Peptidase pode ser considerada, como uma subclasse das enzimas hidrolíticas que ocupam uma posição central em relação às suas aplicações na área fisiológica e também na área comercial. As peptidases representam um dos três maiores grupos de enzimas industriais e são responsáveis por cerca de 60% da venda mundial de enzimas. O presente trabalho visa avaliar a produção de peptidase em fermentação submersa (FSm) pelo fungo Scopulariopsis koningii, utilizando como substrato farinha de pena (FP), a caracterização bioquímica parcial, secagem do extrato bruto, purificação da peptidase e determinação de especificidade da peptidase isolada. Para avaliar a influência da farinha de pena (FP), na produção de peptidases, foram adicionadas às porcentagens de 0,2; 0,4 e 0,8% no meio de cultura. Os melhores níveis de produção de peptidases pelo fungo Scopulariopsis koningii foram obtidos nas concentrações de 0,4% e 0,8% de FP em 48h de fermentação com 1.427 U/mL. A caracterização bioquímica parcial do extrato bruto foi realizada com azocaseína 1% preparada em tampão com pH adequado. A peptidase presente no extrato enzimático apresentou atividade ótima em pH 6,5 e temperatura ótima de 55°C. A peptidase foi inibida por PMSF, indicando a presença de resíduo de aminoácido serino no sítio catalítco, e desta forma sendo classificada como serino peptidase. Entretanto, também observamos uma inibição por EDTA, sugerindo a presença de uma metalo peptidase presente no extrato bruto, desta forma podemos sugerir que o fungo S. koningii na presença do meio contendo FP secreta duas subclasses; serino e metalo peptidase, ou secreta uma serino peptidase dependente de íon. Zimograma constatou a presença de duas enzimas. A atividade enzimática do extrato bruto diminuiu significativamente quando exposta os íons Al+3, Ni2+ e Cu2+ e aumentada quando adicionados os íons Ba2+, Ca2+ e Mg2+. A purificação da metalo peptidase presente no extrato enzimático envolveu sete etapas de purificação, sendo cromatografia de troca-iônica e gel filtração determinantes, com recuperação de 6% e purificação de 3,4 vezes. Utilizando a peptidase pura (metalo) realizouse a caracterização bioquímica funcional e a determinação dos parâmetros cinéticos, ambos utilizando o substrato peptídico de supressão intramolecular de fluorescência. A peptidase pura apresentou atividade ótima em pH 6,0 e temperatura ótima de 40°C e mostrou-se estável em ampla faixa de pH e temperatura. Foi modulada positivamente pelos íons Na+ e K+ e negativamente por Al3+ e Cu2+. A análise dos parâmetros cinéticos revelou uma grande influência de aminoácidos apolares do lado \"linha\" do substrato sintético na eficiência catalítica e no lado não \"linha\" grande influência de aminoácidos polares neutros e apolares. A secagem foi realizada por liofilização foram utilizados três tipos de adjuvantes: maltodextrina, manitol e glicina, em diferentes concentrações. Após a secagem foi realizado estudo da estabilidade nas temperaturas 4, 25 e 60°C por 32 dias para avaliar o desempenho dos adjuvantes na manutenção da atividade do extrato enzimático liofilizado. O adjuvante considerado mais eficaz foi a maltodextrina na concentração de 4,5% que manteve cerca de 93% da atividade do extrato enzimático liofilizado por 32 dias. / Peptidase can be considered as a subclass of hydrolytic enzymes which occupy a central position in relation to their applications in physiological area and also in the commercial area. Peptidases represent one of the three largest groups of industrial enzymes and account for about 60% of worldwide sales of enzymes. This study aims to evaluate the production of peptidase in submerged fermentation (FSm) by the fungus Scopulariopsis koningii, using as substrate feather meal (FP), the partial biochemical characterization, drying the crude extract, purification and determination of specific peptidase peptidase isolated. To evaluate the influence of feather meal (FM), in the production of peptidases, were added to the percentages of 0.2, 0.4 and 0.8% in the culture medium. The best levels of production of peptidases by the fungus Scopulariopsis koningii were obtained at concentrations of 0.4% and 0.8% of FP 48h fermentation with 1,427 U/mL. Partial biochemical characterization of the crude extract was performed with 1% azocasein prepared in buffer with appropriate pH. This in peptidase enzyme extract showed optimal activity at pH 6.5 and optimum temperature of 55°C. The peptidase was inhibited by PMSF, indicating the presence of a serine residue at amino acid catalytic site, and thus being classified as serine peptidase. However, we also observed an inhibition by EDTA, suggesting the presence of a metallo peptidase present in the crude extract, thus we suggest that the fungus S. koningii in the presence of medium containing secret FP two subclasses; serine peptidase and metal, or secretes a serine peptidase dependent ion. Zymogram found the presence of two enzymes. The enzymatic activity of the crude extract decreased significantly when exposed the Al 3+, Ni 2+ and Cu2+ ions and increased when added to Ba2+, Ca2+ and Mg2+ ions. The purification of metallo peptidase present in the enzyme extract involved seven stages of purification, and ion-exchange chromatography and gel filtration determinants, with recovery and purification of 6 % from 3.4 times. Using pure peptidase (metallo) held functional biochemical characterization and determination of kinetic parameters using both the intramolecular peptide substrate fluorescence suppression. Pure peptidase showed optimal activity at pH 6.0 and optimum temperature of 40°C and was stable in a wide range of pH and temperature. The positively modulated by Na+ and K+ and negatively by Al3+ and Cu2+. Analysis of kinetic parameters revealed a strong influence of nonpolar amino acid side \"line\" of the synthetic substrate in the catalytic efficiency and not on the side \"line\" great influence of nonpolar and polar neutral amino acids. The freeze drying was performed by three types of additives were used: maltodextrin, mannitol and glycine in different concentrations. After drying stability study was conducted at the temperatures 4, 25 and 60°C for 32 days to evaluate the performance of additives in maintaining the activity of the enzyme extract lyophilized. The adjuvant was found more efficacious maltodextrin in a concentration of 4.5%, which retained about 93% of the extract lyophilized enzyme activity for 32 days.

