RESUMO
Many recombinant proteins are products of great value in biomedical and industrial fields. The use of solubility and affinity tags are commonly used to increase yields and facilitate the purification process. However, it is of paramount importance in several applications to remove the fusion tag from the final product. In this regard, the Tobacco Etch Virus protease (TEV) is one of the most widely used for tag removal. The presence in the TEV of the same tag to be removed facilitates the separation of TEV and the tag from the cleaved recombinant protein in a single purification step. We generated a double-tagged (StrepTagII and HisTag) TEV variant with reported mutations that improve the activity, the expression yield in E.coli, and that decrease the auto-proteolysis. This TEV can be easily purified by two consecutive affinity chromatography steps with high yields and purity. The cleavage reaction can be done to almost completeness in as fast as 15 min at room temperature and the removal of the protease and tags is performed in a single purification step, independent of the previous presence of a StrepTagII or a HisTag on the target.
Assuntos
Endopeptidases , Escherichia coli , Proteínas Recombinantes de Fusão , Endopeptidases/biossíntese , Endopeptidases/química , Endopeptidases/genética , Endopeptidases/isolamento & purificação , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
Bacillus pumilus is able to secrete subtilisin-like prote-ases, one of which has been purified and characterized biochemically, demonstrating great potential for use in industrial applications. In the current study, the biosynthesis and transcription of extracellular pro-teases in B. pumilus (BA06) under salt stress were investigated using various methods, including a proteolytic assay, zymogram analysis, and real-time PCR. Our results showed that total extracellular proteolytic activity, both in fermentation broth and on milk-containing agar plates, was considerably repressed by salt in a dosage-dependent manner. As Bacillus species usually secret multiple extracellular proteases, a vari-ety of individual extracellular protease encoding genes were selected for real-time PCR analysis. It was shown that proteases encoded by the aprE and aprX genes were the major proteases in the fermentation broth in terms of their transcripts in B. pumilus. Further, transcription of aprE, aprX, and epr genes was indeed repressed by salt stress. In con-trast, transcription of other genes (e.g., vpr and wprA) was not repressed or significantly affected by the salt. Conclusively, salt stress represses total extracellular proteolytic activity in B. pumilus, which can largely be ascribed to suppression of the major protease-encoding genes (aprE, aprX) at the transcriptional level. In contrast, transcription of other pro-tease-encoding genes (e.g., vpr, wprA) was not repressed by salt stress.
Assuntos
Bacillus/enzimologia , Endopeptidases/biossíntese , Estresse Fisiológico/efeitos dos fármacos , Bacillus/efeitos dos fármacos , Endopeptidases/genética , Fermentação , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Cloreto de Sódio/farmacologiaRESUMO
Daily intake of conjugated linoleic acid (CLA) has been shown to reduce body fat accumulation and to increase body metabolism; this latter effect has been often associated with the up-regulation of uncoupling proteins (UCPs). Here we addressed the effects of a CLA-supplemented murine diet (~2 % CLA mixture, cis-9, trans-10 and trans-10, cis-12 isomers; 45 % of each isomer on alternating days) on mitochondrial energetics, UCP2 expression/activity in the liver and other associated morphological and functional parameters, in C57BL/6 mice. Diet supplementation with CLA reduced both lipid accumulation in adipose tissues and triacylglycerol plasma levels, but did not augment hepatic lipid storage. Livers of mice fed a diet supplemented with CLA showed high UCP2 mRNA levels and the isolated hepatic mitochondria showed indications of UCP activity: in the presence of guanosine diphosphate, the higher stimulation of respiration promoted by linoleic acid in mitochondria from the CLA mice was almost completely reduced to the level of the stimulation from the control mice. Despite the increased generation of reactive oxygen species through oxi-reduction reactions involving NAD(+)/NADH in the Krebs cycle, no oxidative stress was observed in the liver. In addition, in the absence of free fatty acids, basal respiration rates and the phosphorylating efficiency of mitochondria were preserved. These results indicate a beneficial and secure dose of CLA for diet supplementation in mice, which induces UCP2 overexpression and UCP activity in mitochondria while preserving the lipid composition and redox state of the liver.
