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1.
Braz. j. biol ; 82: 1-7, 2022. tab, ilus, graf
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1468429

RESUMO

This work aimed to obtain aspartic proteases of industrial and biotechnological interest from the stomach of the crevalle jack fish (Caranx hippos). In order to do so, a crude extract (CE) of the stomach was obtained and subjected to a partial purification by salting-out, which resulted in the enzyme extract (EE) obtainment. EE proteases were characterized physicochemically and by means of zymogram. In addition, the effect of chemical agents on their activity was also assessed. By means of salting-out it was possible to obtain a purification of 1.6 times with a yield of 49.4%. Two acid proteases present in the EE were observed in zymogram. The optimum temperature and thermal stability for EE acidic proteases were 55 ºC and 45 °C, respectively. The optimum pH and pH stability found for these enzymes were pH 1.5 and 7.0, respectively. Total inhibition of EE acid proteolytic activity was observed in the presence of pepstatin A. dithiothreitol (DTT) and Ca2+ did not promote a significant effect on enzyme activity. In the presence of heavy metals, such as Al3+, Cd2+ and Hg2+, EE acidic proteases showed more than 70% of their enzymatic activity. The results show that it is possible to obtain, from the stomach of C. hippos, aspartic proteases with high proteolytic activity and characteristics that demonstrate potential for industrial and biotechnological applications.


Este trabalho objetivou obter proteases aspárticas de interesse industrial e biotecnológico a partir do estômago do peixe xaréu (Caranx hippos). Para isso, foi obtido um extrato bruto do estômago, o qual foi submetido a uma purificação parcial por salting-out onde se obteve o extrato enzimático (EE). As proteases do EE foram caracterizadas físico-quimicamente e através de zimograma. Além disso, o efeito de agentes químicos sobre sua atividade também foi avaliado. Através de salting-out foi possível obter uma purificação de 1,6 vezes com rendimento de 49,4%. Foram observadas duas proteases ácidas presentes no EE através de zimograma. A temperatura ótima e a estabilidade térmica para as proteases ácidas do EE foram de 55 ºC e 45 °C, respectivamente. O pH ótimo e a estabilidade ao pH encontrados para estas enzimas foram o pH 1,5 e 7,0, respectivamente. Observou-se a inibição total da atividade proteolítica ácida do EE na presença de pepstatina A. O ditiotreitol (DTT) e o Ca2+ não promoveram efeito significativo na atividade enzimática. Na presença de metais pesados, como Al3+, Cd2+ e Hg2+, o EE manteve mais de 70% de atividade enzimática do EE. Os resultados mostram que é possível obter, a partir do estômago de C. hippos, proteases aspárticas com alta atividade proteolítica e características que demonstram potencial para aplicações industriais e biotecnológicas.


Assuntos
Animais , Estômago/enzimologia , Estômago/química , Peixes , Ácido Aspártico Proteases/análise , Ácido Aspártico Proteases/economia
2.
Braz. j. biol ; 82: 1-8, 2022. tab, ilus, graf
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1468480

RESUMO

The viscera and other residues from fish processing are commonly discarded by the fishing industry. These by products can be a source of digestive enzymes with industrial and biotechnological potential. In this study, we aimed at the extraction, characterization, and application of acidic proteases from the stomach of Carangoides bartholomaei (Cuvier, 1833). A crude extract from the stomachs was obtained and submitted to a partial purification process by salting-out, which obtained a Purified Extract (PE) with a specific proteolytic activity of 54.0 U·mg-¹. A purification of 1.9 fold and a yield of 41% were obtained. The PE presents two isoforms of acidic proteases and a maximum proteolytic activity at 45 °C and pH 2.0. The PE acidic proteolytic activity was stable in the pH range of 1.5 to 7.0 and temperature from 25 °C to 50 °C. Purified Extract kept 35% of its proteolytic activity at the presence of NaCl 15% (m/v) but was totally inhibited by pepstatin A. Purified Extract aspartic proteases presented high activity in the presence of heavy metals such as Cd2+, Hg2+, Pb2+, Al3+, and Cu2+. The utilization of PE as an enzymatic addictive in the collagen extraction from Nile tilapia scales has doubled the process yield. The results indicate the potential of these aspartic proteases for industrial and biotechnological applications.