Peptidase pode ser considerada, como uma subclasse das enzimas hidrolíticas que ocupam uma posição central em relação às suas aplicações na área fisiológica e também na área comercial. As peptidases representam um dos três maiores grupos de enzimas industriais e são responsáveis por cerca de 60% da venda mundial de enzimas. O presente trabalho visa avaliar a produção de peptidase em fermentação submersa (FSm) pelo fungo Scopulariopsis koningii, utilizando como substrato farinha de pena (FP), a caracterização bioquímica parcial, secagem do extrato bruto, purificação da peptidase e determinação de especificidade da peptidase isolada. Para avaliar a influência da farinha de pena (FP), na produção de peptidases, foram adicionadas às porcentagens de 0,2; 0,4 e 0,8% no meio de cultura. Os melhores níveis de produção de peptidases pelo fungo Scopulariopsis koningii foram obtidos nas concentrações de 0,4% e 0,8% de FP em 48h de fermentação com 1.427 U/mL. A caracterização bioquímica parcial do extrato bruto foi realizada com azocaseína 1% preparada em tampão com pH adequado. A peptidase presente no extrato enzimático apresentou atividade ótima em pH 6,5 e temperatura ótima de 55°C. A peptidase foi inibida por PMSF, indicando a presença de resíduo de aminoácido serino no sítio catalítco, e desta forma sendo classificada como serino peptidase. Entretanto, também observamos uma inibição por EDTA, sugerindo a presença de uma metalo peptidase presente no extrato bruto, desta forma podemos sugerir que o fungo S. koningii na presença do meio contendo FP secreta duas subclasses; serino e metalo peptidase, ou secreta uma serino peptidase dependente de íon. Zimograma constatou a presença de duas enzimas. A atividade enzimática do extrato bruto diminuiu significativamente quando exposta os íons Al+3, Ni2+ e Cu2+ e aumentada quando adicionados os íons Ba2+, Ca2+ e Mg2+. A purificação da metalo peptidase presente no extrato enzimático envolveu sete etapas de purificação, sendo cromatografia de troca-iônica e gel filtração determinantes, com recuperação de 6% e purificação de 3,4 vezes. Utilizando a peptidase pura (metalo) realizouse a caracterização bioquímica funcional e a determinação dos parâmetros cinéticos, ambos utilizando o substrato peptídico de supressão intramolecular de fluorescência. A peptidase pura apresentou atividade ótima em pH 6,0 e temperatura ótima de 40°C e mostrou-se estável em ampla faixa de pH e temperatura. Foi modulada positivamente pelos íons Na+ e K+ e negativamente por Al3+ e Cu2+. A análise dos parâmetros cinéticos revelou uma grande influência de aminoácidos apolares do lado \"linha\" do substrato sintético na eficiência catalítica e no lado não \"linha\" grande influência de aminoácidos polares neutros e apolares. A secagem foi realizada por liofilização foram utilizados três tipos de adjuvantes: maltodextrina, manitol e glicina, em diferentes concentrações. Após a secagem foi realizado estudo da estabilidade nas temperaturas 4, 25 e 60°C por 32 dias para avaliar o desempenho dos adjuvantes na manutenção da atividade do extrato enzimático liofilizado. O adjuvante considerado mais eficaz foi a maltodextrina na concentração de 4,5% que manteve cerca de 93% da atividade do extrato enzimático liofilizado por 32 dias. / Peptidase can be considered as a subclass of hydrolytic enzymes which occupy a central position in relation to their applications in physiological area and also in the commercial area. Peptidases represent one of the three largest groups of industrial enzymes and account for about 60% of worldwide sales of enzymes. This study aims to evaluate the production of peptidase in submerged fermentation (FSm) by the fungus Scopulariopsis koningii, using as substrate feather meal (FP), the partial biochemical characterization, drying the crude extract, purification and determination of specific peptidase peptidase isolated. To evaluate the influence of feather meal (FM), in the production of peptidases, were added to the percentages of 0.2, 0.4 and 0.8% in the culture medium. The best levels of production of peptidases by the fungus Scopulariopsis koningii were obtained at concentrations of 0.4% and 0.8% of FP 48h fermentation with 1,427 U/mL. Partial biochemical characterization of the crude extract was performed with 1% azocasein prepared in buffer with appropriate pH. This in peptidase enzyme extract showed optimal activity at pH 6.5 and optimum temperature of 55°C. The peptidase was inhibited by PMSF, indicating the presence of a serine residue at amino acid catalytic site, and thus being classified as serine peptidase. However, we also observed an inhibition by EDTA, suggesting the presence of a metallo peptidase present in the crude extract, thus we suggest that the fungus S. koningii in the presence of medium containing secret FP two subclasses; serine peptidase and metal, or secretes a serine peptidase dependent ion. Zymogram found the presence of two enzymes. The enzymatic activity of the crude extract decreased significantly when exposed the Al 3+, Ni 2+ and Cu2+ ions and increased when added to Ba2+, Ca2+ and Mg2+ ions. The purification of metallo peptidase present in the enzyme extract involved seven stages of purification, and ion-exchange chromatography and gel filtration determinants, with recovery and purification of 6 % from 3.4 times. Using pure peptidase (metallo) held functional biochemical characterization and determination of kinetic parameters using both the intramolecular peptide substrate fluorescence suppression. Pure peptidase showed optimal activity at pH 6.0 and optimum temperature of 40°C and was stable in a wide range of pH and temperature. The positively modulated by Na+ and K+ and negatively by Al3+ and Cu2+. Analysis of kinetic parameters revealed a strong influence of nonpolar amino acid side \"line\" of the synthetic substrate in the catalytic efficiency and not on the side \"line\" great influence of nonpolar and polar neutral amino acids. The freeze drying was performed by three types of additives were used: maltodextrin, mannitol and glycine in different concentrations. After drying stability study was conducted at the temperatures 4, 25 and 60°C for 32 days to evaluate the performance of additives in maintaining the activity of the enzyme extract lyophilized. The adjuvant was found more efficacious maltodextrin in a concentration of 4.5%, which retained about 93% of the extract lyophilized enzyme activity for 32 days.