Assuntos
Suplementos Nutricionais , Endopeptidases/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Ácidos Linoleicos Conjugados/farmacologia , Fígado/metabolismo , Mitocôndrias Hepáticas/metabolismo , Animais , Masculino , Camundongos , NAD/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteases Específicas de UbiquitinaRESUMO
This investigation provides the enhanced production of thrombinase, a fibrinolytic enzyme using mutant Streptomyces venezuelae. Initially the mutagenesis of the marine isolate was done by UV and Ethyl methane sulfonate (EMS) and their mutational efficiencies were compared. The mutants were selected based on their high thrombinase activity and used for further studies. The mutant was found to be more halo and thermo tolerant comparing to wild. The effect of Dissolved oxygen level was also determined and the mutant offered the maximum specific growth rate as 0.2404 (h(-1)). The mutant showed high resistance to higher initial lactose concentration and the inhibition concentration was found to be 155.1mg/mL. The effect of S(0)/X(0) ratio on specific substrate consumption and production rate were also investigated. Both mutant and wild showed increase in specific substrate consumption and production rate at higher S(0)/X(0) ratio but the mutant showed better values than the wild strain.
Assuntos
Endopeptidases/biossíntese , Mutação , Streptomyces/enzimologia , Concentração de Íons de Hidrogênio , Cinética , TemperaturaRESUMO
The effects of nutritional conditions on growth and protease production by the feather-degrading Chryseobacterium sp. kr6 were investigated. Higher growth was observed on feather-containing or tryptone (TR) medium when compared to casein (CA) or glucose-nitrogen (GN) base medium. Protease production occurred during growth on feather-containing and TR media, whereas no protease activity was detected on CA or GN medium, indicating that protease production is not constitutive, depending on the presence of specific complex nitrogen sources. Supplementation of whole feathers (WF) medium with glucose (WFG) or NH(4)Cl (WFN) did not result in major differences in growth and protease production, whereas soluble protein was lower in supplemented media. Glucose consumption and growth were higher on WFG than on GN medium, suggesting that the absence of a specific complex nitrogen source limited bacterial growth. On WF medium, this strain grew closely attached to the feather structures, initially on the barbules and subsequently on the feather rachis. It was observed, through zymogram analysis, that strain kr6 produced diverse proteolytic enzymes in response to different growth substrates. These results were confirmed by the differential behaviors of crude proteases towards protease inhibitors.
Assuntos
Chryseobacterium/enzimologia , Chryseobacterium/crescimento & desenvolvimento , Endopeptidases/biossíntese , Plumas/microbiologia , Plumas/patologia , Animais , Proteínas de Bactérias/biossíntese , Meios de Cultura/química , Inibidores Enzimáticos/metabolismo , Plumas/ultraestruturaRESUMO
In the present work, Aspergillus fumigatus is described as a higher producer of hydrolytic enzymes secreted in response to the presence of the Callosobruchus maculatus bruchid pest. This fungus was able to grow over cowpea weevil shells as a unique carbon source, secreting alkaline proteolytic and chitinolytic enzymes. Enzyme secretion in A. fumigatus was induced by both C. maculatus exoskeleton as well as commercial chitin, and alkaline proteolytic and chitinolytic activities were detected after 48 hours of growth. Furthermore, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the production of specific proteins. Among them, two extracellular alkaline proteinases from culture enriched with C. maculatus exoskeleton were purified after chromatographic procedures using ion exchange and affinity columns. These proteins, named AP15 and AP30, had apparent molecular masses of 15,500 and 30,000 Da, respectively, as estimated by SDS-PAGE electrophoresis and mass spectrometry. AP30 was classified as a serine proteinase because it was inhibited by 5 mM: phenylmethylsulfonyl fluoride (100%) and 50 microM leupeptin (67.94%).
