologia , Betacoronavirus/genética
8.
Viruses ; 16(6)2024 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-38932275

RESUMO

Viral tropism is most commonly linked to receptor use, but host cell protease use can be a notable factor in susceptibility to infection. Here we review the use of host cell proteases by human viruses, focusing on those with primarily respiratory tropism, particularly SARS-CoV-2. We first describe the various classes of proteases present in the respiratory tract, as well as elsewhere in the body, and incorporate the targeting of these proteases as therapeutic drugs for use in humans. Host cell proteases are also linked to the systemic spread of viruses and play important roles outside of the respiratory tract; therefore, we address how proteases affect viruses across the spectrum of infections that can occur in humans, intending to understand the extrapulmonary spread of SARS-CoV-2.


Assuntos
Peptídeo Hidrolases , Infecções Respiratórias , SARS-CoV-2 , Humanos , Infecções Respiratórias/virologia , Infecções Respiratórias/tratamento farmacológico , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , SARS-CoV-2/enzimologia , Peptídeo Hidrolases/metabolismo , Tropismo Viral , COVID-19/virologia , Viroses/tratamento farmacológico , Viroses/virologia , Antivirais/farmacologia , Interações Hospedeiro-Patógeno , Inibidores de Proteases/farmacologia
9.
New Phytol ; 243(3): 1034-1049, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38853453

RESUMO

Processing by proteases irreversibly regulates the fate of plant proteins and hampers the production of recombinant proteins in plants, yet only few processing events have been described in agroinfiltrated Nicotiana benthamiana, which has emerged as the main transient protein expression platform in plant science and molecular pharming. Here, we used in-gel digests and mass spectrometry to monitor the migration and topography of 5040 plant proteins within a protein gel. By plotting the peptides over the gel slices, we generated peptographs that reveal where which part of each protein was detected within the protein gel. These data uncovered that 60% of the detected proteins have proteoforms that migrate at lower than predicted molecular weights, implicating extensive proteolytic processing. This analysis confirms the proteolytic removal and degradation of autoinhibitory prodomains of most but not all proteases, and revealed differential processing within pectinemethylesterase and lipase families. This analysis also uncovered intricate processing of glycosidases and uncovered that ectodomain shedding might be common for a diverse range of receptor-like kinases. Transient expression of double-tagged candidate proteins confirmed processing events in vivo. This large proteomic dataset implicates an elaborate proteolytic machinery shaping the proteome of N. benthamiana.


Assuntos
Nicotiana , Proteínas de Plantas , Proteólise , Proteoma , Nicotiana/genética , Nicotiana/metabolismo , Proteoma/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Proteômica , Hidrolases de Éster Carboxílico/metabolismo , Hidrolases de Éster Carboxílico/genética , Lipase/metabolismo , Lipase/genética , Peptídeo Hidrolases/metabolismo , Glicosídeo Hidrolases/metabolismo , Glicosídeo Hidrolases/genética
10.
J Med Chem ; 67(13): 10891-10905, 2024 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-38934239

RESUMO

Antifungal peptides are an appealing alternative to standard antifungal medicines due to their unique mechanism of action and low-level resistance. However, their susceptibility to protease degradation keeps hindering their future development. Herein, a library was established to design peptides with protease resistance and high antifungal activity. The peptides were incorporated with minimal D-amino acids to further improve the protease stability. The most active peptide, IR3, demonstrated good antifungal activity and low toxicity, and its molecular integrity was maintained after protease hydrolysis for 8 h at 2 mg/mL. Furthermore, IR3 could permeate the fungal cell wall, disrupt the cell membrane, produce reactive oxygen species, and induce apoptosis in fungal cells. In vivo experiments confirmed that IR3 could effectively treat fungal keratitis. Collectively, these findings suggest that IR3 is a promising antifungal agent and may be beneficial in the design and development of protease-resistant antifungal peptides.