Preliminary experiments by API ZYM enzyme system showed that Lactobacillus casei (Lb. casei) subspecies contained low proteinase and high aminopeptidase and esterase-lipase activities, which are the desirable traits of microorganisms to be used as starter adjuncts in Cheddar cheese-making. Six strains of Lb. casei (ssp. casei, ssp. rhamnosus, and ssp. pseudoplantarum), selected from superior peptidase and esterase-lipase profiles, were further studied for their amino-, di-, and carboxy-peptidase activities using thirty synthetic substrates. This study revealed useful information towards improving our understanding of the peptidase profiles and probable role of Lb. casei in Cheddar cheese ripening. Although individual strains varied in their specific activities against different substrates, Lactobacillus subspecies generally exhibited high amino- and di-peptidase, relatively weak tripeptidase, but no carboxypeptidase activities. The knowledge gained from these studies helped us selecting two strains (Lb. casei ssp. casei LLG and Lb. casei ssp. rhamnosus S93) with highest amino- and di-peptidase activities for further research. In order to study their enzymatic characteristics and kinetics, aminopeptidase of these two strains were purified to homogeneity by Fast Protein Liquid Chromatography (FPLC). A single monomeric enzyme was shown to be responsible for the entire aminopeptidase activity of the cell-free extracts. This investigation provided new insights and revealed fundamental knowledge about the peptidases of Lb. casei group. In addition, new methodologies were developed for rapid enzyme purification using FPLC system, and evaluation of peptidases by API ZYM enzyme system.