As vísceras e outros resíduos do processamento de peixes são geralmente descartados pela indústria pesqueira. Esses resíduos podem ser uma fonte de enzimas digestivas com potencial industrial e biotecnológico. Neste estudo, objetivamos a extração, caracterização e aplicação de proteases aspárticas do estômago de Carangoides bartholomaei (Cuvier, 1833). Um extrato bruto do estômago foi obtido e submetido a um processo de purificação parcial, que obteve um Extrato Purificado (EP) com uma atividade proteolítica específica de 54,0 U·mg-¹. Foi obtida uma purificação de 1,9 vezes e um rendimento de 41%. O EP apresenta duas isoformas de proteases ácidas e atividade proteolítica máxima a 45 °C e pH 2,0. A atividade proteolítica do EP foi estável na faixa de pH de 1,5 a 7,0 e temperatura de 25 °C a 50 °C. O EP manteve 35% de sua atividade proteolítica na presença de NaCl a 15% (m/v), mas foi totalmente inibida pela pepstatina A. As proteases ácidas do EP apresentaram alta atividade na presença de metais pesados como o Cd2+, Hg2+, Pb2+, Al3+ e Cu2+. A utilização de EP como aditivo enzimático na extração de colágeno a partir de escamas de tilápia do Nilo dobrou o rendimento do processo. Os resultados indicam um potencial dessas proteases para aplicações industriais e biotecnológicas.


Assuntos
Animais , Colágeno/análise , Estômago , Pepsina A/análise , Perciformes , Vísceras/enzimologia , Ácido Aspártico Proteases/análise
3.
Rev. Soc. Bras. Med. Trop ; 53: e20190214, 2020. tab, graf
Artigo em Inglês | LILACS | ID: biblio-1057290

RESUMO

Abstract INTRODUCTION: The aim of this study was to evaluate some virulence factors in Candida albicans isolates from patients with onychomycosis and determine the correlation between these factors and the antifungal resistance profile. METHODS: Seventy species of C. albicans were confirmed using polymerase chain reaction amplification of the HWP1 gene. According to the Clinical & Laboratory Standards Institute guidelines, the susceptibility profile of four antifungal agents was investigated, and the production of aspartyl protease, phospholipase, haemolysin, and biofilm was determined. The correlation between these profiles was also investigated. RESULTS: The isolates indicated different levels of resistance and production of virulence factors. Significant correlations were observed between the minimum inhibitory concentration (MIC) of fluconazole/itraconazole and biofilm production, between phospholipase production and fluconazole/itraconazole MIC, and between fluconazole MIC and hemolytic activity in C. albicans isolates. The results also showed significant correlations between phospholipase activity and biofilm production. CONCLUSIONS: Our findings will contribute to a better understanding of the pathogenesis of C. albicans and characterize the relationship between virulence factors and antifungal resistance, which may suggest new therapeutic strategies considering the possible involvement of the virulence mechanism in the effectiveness of treatment.


Assuntos
Humanos , Candida albicans/patogenicidade , Onicomicose/microbiologia , Fatores de Virulência , Antifúngicos/farmacologia , Unhas/microbiologia , Fosfolipases/biossíntese , Candida albicans/efeitos dos fármacos , Candida albicans/ultraestrutura , Microscopia Eletrônica de Varredura , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase , Biofilmes/crescimento & desenvolvimento , Farmacorresistência Fúngica , Ácido Aspártico Proteases/biossíntese , Hemólise
4.
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-976031

RESUMO

Lachesis muta rhombeata (Lmr) is the largest venomous snake in Latin America and its venom contains mainly enzymatic components, such as serine and metalloproteases, L-amino acid oxidase and phospholipases A2. Metalloproteases comprise a large group of zinc-dependent proteases that cleave basement membrane components such as fibronectin, laminin and collagen type IV. These enzymes are responsible for local and systemic changes, including haemorrhage, myonecrosis and inflammation. This study aimed the isolation and enzymatic characterization of the first metalloprotease (Lmr-MP) from Lmr venom (LmrV). Methods and results: Lmr-MP was purified through two chromatographic steps and submitted to enzymatic characterization. It showed proteolytic activity on azocasein with maximum activity at pH 7.0-9.0. It was inhibited by EDTA (a metal chelator that removes zinc, which is essential for enzymatic activity) and no effect was observed with PMSF, iodoacetic acid or pepstatin (inhibitors of serine, cysteine and aspartyl proteases, respectively). Ca2+, Mg2+ and Ba2+ ions increased its activity, while Al3+, Cu2+, Ni2+ and Zn2+ inhibited it. Additionally, ZnCl2 showed a dose dependent inhibition of the enzyme. Lmr-MP activity was also evaluated upon chromogenic substrates for plasma kallikrein (S-2302), plasmin and streptokinase-activated plasminogen (S-2251) and Factor Xa (S-2222) showing the highest activity on S-2302. The activity in different solutions (5 mM or 50 mM ammonium bicarbonate, pH 7.8; 0.1% trifluoroacetic acid + 50% acetonitrile; phosphate buffer saline, pH 7.4; 50 mM sodium acetate, pH 4.0 or ammonium acetate pH 4.5) was also evaluated and the results showed that its activity was abolished at acidic pHs. Its molecular mass (22,858 Da) was determined by MALDI-TOF and about 90% of its primary structure was verified by high-resolution mass spectrometry using HCD and ETD fragmentations and database search against the sequence of closely related species. It is a novel enzyme which shared high identity with other snake venom metalloproteases (svMPs) belonging to the P-I group. Conclusion: The purification procedure achieved a novel pure highly active metalloprotease from LmrV. This new molecule can help to understand the metalloproteases mechanisms of action, the Lachesis envenoming, as well as to open new perspectives for its use as therapeutic tools.(AU)