Assuntos
Aminoácidos , Antifúngicos , Testes de Sensibilidade Microbiana , Antifúngicos/farmacologia , Antifúngicos/síntese química , Antifúngicos/química , Aminoácidos/química , Aminoácidos/farmacologia , Aminoácidos/metabolismo , Desenho de Fármacos , Animais , Proteólise/efeitos dos fármacos , Peptídeos/farmacologia , Peptídeos/química , Peptídeos/metabolismo , Candida albicans/efeitos dos fármacos , Ceratite/tratamento farmacológico , Ceratite/microbiologia , Peptídeo Hidrolases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Camundongos , Apoptose/efeitos dos fármacos , Farmacorresistência Fúngica/efeitos dos fármacos , Relação Estrutura-Atividade
11.
Cell Biol Toxicol ; 40(1): 45, 2024 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-38864940

RESUMO

MALT1 has been implicated as an upstream regulator of NF-κB signaling in immune cells and tumors. This study determined the regulatory mechanisms and biological functions of MALT1 in non-small cell lung cancer (NSCLC). In cell culture and orthotopic xenograft models, MALT1 suppression via gene expression interference or protein activity inhibition significantly impaired malignant phenotypes and enhanced radiation sensitivity of NSCLC cells. CSN5, the core subunit of COP9 signalosome, was firstly verified to stabilize MALT1 via disturbing the interaction with E3 ligase FBXO3. Loss of FBXO3 in NSCLC cells reduced MALT1 ubiquitination and promoted its accumulation, which was reversed by CSN5 interference. An association between CSN5/FBXO3/MALT1 regulatory axis and poor prognosis in NSCLC patients was identified. Our findings revealed the detail mechanism of continuous MALT1 activation in NF-κB signaling, highlighting its significance as predictor and potential therapeutic target in NSCLC.


Assuntos
Complexo do Signalossomo COP9 , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , NF-kappa B , Transdução de Sinais , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/genética , Humanos , Complexo do Signalossomo COP9/metabolismo , Complexo do Signalossomo COP9/genética , NF-kappa B/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Animais , Linhagem Celular Tumoral , Camundongos , Camundongos Nus , Ubiquitinação , Peptídeo Hidrolases/metabolismo , Peptídeo Hidrolases/genética , Progressão da Doença , Camundongos Endogâmicos BALB C , Feminino , Proteínas F-Box/metabolismo , Proteínas F-Box/genética , Peptídeos e Proteínas de Sinalização Intracelular
12.
Int J Mol Sci ; 25(11)2024 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-38891925

RESUMO

Stress exposure worsens allergic inflammatory diseases substantially. Mast cells (MCs) play a key role in peripheral immune responses to neuroendocrine stress mediators such as nerve growth factor (NGF) and substance P (SP). Mast cell proteases (MCPs) and cholinergic factors (Chrna7, SLURP1) were recently described to modulate MC stress response. We studied MCPs and Chrna7/SLURP1 and their interplay in a mouse model for noise induced stress (NiS) and atopic dermatitis-like allergic inflammation (AlD) and in cultured MC lacking Chrna7. We found that the cholinergic stress axis interacts with neuroendocrine stress mediators and stress-mediator cleaving enzymes in AlD. SP-cleaving mMCP4+ MC were upregulated in AlD and further upregulated by stress in NiS+AlD. Anti-NGF neutralizing antibody treatment blocked the stress-induced upregulation in vivo, and mMCP4+ MCs correlated with measures of AlD disease activity. Finally, high mMCP4 production in response to SP depended on Chrna7/SLURP1 in cultured MCs. In conclusion, mMCP4 and its upstream regulation by Chrna7/SLURP1 are interesting novel targets for the treatment of allergic inflammation and its aggravation by stress.