Assuntos
Aspergillus fumigatus/enzimologia , Proteínas de Bactérias/biossíntese , Quitinases/química , Endopeptidases/biossíntese , Gorgulhos/microbiologia , Animais , Aspergillus fumigatus/metabolismo , Proteínas de Bactérias/isolamento & purificação , Quitinases/biossíntese , Endopeptidases/isolamento & purificação , Controle Biológico de Vetores/métodos , Serina Endopeptidases/biossínteseRESUMO
A disponibilidade da terapia anti-retroviral tem resultado em um enorme declínio nas taxas de morbidade e mortalidade dos pacientes infectados pelo vírus da imunodeficiência humana. entretanto, tal terapia tem sido associada a efeitos metabólicos adversos, como a dislipidemia; sendo assim, indivíduos com doença arterial coronariana devem ser identificados e tratados. Este artigo faz uma revisão das anormalidades dos lipídios e das lipoproteínas associadas ao uso dos inibidores de proteases, sobre o possível mecanismo da dislipidemia associada a estes inibidores e usa o protocolo do programa de educação para tratamento do colesterol em adultos(National Cholesterol Education Program Adult Treatment Panel III Guidelines) para o acompanhamento do paciente com dislipidemia.
Assuntos
Terapia Antirretroviral de Alta Atividade , Hiperlipidemias , Síndrome da Imunodeficiência Adquirida/diagnóstico , Síndrome da Imunodeficiência Adquirida/fisiopatologia , Síndrome da Imunodeficiência Adquirida/terapia , Endopeptidases/biossíntese , Endopeptidases/metabolismo , Lipídeos/biossíntese , Lipoproteínas/biossíntese , Lipoproteínas/metabolismoAssuntos
Endopeptidases/biossíntese , Proteínas de Membrana/biossíntese , Lipofuscinoses Ceroides Neuronais/diagnóstico , Saliva/enzimologia , Adolescente , Adulto , Aminopeptidases , Argentina , Criança , Pré-Escolar , Dipeptidil Peptidases e Tripeptidil Peptidases , Saúde da Família , Feminino , Humanos , Lactente , Pessoa de Meia-Idade , Valores de Referência , Serina Proteases , Tioléster Hidrolases , Tripeptidil-Peptidase 1RESUMO
As candidíases bucais (também chamadas sapinhos) que ocorrem em crianças são causadas por uma deficiência imunológica, bem como por outros fatores tais como má higiene bucal e esterilização inadequada dos utensílios utilizados pelas mesmas, que potencializam a ocorrência dessa infecção fúngica. Considerando esse fato, foram avaliadas a freqüência e a atividade enzimática de Candida sp. isoladas em crianças de uma creche pública (Aprisco) na cidade de Fortaleza, Ceará. Foram coletadas amostras da mucosa bucal de 364 alunos de 1 a 5 anos de idade. Elas foram semeadas em ágar Sabouraud dextrose com cloranfenicol, incubadas por 72 horas a 37ºC e identificadas por testes micológicos. Verificou-se que 67 (18 por cento) apresentaram leveduras do gênero Candida. A Candida albicans foi a mais freqüente, com 30 isolados (45 por cento), seguida pelas C. tropicalis (31 por cento), C. guilliermondii (17 por cento), C. glabrata (4,5 por cento) e C. stellatoidea (1,5 por cento). Com relação às atividades enzimáticas das cepas de Candida albicans, 20 por cento produziram a enzima proteinase e 33 por cento, a fosfolipase. As Candida albicans isoladas da mucosa bucal de crianças dessa creche da prefeitura apresentaram uma fraca atividade enzimática. Assim, conclui-se que essas cepas parecem ter uma baixa virulência.