Assuntos
Animais , Peptídeo Hidrolases , Venenos de Serpentes , Lachesis muta , Metaloproteases , Ácido Aspártico Proteases
5.
Electron. j. biotechnol ; 17(2): 89-94, Mar. 2014. ilus, graf, tab
Artigo em Inglês | LILACS | ID: lil-714278

RESUMO

Background Aspartic proteases are a subfamily of endopeptidases that are useful in a variety of applications, especially in the food processing industry. Here we describe a novel aspartic protease that was purified from Peptidase R, a commercial protease preparation derived from Rhizopus oryzae. Results An aspartic protease sourced from Peptidase R was purified to homogeneity by anion exchange chromatography followed by polishing with a hydrophobic interaction chromatography column, resulting in a 3.4-fold increase in specific activity (57.5 × 10³ U/mg) and 58.8% recovery. The estimated molecular weight of the purified enzyme was 39 kDa. The N-terminal sequence of the purified protein exhibited 63-75% identity to rhizopuspepsins from various Rhizopus species. The enzyme exhibited maximal activity at 75°C in glycine-HCl buffer, pH 3.4 with casein as the substrate. The protease was stable at 35°C for 60 min and had an observed half-life of approximately 30 min at 45°C. Enzyme activity was not significantly inhibited by chelation with ethylenediamine tetraacetic acid (EDTA), and the addition of metal ions to EDTA-treated protease did not significantly change enzyme activity, indicating that proteolysis is not metal ion-dependent. The purified enzyme was completely inactivated by the aspartic protease inhibitor Pepstatin A. Conclusion Based on the observed enzyme activity, inhibition profile with Pepstatin A, and sequence similarity to other rhizopuspepsins, we have classified this enzyme as an aspartic protease.


Assuntos
Ácido Aspártico Proteases/isolamento & purificação , Ácido Aspártico Proteases/metabolismo , Rhizopus oryzae/enzimologia , Rhizopus oryzae/química , Endopeptidases , Temperatura , Indústria Alimentícia , Cromatografia , Sequência de Aminoácidos , Concentração de Íons de Hidrogênio , Peso Molecular
6.
Arch. oral res. (Impr.) ; 9(2): 177-184, May-Aug. 2013. graf
Artigo em Inglês | LILACS | ID: lil-754541

RESUMO

Among other non-bacterial organisms, yeasts have been isolated from subgingival sites with relative frequency. Candida albicans is the species most commonly isolated although its role in periodontal disease has not been established. Objective: This study evaluated the secretion patterns of aspartyl-protease (Sap) by periodontal and nonperiodontal Candida albicans strains in normoxic and anoxic conditions. Material and methods: Periodontal strains (n=10; periodontal pockets ≥3.00 mm) and nonperiodontal Candida albicans strains (n=10) were grown under normoxic and anoxic conditions in protease-inducible broth. Sap activities were quantified in supernatants using azocasein as substrate. Whole-protein contents in supernatants were determined by Bradford’s method. Specific protease activities (Sap activity.protein-1) were assessed and compared. Results: While nonperiodontal strains secrete similar amounts of Sap under both atmospheric conditions, periodontal strains secrete reduced amounts in the presence of molecular oxygen. Conclusion: Despite the limited number of assayed isolates, the possibilities of adaptation or selection of candidal strains to periodontal microenvironment may be considered...