No description available.

The objectives of this study were (l) to screen out active proline-specific peptidases from Lactobacillus casei subspecies, (2) to study growth kinetic and enzyme production from enriched medium (MRS) and cheese whey medium, (3) to purify and characterize two active proline-specific enzymes, and (4) to investigate the action of purified enzyme on bitter tryptic digests of $ beta$-casein as well as bitter enzyme-modified cheese. Lactobacillus casei subsp. casei LLG and Lactobacillus casei subsp. rhamnosus S93 were examined for extra- and intra-cellular proline-specific peptidase activities. Both strains showed strong activity for x-prolyl dipeptidyl peptidase and proline iminopeptidase but had weak activities for prolidase, prolinase, and post proline endopeptidase. Histochemical staining of crude enzyme extract from Lactobacillus casei ssp. casei LLG with different substrates revealed a distinct protein band for x-prolyl dipeptidyl peptidase as well as for proline iminopeptidase. The growth kinetics showed that the intracellular proline-specific peptidases increased gradually at the beginning of the exponential phase and reached a maximum at the beginning of stationary phase. / Storage stability of x-prolyl dipeptidyl peptidase and proline iminopeptidase in crude extract, with and without stabilizers showed no significant loss in activity of these two enzymes at 4$ sp circ$C for 9 days without adding any stabilizers. The levels of x-prolyl dipeptidyl peptidase, proline iminopeptidase, and post proline endopeptidase activities of cells grown in whey did not vary markedly from cells grown in MRS broth. X-prolyl dipeptidyl peptidase and proline iminopeptidase were purified from crude cell-free extract of Lactobacillus casei ssp. casei LLG by Fast Protein Liquid Chromatography (FPLC) equipped with ion-exchange and gel-filtration columns. X-prolyl dipeptidyl peptidase was found to be a serine-dependent enzyme with molecular mass of 79 kDa. The pH and the temperature optima by the purified enzyme were 7.0 and 50$ sp circ$C, respectively. Proline iminopeptidase was sulfhydryl enzyme with molecular mass of 46 kDa. The maximum enzyme activity was observed at pH 7.5 and 40$ sp circ$C. This is the first report describing the purification and characterization of x-prolyl dipeptidyl peptidase and proline iminopeptidase from Lactobacillus casei to homogeneity. / The debittering of tryptic digests from $ beta$-casein by x-prolyl dipeptidyl peptidase was studied by reversed phase high performance liquid chromatography (RP-HPLC) and liquid chromatography/mass spectrometry. The results showed that two bitter peptides (f53-97 and f03-209) containing X-Pro-Y-Pro in their amino acid residues were completely hydrolyzed and many other peptides with high hydrophobicity were decreased in peak area. The addition of purified x-prolyl dipeptidyl peptidase on bitter enzyme-modified cheese (EMC) also showed that at least one bitter peptide with X-Pro-Y derived from $ alpha$-casein hydrolysis was removed.

No description available.

No description available.