Assuntos
Humanos , Masculino , Feminino , Pré-Escolar , Brasil , Candida albicans/enzimologia , Candida albicans/isolamento & purificação , Candidíase Bucal/imunologia , Candidíase Bucal/microbiologia , Endopeptidases/biossíntese , Fosfolipases/biossíntese , Mucosa Bucal/microbiologiaRESUMO
Isogenic strains (with and without dsRNA) of the entomogenous fungi Metarhizium anisopliae var. acridum and Paecilomyces fumosoroseus were investigated for correlation between the presence of dsRNA and the production of cuticle-degrading proteases that play an important role in host parasitism, total secreted protein, and conidia production. Similar levels of cuticle-degrading subtilisin-like (Pr1) protease were observed for isogenic strains of M. anisopliae var. acridum after growth in medium supplemented with the cuticle of the grasshopper Rhammatocerus schistocercoides. Similarly, no statistical differences were observed for protease production, detected using the chromogenic substrate azocasein. For P. fumosoroseus isogenic strains, no significant differences in protease activity were observed after growth in the presence of either Euschistus heros or Nezara viridula (Hemiptera: Pentatomidae) cuticle. Similarly, no statistical differences were observed in virulence against E. heros. A comparison of mean conidia production showed a significantly higher production in the dsRNA-free isogenic strains of M. anisopliae var. acridum. Although, for most of the fungal phenotypes analysed, no overt effects were associated with the presence of these dsRNA infections, the reduction in conidia production by the isogenic strains of M. anisopliae var. acridum with dsRNA suggested that it may not be entirely accurate to describe these infections as latent.
Assuntos
Endopeptidases/metabolismo , Hypocreales/virologia , Paecilomyces/virologia , RNA de Cadeia Dupla/fisiologia , Animais , Caseínas/metabolismo , Endopeptidases/biossíntese , Proteínas Fúngicas/biossíntese , Regulação Fúngica da Expressão Gênica , Gafanhotos/química , Hemípteros/química , Hypocreales/citologia , Hypocreales/metabolismo , Hypocreales/patogenicidade , Proteínas de Insetos/metabolismo , Paecilomyces/citologia , Paecilomyces/metabolismo , Paecilomyces/patogenicidade , RNA de Cadeia Dupla/genética , Esporos Fúngicos/crescimento & desenvolvimento , VirulênciaRESUMO
The potential for thermal denaturation to cause enzyme losses during solid-state fermentation processes for the production of enzymes was examined, using the protease of Penicillium fellutanum as a model system. The frequency factor and activation energies for the first-order denaturation of this enzyme were determined as 3.447 x 10(59) h(-1) and 364,070 Jmol(-1), respectively. These values were incorporated into a mathematical model of enzyme deactivation, which was used to investigate the consequences of subjecting this protease to temporal temperature profiles reported in the literature for mid-height in a 34 cm high packed-bed bioreactor of 150 mm diameter. In this literature source, temperature profiles were measured for 5, 15 and 25 liters per minute of air and enzyme activities were measured as a function of time. The enzyme activity profiles predicted by the model were distributed similarly, one relative to the other, as had been found in the experimental study, with substantial amounts of denaturation being predicted when the substrate temperature exceeded 40 degrees C, which occurred at the lower two airflow rates. A mathematical model of a well-mixed bioreactor was used to explore the difficulties that would be faced at large scale. It suggests that even with airflows as high as one volume per volume per minute, up to 85% of the enzyme produced by the microorganism can be denatured by the end of the fermentation. This work highlights the extra care that must be taken in scaling up solid-state fermentation processes for the production of thermolabile products.
Assuntos
Reatores Biológicos , Endopeptidases/biossíntese , Helianthus/química , Modelos Químicos , Penicillium/enzimologia , Sementes/química , Endopeptidases/química , Fermentação , Cinética , Temperatura , Fatores de TempoRESUMO
A comparative study of proteolytic enzymes and cell-surface protein composition in virulent and avirulent Leishmania (Leishmania) amazonensis promastigote forms was carried out using one- and two-dimensional dodecyl sulfate sodium-polyacrylamide gel electrophoresis (SDS-PAGE). The surface iodinated protein profiles showed two major polypeptides of 65-60 and 50-47 kDa that were expressed in both virulent and avirulent promastigote forms. However, minor quantitative differences were observed in the cell-surface profile between the avirulent and virulent promastigotes. These included polypeptides of 115, 52, 45, 32, and 25 kDa that were preferentially expressed in the virulent forms. Two-dimensional SDS-PAGE showed an accentuated expression of acidic polypeptides; some of them differentially expressed in the promastigote forms analyzed. Live parasites treated with glycosylphosphatidylinositol (GPI)-specific phospholipase C (PLC) from Trypanosoma brucei and immunoprecipitated with the cross-reacting determinant (CRD) antibody recognized three major polypeptides of 65-60, 52, and 50-47 kDa, hence suggesting that these peptides were anchored to the plasma membrane domains through GPI anchor. Moreover, the polypeptides of 65-60 and 52 kDa were also recognized by the gp63 antiserum. Several metalloproteinase activities were similar in both virulent and avirulent promastigote forms, whereas cysteine proteinase activities, sensitive to E-64, were preferentially expressed in virulent promastigotes. These results suggest that cell-surface polypeptides and intracellular cysteine proteinases might play an important role in the virulence of L. (L.) amazonensis.