Assuntos
Dermatite Atópica , Modelos Animais de Doenças , Mastócitos , Pele , Receptor Nicotínico de Acetilcolina alfa7 , Animais , Mastócitos/metabolismo , Mastócitos/imunologia , Dermatite Atópica/metabolismo , Dermatite Atópica/patologia , Dermatite Atópica/imunologia , Camundongos , Pele/metabolismo , Pele/patologia , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Peptídeo Hidrolases/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Substância P/metabolismo , Estresse Fisiológico , Camundongos Endogâmicos C57BL , Fator de Crescimento Neural/metabolismo
13.
Mol Biol Rep ; 51(1): 713, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38824247

RESUMO

BACKGROUND: Protease S (PrtS) from Photorhabdus laumondii belongs to the group of protealysin-like proteases (PLPs), which are understudied factors thought to play a role in the interaction of bacteria with other organisms. Since P. laumondii is an insect pathogen and a nematode symbiont, the analysis of the biological functions of PLPs using the PrtS model provides novel data on diverse types of interactions between bacteria and hosts. METHODS AND RESULTS: Recombinant PrtS was produced in Escherichia coli. Efficient inhibition of PrtS activity by photorin, a recently discovered emfourin-like protein inhibitor from P. laumondii, was demonstrated. The Galleria mellonella was utilized to examine the insect toxicity of PrtS and the impact of PrtS on hemolymph proteins in vitro. The insect toxicity of PrtS is reduced compared to protease homologues from non-pathogenic bacteria and is likely not essential for the infection process. However, using proteomic analysis, potential PrtS targets have been identified in the hemolymph. CONCLUSIONS: The spectrum of identified proteins indicates that the function of PrtS is to modulate the insect immune response. Further studies of PLPs' biological role in the PrtS and P. laumondii model must clarify the details of PrtS interaction with the insect immune system during bacterial infection.


Assuntos
Mariposas , Peptídeo Hidrolases , Photorhabdus , Animais , Mariposas/microbiologia , Peptídeo Hidrolases/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Hemolinfa/metabolismo , Proteômica/métodos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Escherichia coli/genética , Escherichia coli/metabolismo
14.
Curr Microbiol ; 81(8): 227, 2024 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-38879855

RESUMO

Microbial degradation of keratin is characterized by its inherent safety, remarkable efficiency, and the production of copious degradation products. All these attributes contribute to the effective management of waste materials at high value-added and in a sustainable manner. Microbial degradation of keratin materials remains unclear, however, with variations observed in the degradation genes and pathways among different microorganisms. In this study, we sequenced the transcriptome of Purpureocillium lilacinum GZAC18-2JMP mycelia on control medium and the medium containing 1% feather powder, analyzed the differentially expressed genes, and revealed the degradation mechanism of chicken feathers by P. lilacinum GZAC18-2JMP. The results showed that the chicken feather degradation rate of P. lilacinum GZAC18-2JMP reached 64% after 216 h of incubation in the fermentation medium, reaching a peak value of 148.9 µg·mL-1 at 192 h, and the keratinase enzyme activity reached a peak value of 211 U·mL-1 at 168 h, which revealed that P. lilacinum GZAC18-2JMP had a better keratin degradation effect. A total of 1001 differentially expressed genes (DEGs) were identified from the transcriptome database, including 475 upregulated genes and 577 downregulated genes. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis of the DEGs revealed that the metabolic pathways related to keratin degradation were mainly sulfur metabolism, ABC transporters, and amino acid metabolism. Therefore, the results of this study provide an opportunity to gain further insight into keratin degradation and promote the biotransformation of feather wastes.