The cytosolic protein degradation pathway, performed by ATP-dependent proteases and ATP-independent peptidases, plays important roles in several cellular activities, e.g. cell division, cell cycle progression, intracellular signaling, MHC class I antigen presentation, host-pathogen interactions, etc. The roles of ATP-dependent proteases during stress and infection have been studied in great detail but the functional roles of ATP-independent peptidases are not clearly understood. In this study, the functional roles of E. coli or S. typhimurium encoded Peptidase N (PepN), an ATP-independent enzyme belonging to theM1 family of metallopeptidases, were investigated. The thesis will address four different aspects. (i) In the first part, the utility of using E coli ∆pepN to identify and characterize novel peptidases will be shown. It is known that deletion of pepN leads to inability to cleave the majority of in vitro peptidase substrates in E. coli and S. typhimurium. To study the differences between two closely related paralogs of the M17 family, E. coli encoded pepA and pepB were cloned in pBAD24 vector and introduced in E. coli ∆pepN. Peptidase A (PepA) and Peptidase B (PepB) expression increases the cleavage of several aminopeptidase substrates and partially rescues growth of ∆pepN during nutritional downshift and high temperature stress (NDHT), a dual stress involving growth in minimal media at 42°C. Purified PepA and PepB enzymes display broad substrate specificity; however, distinct differences are observed between these two paralogs: PepA is more stable at high temperature whereas PepB displays broader substrate specificity as it cleaves Asp and Insulin B chain peptide. The strategy utilized in this study, i.e. overexpression of peptidases in ∆pepN followed by screening for substrate specificities in total cell extracts, may be used to rapidly identify the substrate preferences of novel peptidases encoded in genomes of different organisms. (ii) The second aspect investigates the functional roles of PepN during stress and infection in S. typhimurium. PepN has two conserved signature motifs of the M1 family, GAMEN and HEXXH, which play roles in substrate recognition and catalysis. To address the roles of catalytic activity of PepN, the residue E-298, which is present in the HEXXH motif and acts as a general base during catalysis, was mutated to A-298 by site-specific mutagenesis and introduced into ∆pepN (pBR322/pepNE298A). Biochemical and biophysical analysis of purified PepN (WT and E298A) revealed loss of catalytic activity of E298A but no major structural changes were observed in comparison to the WT protein. The functional roles of this mutation using ∆pepN expressing pBR322/pepN or pBR322/pepNE298A were investigated using two conditions: (i) Nutritional downshift high temperature (NDHT)stress and (ii) systemic infection in mice. Monitoring growth profiles of different strains demonstrated the requirement of the enzymatic activity of PepN for adaptation and growth to NDHT stress. Earlier studies have shown that S. typhimurium ∆pepN hyper proliferates in peripheral organs during systemic infection in mice. However, expression of wild type (WT)or E298A PepN led to lower colony forming units (CFU), demonstrating that the decrease in CFU is independent of catalytic activity. These observations are consistent with lower serum amounts of inflammatory cytokines, lower tissue damage and increase in survival of mice infected with S. typhimurium expressing WT or E298A PepN. (iii) Although pathogen encoded peptidases are known to be important during infection, their roles in modulating host responses in immunocompromised individuals are not well studied. In the third part of this thesis, the roles of S. typhimurium encoded PepN were studied in mice lacking Interferon-γ (Ifnγ), a cytokine important for immunity. S. typhimurium lacking pepN displays enhanced CFU compared to WT in peripheral organs during systemic infection in C57BL/6 mice. However, Ifnγ-/-mice show higher CFU compared to C57BL/6 mice, resulting in lower fold differences between WT and ∆pepN. Concomitantly, reintroduction of pepN in ∆pepN reduces CFU, demonstrating pepN dependence. In addition, three distinct differences were observed between infection ofC57BL/6 and Ifnγ-/-mice upon infection with different S. typhimurium strains: (i) cytokine profiles, (ii) histological analysis and (iii) mice survival. Overall, the roles of the host encoded Ifnγ during infection with S. typhimurium strains with varying degrees of virulence will be highlighted. (iv) The final aspect of this study reveals differences in gene expression between S. typhimurium grown in rich medium (Luria-Bertani) versus NDHT stress. This adaptation affects several pathways and the gene expression of secretory proteins that are important for virulence in S. typhimurium are greatly reduced during NDHT stress. Also, analysis of secretory protein amounts in different media conditions shows reduction during growth in minimal media plus high temperature stress. The functional consequences of this reduction in secretory protein amounts lead to lower bacterial replication after infection of RAW cells or mice infected via the oral route. In addition, the differences in gene expression between WT and ∆pepN during these conditions were studied. Interestingly, there is reduction in expression of flagellar genes whereas the genes involved in nitrogen metabolism are upregulated in ∆pepN upon exposure to NDHT stress. Further studies were performed by quantifying the motility of different S. typhimurium strains grown in a variety of culture conditions. Overall, this part of the study attempts to compare and contrast the possible adaptive responses of WT and ∆pepN to NDHT stress. Together, this thesis addresses multiple aspects of the biochemistry and roles of the enigmatic PepN during stress and infection.

No description available.

No description available.