Assuntos
Endopeptidases/biossíntese , Leishmania mexicana/patogenicidade , Proteínas de Membrana/biossíntese , Proteínas de Protozoários/biossíntese , Animais , Cricetinae , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Endopeptidases/análise , Humanos , Leishmania mexicana/enzimologia , Leishmania mexicana/metabolismo , Proteínas de Membrana/análise , Camundongos , Testes de Precipitina , Proteínas de Protozoários/análise , VirulênciaRESUMO
The aim of this work was to investigate the role played by iron during interaction of Tritrichomonas foetus with cultured epithelial cells. We have observed that the growth rate of T. foetus is influenced by the amount of iron available into culture medium. When organisms maintained for 24h in iron-depleted medium were transferred to an iron-rich one, many protozoan cells exhibited a cytokinesis blockage. Parasites maintained in iron-depleted medium exhibited a significant increase in cytoadhesion when compared with both controls and parasites that had been cultured in medium in which iron was replaced. T. foetus collected from iron-depleted medium also exhibited a reduction in its ability to destroy epithelial cell monolayers and a reduction in the activity of several cysteine proteases. Taken together, the results presented here demonstrate that iron may be an extracellular signal, which seems to modulate the ability of T. foetus to interact with host epithelial cells.
Assuntos
Células Epiteliais/parasitologia , Ferro/fisiologia , Tritrichomonas foetus/crescimento & desenvolvimento , 2,2'-Dipiridil/farmacologia , Animais , Adesão Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Meios de Cultura , Relação Dose-Resposta a Droga , Endopeptidases/biossíntese , Endopeptidases/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Compostos Ferrosos/farmacologia , Células HeLa , Humanos , Indicadores e Reagentes/farmacologia , Ferro/farmacologia , Inibidores de Proteases/farmacologia , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/efeitos dos fármacos , Tritrichomonas foetus/citologia , Tritrichomonas foetus/efeitos dos fármacos , Tritrichomonas foetus/metabolismoRESUMO
The oral fungal microbiota of 30 children with AIDS, of both genders, aged from two to six years, receiving outpatient treatment, was evaluated and compared with that of a control group composed of 30 healthy subjects with matching ages and genders. Virulence factors, such as exoenzyme production, and susceptibility to five antifungal agents using an E-Test kit were evaluated. C. albicans predominated over other species in the AIDS group, showing a higher production of proteinase and phospholipase when compared with that observed in the control group. In this study few clinical manifestations of and low selectivity for C. albicans (23.3%) were observed in the AIDS group. The enzymatic studies showed that 53.8% of the AIDS strains were strongly positive whereas only 33.3% of the non-AIDS strains were positive. Amphotericin B was the most effective drug among the antifungal agents tested against C. albicans. The frequency, selectivity and level of exoenzyme production by C. albicans suggest a higher pathogenicity in the AIDS children than in the control children.