Assuntos
Plumas , Hypocreales , Queratinas , Transcriptoma , Queratinas/metabolismo , Hypocreales/genética , Hypocreales/metabolismo , Animais , Plumas/metabolismo , Galinhas , Perfilação da Expressão Gênica , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Peptídeo Hidrolases/metabolismo , Peptídeo Hidrolases/genética , Micélio/genética , Micélio/metabolismo , Micélio/crescimento & desenvolvimento , Fermentação , Biodegradação Ambiental
15.
Curr Microbiol ; 81(7): 217, 2024 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-38852107

RESUMO

The application of enzymes in agricultural fields has been little explored. One potential application of fungal lytic enzymes (chitinases, lipases, and proteases) is as an additive to current biopesticides to increase their efficacy and reduce the time of mortality. For this, a screening of lytic overproducer fungi under submerged fermentation with a chemical-defined medium was performed. Then, the enzymatic crude extract (ECE) was concentrated and partially characterized. This characterization consisted of measuring the enzymatic activity (lipase, protease and, chitinase) and determining the enzyme stability after storage at temperatures of - 80, - 20 and, 4 °C. And lastly, the application of these concentrated enzymatic crude extracts (C-ECE) as an enhancer of spores-based fungal biopesticide was proven. Beauveria were not as good producers of lytic enzymes as the strains from Trichoderma and Metarhizium. The isolate M. robertsii Mt015 was selected for the co-production of chitinases and proteases; and the isolate T. harzianum Th180 for co-production of chitinases, lipases, and proteases. The C-ECE of Mt015 had a protease activity of 18.6 ± 1.1 U ml-1, chitinase activity of 0.28 ± 0.01 U ml-1, and no lipase activity. Meanwhile, the C-ECE of Th180 reached a chitinase activity of 0.75 U ml-1, lipase activity of 0.32 U ml-1, and protease activity of 0.24 U ml-1. Finally, an enhancing effect of the enzymatic extracts of M. robertsii (66.7%) and T. harzianum (43.5%) on the efficacy of B. bassiana Bv064 against Diatraea saccharalis larvae was observed. This work demonstrates the non-species-specific enhancing effect of enzymatic extracts on the insecticidal activity of conidial-based biopesticides, which constitutes a contribution to the improvement of biological control agents' performance.


Assuntos
Quitinases , Fermentação , Peptídeo Hidrolases , Quitinases/metabolismo , Peptídeo Hidrolases/metabolismo , Animais , Lipase/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Agentes de Controle Biológico/farmacologia , Agentes de Controle Biológico/metabolismo , Fungos/metabolismo , Controle Biológico de Vetores/métodos , Beauveria/enzimologia , Beauveria/metabolismo , Estabilidade Enzimática
16.
Environ Microbiol Rep ; 16(3): e13282, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38923398

RESUMO

The global landscape of Candida infections has seen a significant shift. Previously, Candida albicans was the predominant species. However, there has been an emergence of non-albicans Candida species, which are often less susceptible to antifungal treatment. Candida kefyr, in particular, has been increasingly associated with infections. This study aimed to investigate the profiles of enzymatic activity and biofilm formation in both clinical and non-clinical isolates of C. kefyr. A total of 66 C. kefyr isolates were analysed. The activities of proteinase and phospholipase were assessed using bovine serum albumin and egg yolk agar, respectively. Haemolysin, caseinolytic and esterase activities were evaluated using specific methods. Biofilm formation was investigated using crystal violet staining. The findings indicated that biofilm and proteinase activity were detected in 81.8% and 93.9% of all the isolates, respectively. Haemolysin activity was observed with the highest occurrence (95.5%) among normal microbiota isolates. Esterase activity was predominantly identified in dairy samples and was absent in hospital samples. Caseinase production was found with the highest occurrence (18.2%) in normal microbiota and hospital samples. Phospholipase activity was limited, found in only 3% of all the isolates. These findings reveal variations in enzyme activity between clinical and non-clinical C. kefyr isolates. This sheds light on their pathogenic potential and has implications for therapeutic strategies.