Entre organismos não bacterianos, as leveduras têm sido isoladas de sítios subgengivais com relativa frequência. Candida albicans é a espécie mais comumente isolada, embora seu papel na doença periodontal não esteja estabelecido. Objetivo: Este estudo avaliou os padrões de secreção de aspartil-protease (Sap) por cepas periodontais e não periodontais de Candida albicans em situações de normóxia e anóxia. Material e métodos: Cepas periodontais (n=10; bolsas periodontais ≥3,00 milímetros) e cepas de não periodontais (n=10) Candida albicans foram cultivadas sob condições normóxicas e anóxicas em caldo de protease-induzida. A atividade Sap foi quantificada em sobrenadantes utilizando azocaseína como substrato. O conteúdo de proteínas totais nos sobrenadantes foi determinado pelo método de Bradford. Atividades de protease específica (atividade de proteína Sap-1) foram avaliadas e comparadas. Resultados: Apesar das cepas não periodontais secre¬tarem quantidades semelhantes de Sap em ambas as condições atmosféricas, as cepas periodontais secretam quantidades reduzidas na presença de oxigênio molecular. Conclusão: Apesar do número limitado de amostras analisadas, as possibilidades de adaptação ou seleção de cepas de Candida no microambiente periodontal pode ser considerada...


Assuntos
Humanos , Ácido Aspártico Proteases , Candida albicans/isolamento & purificação , Candida albicans/metabolismo , Células Cultivadas , Hipóxia , Periodonto/microbiologia , Valores de Referência , Estatísticas não Paramétricas
7.
Rev. Inst. Med. Trop. Säo Paulo ; 52(3): 139-143, May-June 2010. tab
Artigo em Inglês | LILACS | ID: lil-550344

RESUMO

Phospholipase and proteinase production and the ability of adhesion to buccal epithelial cells (BEC) of 112 Candida isolates originated from oral cavity of HIV infected patients and from blood and catheter of intensive care unit patients were investigated. The proteinase production was detected by inoculation into bovine serum albumin (BSA) agar and the phospholipase activity was performed using egg yolk emulsion. A yeast suspension of each test strain was incubated with buccal epithelial cells and the number of adherence yeast to epithelial cells was counted. A percentage of 88.1 percent and 55.9 percent of Candida albicans and 69.8 percent and 37.7 percent of non-albicans Candida isolates produced proteinase and phospholipase, respectively. Non-albicans Candida isolated from catheter were more proteolytic than C. albicans isolates. Blood isolates were more proteolytic than catheter and oral cavity isolates while oral cavity isolates produced more phospholipase than those from blood and catheter. C. albicans isolates from oral cavity and from catheter were more adherent to BEC than non-albicans Candida isolates, but the adhesion was not different among the three sources analyzed. The results indicated differences in the production of phospholipase and proteinase and in the ability of adhesion to BEC among Candida spp. isolates from different sources. This study suggests that the pathogenicity of Candida can be correlated with the infected site.


A produção de proteinase e fosfolipase e habilidade de adesão à célula epitelial bucal de 112 isolados de Candida originadas da cavidade bucal de pacientes infectados pelo HIV e de sangue e cateter de pacientes hospitalizados foram investigados. A produção de proteinase foi detectada por inoculação em ágar soro albumina bovina e a atividade de fosfolipase foi realizada usando emulsão de gema de ovo. A suspensão de levedura de cada isolado foi incubada com célula epitelial e o número de leveduras aderidas a célula epitelial foi contada. Uma porcentagem de 88,1 e 55,9 por cento de C. albicans e 69,8 e 37,7 por cento de isolados de Candida não albicans produziram proteinase e fosfolipase, respectivamente. Candida não albicans obtidas do cateter foram mais proteolíticos que isolados de Candida albicans (p < 0,001). Isolados do sangue foram mais proteolíticos do que isolados do cateter e cavidade bucal, enquanto isolados da cavidade bucal produziram mais fosfolipase do que aqueles isolados do sangue e cateter. C. albicans isoladas da cavidade bucal e do cateter foram mais aderentes à célula epitelial bucal do que isolados de Candida não albicans, mas não houve diferença na adesão entre os três locais analisados. Os resultados indicaram diferenças na produção de fosfolipase e proteinase e na habilidade de adesão à célula epitelial bucal entre os isolados de Candida das diferentes fontes. Este estudo sugere que a patogenicidade de Candida spp pode estar correlacionada ao local infectado.


Assuntos
Humanos , Ácido Aspártico Proteases/biossíntese , Aderência Bacteriana/fisiologia , Candida/enzimologia , Candida/fisiologia , Fosfolipases/biossíntese , Candida/isolamento & purificação , Cateteres de Demora/microbiologia , Células Epiteliais/microbiologia , Infecções por HIV/microbiologia , Boca/microbiologia
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