Assuntos
Síndrome da Imunodeficiência Adquirida/microbiologia , Antifúngicos/farmacologia , Farmacorresistência Fúngica , Hidrolases/biossíntese , Boca/microbiologia , Leveduras , Infecções Oportunistas Relacionadas com a AIDS/imunologia , Infecções Oportunistas Relacionadas com a AIDS/microbiologia , Síndrome da Imunodeficiência Adquirida/imunologia , Contagem de Linfócito CD4 , Candida albicans/efeitos dos fármacos , Candida albicans/enzimologia , Candida albicans/isolamento & purificação , Candidíase Bucal/imunologia , Candidíase Bucal/microbiologia , Estudos de Casos e Controles , Criança , Pré-Escolar , Endopeptidases/biossíntese , Feminino , Humanos , Masculino , Fosfolipases/biossíntese , Leveduras/efeitos dos fármacos , Leveduras/enzimologia , Leveduras/isolamento & purificaçãoRESUMO
The tobacco budworm Heliothis virescens is adapted to feed on tobacco leaves that have proteinase protein inhibitors (PIs). To study this adaptation, the midgut proteinases of Heliothis virescens larvae reared on artificial PI-free diet and on tobacco leaves were compared using ion exchange chromatography, hydrophobic chromatography, gel filtration and polyacrylamide gel electrophoresis at different conditions. SDS polyacrylamide-gradient gel electrophoresis (SDS-PAGE) and kinetic studies shown that leaf-fed larvae have a chymotrypsin (M(r) 26000) and four trypsins (T1-T4) with the following properties: T1, K(m) 0.3 microM, M(r) 70000; T2, K(m) 0.4 microM, M(r) 67000; T3, K(m) 2.4 microM, M(r) 29000; T4, K(m) 15 microM, M(r) 17000. Diet-fed larvae have a chymotrypsin (M(r) 26000) and a major trypsin (K(m) 2.9 microM, M(r) 29000). Native PAGE at different gel concentrations showed that in these conditions, only T1 and T2 occur in leaf-fed larvae, whereas gel filtration in the absence and presence of SDS revealed that T1 and T2 might arise by polymerization of T3 and T4, respectively. The data suggest that, in the presence of PI-containing food, H. virescens larvae express new trypsin molecules that form oligomers and are apparently less affected by PIs because of tighter binding to the substrate (lower K(m) values) and a putative decreased affinity for PIs.
Assuntos
Endopeptidases/biossíntese , Endopeptidases/química , Endopeptidases/isolamento & purificação , Inibidores de Proteases/farmacologia , Animais , Cromatografia , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Quimotripsina/química , Sistema Digestório/enzimologia , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Hidrolases/metabolismo , Cinética , Lepidópteros , Tripsina/metabolismoRESUMO
The general use of refrigeration of raw milk has contributed to maintenance of its quality, but has induced the selection of a psychotrophic bacteria which during its growth produces heat-resistant enzymes responsible, in part, for the deterioration of long-life products. Given the condition of the prolonged refrigeration of the milk before the process, it was necessary to determine the growth curves for bacteria at temperatures between 2 degrees C and 10 degrees C and the kinetics of production of proteases in raw fresh milk, inoculated with Pseudomonas fluorescens RV1O, using an automatic fermentation system, with minimal agitation under conditions of controlled temperature and pH. The results show a development over 10(5) ufc/mL in the cultures at 6 degrees, 8 degrees and 10 degrees C during the first 30 h and proteasic activities over 30 microM p-NA/2 h getting levels of de 80-180 microM p-NA/2 h over 50 h of cultivation. Only the cultures at 2 degrees C appeared stable with inferior cell counts and without inducing enzymatic activity. At 4 degrees C an intermediate situation occurs.