Assuntos
Biofilmes , Candida , Candidíase , Fosfolipases , Biofilmes/crescimento & desenvolvimento , Candida/isolamento & purificação , Candida/enzimologia , Candida/fisiologia , Candida/classificação , Humanos , Candidíase/microbiologia , Fosfolipases/metabolismo , Esterases/metabolismo , Proteínas Hemolisinas/metabolismo , Peptídeo Hidrolases/metabolismo , Microbiologia Ambiental
17.
ACS Nano ; 18(26): 17018-17030, 2024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-38845136

RESUMO

The advantageous optical properties of quantum dots (QDs) motivate their use in a wide variety of applications related to imaging and bioanalysis, including the detection of proteases and their activity. Recent studies have shown that surface chemistry on QDs is able to modulate protease activity, but only nonspecifically. Here, we present a strategy to selectively accelerate the activity of a particular target protease by as much as two orders of magnitude. Exosite-binding "bait" peptides were derived from proteins that span a range of biological roles─substrate, receptor, and inhibitor─and were used to increase the affinity of the QD-peptide conjugates for either thrombin or factor Xa, resulting in increased rates of proteolysis for coconjugated substrates. Unlike effects from QD surface chemistry, the acceleration was specific to the target protease with negligible acceleration of other proteases. Benefits of this "bait and cleave" sensing approach included detection limits that improved by more than an order of magnitude, reenabled detection of target protease against an overwhelming background of nontarget proteolysis, and mitigation of the action of inhibitors. The cumulative results point to a generalizable strategy, where the mechanism of acceleration, considerations for the design of bait peptides and conjugates, and routes to expanding the scope of this approach are discussed. Overall, this research represents a major step forward in the rational design of nanoparticle-based enzyme sensors that enhance sensitivity and selectivity.


Assuntos
Peptídeos , Pontos Quânticos , Trombina , Pontos Quânticos/química , Peptídeos/química , Peptídeos/metabolismo , Trombina/metabolismo , Trombina/análise , Trombina/química , Fator Xa/metabolismo , Fator Xa/química , Proteólise , Humanos , Propriedades de Superfície , Peptídeo Hidrolases/metabolismo , Peptídeo Hidrolases/química
18.
Int J Biol Macromol ; 273(Pt 2): 133199, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38885866

RESUMO

This study aimed to produce, characterize and purify a protease from Aspergillus heteromorphus URM0269. After production by solid fermentation of wheat bran performed according to a central composite design, protease was characterized in terms of biochemical, kinetic, and thermodynamic parameters for further purification by chromatography. Proteolytic activity achieved a maximum value of 57.43 U/mL using 7.8 g of wheat bran with 40 % moisture. Protease displayed high stability in the pH and temperature ranges of 5.0-10.0 and 20-30 °C, respectively, and acted optimally at pH 7.0 and 50 °C. The enzyme, characterized as a serine protease, followed Michaelis-Menten kinetics with a maximum reaction rate of 140.0 U/mL and Michaelis constant of 11.6 mg/mL. Thermodynamic activation parameters, namely activation Gibbs free energy (69.79 kJ/mol), enthalpy (5.86 kJ/mol), and entropy (-214.39 J/mol.K) of the hydrolysis reaction, corroborated with kinetic modeling showing high affinity for azocasein. However, thermodynamic parameters suggested a reversible mechanism of unfolding. Purification by chromatography yielded a protease purification factor of 7.2, and SDS-PAGE revealed one protein band with a molecular mass of 14.7 kDa. Circular dichroism demonstrated a secondary structure made up of 45.6 % α-helices. These results show the great potential of this protease for future use in the industrial area.