Assuntos
Endopeptidases/biossíntese , Conservação de Alimentos/métodos , Leite/microbiologia , Pseudomonas fluorescens/crescimento & desenvolvimento , Animais , Bovinos , Temperatura Baixa , Meios de Cultura , Feminino , Microbiologia de Alimentos , Cinética , Leite/química , Pseudomonas fluorescens/enzimologia , RefrigeraçãoRESUMO
The production of phospholipase and proteinase exoenzymes was evaluated in seventy nine samples of Candida isolated from the oral cavity of patients with oral lesions characteristic of candidosis and from individuals presenting a clinically normal mouth, attended at the University of Dentistry of Ribeirão Preto USP. Among the strains of C. albicans isolated from oral lesions, the phospholipase and proteinase were detected in 83.3% and 66.7%, respectively. C. tropicalis and C. parapsilosis produced only proteinase. Regarding the isolated strains from niches without lesions, out of a total of 32 C. albicans, 71.9% presented phospholipase and 68.7% proteinase. C. tropicalis only presented the enzyme proteinase, C. glabrata, C. krusei, C. guilliermondii and Candida spp did not present any of the exoenzymes. Among the samples of C. albicans from both groups, the enzymotype 22 (positive phospholipase and proteinase weakly positive), was prevalent. Different enzymotypes of the same species were detected in samples collected from the same patient.
Assuntos
Candida albicans/classificação , Candida albicans/enzimologia , Endopeptidases/biossíntese , Boca/microbiologia , Fosfolipases/biossíntese , Adulto , HumanosRESUMO
The influence of culture medium buffer capacity, the supplementation of culture medium with L-ala and the requirement of calcium for exoprotease production by Antarctic psychrotrophic Stenotrophomonas maltophilia strains ANT-1-1 and ANT-7-1 were examined. When increasing concentrations of calcium chloride (0 to 0.3 g l-1) were added to culture media, maximum protease production yields increased 70-75% (ANT-1-1) and 50% (ANT-7-1), while biomass levels showed little difference. Calcium was also necessary for optimal activity of proteases. L-ala had no effect on protease production. The reduction in buffer capacity, with the consequent change in external pH, had a positive effect, enhancing protease yields. Secretion of proteases into the medium started at the beginning of the stationary phase, corresponding with a rise in pH values up to pH 8.7 and was maximal at 36 h of culture. These results indicate that the regulation of calcium concentration and buffer capacity and also pH monitoring are factors to be considered when the design of an industrial culture medium and the optimisation of protease production processes using these Antarctic strains are concerned.
Assuntos
Endopeptidases/biossíntese , Stenotrophomonas maltophilia/enzimologia , Aminoácidos/metabolismo , Regiões Antárticas , Cálcio/metabolismo , Meios de Cultura , Concentração de Íons de HidrogênioRESUMO
Lipase, protease, and amylase production by Penicillium restrictum in solid-state fermentation was investigated. The basal medium was an industrial waste of babassu oil (Orbignya oleifera) production. It was enriched with peptone, olive oil, and Tween-80. The supplementation positively influenced both enzyme production and fungal growth. Media enriched with Tween-80 provided the highest protease activity (8.6 U/g), whereas those enriched with peptone and olive oil led to the highest lipase (27.8 U/g) and amylase (31.8 U/g) activities, respectively.
Assuntos
Resíduos Industriais , Lipase/biossíntese , Penicillium/crescimento & desenvolvimento , Óleos de Plantas , Gerenciamento de Resíduos/métodos , Amilases/biossíntese , Endopeptidases/biossíntese , Fermentação , Indústria Alimentícia , Azeite de Oliva , Penicillium/enzimologia , Peptonas , PolissorbatosRESUMO
The influence of culture medium buffer capacity, the supplementation of culture medium with L-ala and the requirement of calcium for exoprotease production by Antarctic psychrotrophic Stenotrophomonas maltophilia strains ANT-1-1 and ANT-7-1 were examined. When increasing concentrations of calcium chloride (0 to 0.3 g l-1) were added to culture media, maximum protease production yields increased 70-75 (ANT-1-1) and 50 (ANT-7-1), while biomass levels showed little difference. Calcium was also necessary for optimal activity of proteases. L-ala had no effect on protease production. The reduction in buffer capacity, with the consequent change in external pH, had a positive effect, enhancing protease yields. Secretion of proteases into the medium started at the beginning of the stationary phase, corresponding with a rise in pH values up to pH 8.7 and was maximal at 36 h of culture. These results indicate that the regulation of calcium concentration and buffer capacity and also pH monitoring are factors to be considered when the design of an industrial culture medium and the optimisation of protease production processes using these Antarctic strains are concerned.(AU)