Assuntos
Aspergillus , Temperatura , Termodinâmica , Aspergillus/enzimologia , Cinética , Concentração de Íons de Hidrogênio , Estabilidade Enzimática , Fermentação , Peptídeo Hidrolases/química , Peptídeo Hidrolases/isolamento & purificação , Peptídeo Hidrolases/metabolismo , Hidrólise , Agricultura
19.
Mem Inst Oswaldo Cruz ; 119: e230243, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38775551

RESUMO

BACKGROUND: Leishmania tarentolae is a non-pathogenic species found in lizards representing an important model for Leishmania biology. However, several aspects of this Sauroleishmania remain unknown to explain its low level of virulence. OBJECTIVES: We reported several aspects of L. tarentolae biology including glycoconjugates, proteolytic activities and metabolome composition in comparison to pathogenic species (Leishmania amazonensis, Leishmania braziliensis, Leishmania infantum and Leishmania major). METHODS: Parasites were cultured for extraction and purification of lipophosphoglycan (LPG), immunofluorescence probing with anti-gp63 and resistance against complement. Parasite extracts were also tested for proteases activity and metabolome composition. FINDINGS: Leishmania tarentolae does not express LPG on its surface. It expresses gp63 at lower levels compared to pathogenic species and, is highly sensitive to complement-mediated lysis. This species also lacks intracellular/extracellular activities of proteolytic enzymes. It has metabolic differences with pathogenic species, exhibiting a lower abundance of metabolites including ABC transporters, biosynthesis of unsaturated fatty acids and steroids, TCA cycle, glycine/serine/threonine metabolism, glyoxylate/dicarboxylate metabolism and pentose-phosphate pathways. MAIN CONCLUSIONS: The non-pathogenic phenotype of L. tarentolae is associated with alterations in several biochemical and molecular features. This reinforces the need of comparative studies between pathogenic and non-pathogenic species to elucidate the molecular mechanisms of virulence during host-parasite interactions.


Assuntos
Glicoconjugados , Leishmania , Metaboloma , Peptídeo Hidrolases , Leishmania/enzimologia , Peptídeo Hidrolases/metabolismo , Animais , Glicoesfingolipídeos/metabolismo , Proteínas do Sistema Complemento
20.
Food Res Int ; 183: 114225, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38760144

RESUMO

The aim of this study was to isolate and identify the main milk-clotting proteases from Prinsepia utilis Royle. Protein isolates obtained using precipitation with 20 %-50 % ammonium sulfate (AS) showed higher milk-clotting activity (MCA) at 154.34 + 0.35 SU. Two milk-clotting proteases, namely P191 and P1831, with molecular weight of 49.665 kDa and 68.737 kDa, respectively, were isolated and identified using liquid chromatography-mass spectrometry (LC-MS/MS). Bioinformatic analysis showed that the two identified milk-clotting proteases were primarily involved in hydrolase activity and catabolic processes. Moreover, secondary structure analysis showed that P191 structurally consisted of 40.85 % of alpha-helices, 15.96 % of beta-strands, and 43.19 % of coiled coil motifs, whereas P1831 consisted of 70 % of alpha-helices, 7.5 % of beta-strands, and 22.5 % of coiled coil motifs. P191 and P1831 were shown to belong to the aspartic protease and metalloproteinase types, and exhibited stability within the pH range of 4-6 and good thermal stability at 30-80 °C. The addition of CaCl2 (<200 mg/L) increased the MCA of P191 and P1831, while the addition of NaCl (>3 mg/mL) inhibited their MCA. Moreover, P191 and P1831 preferably hydrolyzed kappa-casein, followed by alpha-casein, and to a lesser extent beta-casein. Additionally, cheese processed with the simultaneous use of the two proteases isolated in the present study exhibited good sensory properties, higher protein content, and denser microstructure compared with cheese processed using papaya rennet or calf rennet. These findings unveil the characteristics of two proteases isolated from P. utilis, their milk-clotting properties, and potential application in the cheese-making industry.


Assuntos
Queijo , Manipulação de Alimentos , Peptídeo Hidrolases , Queijo/análise , Manipulação de Alimentos/métodos , Animais , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Espectrometria de Massas em Tandem , Concentração de Íons de Hidrogênio , Leite/química , Peso Molecular , Estabilidade Enzimática , Cromatografia Líquida
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