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1.
Food Chem ; 306: 125581, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31606636

RESUMO

A comprehensive evaluation was conducted to compare the generation of antioxidative peptides produced by alcalase versus trypsin from Atlantic sea cucumber. The in vitro antioxidative peptides were sequenced by de novo sequencing using LC-MS/MS. Key constituent antioxidative amino acids (KCAAA), i.e., Cys, His, Met, Trp and Tyr in the peptides and the molecular interactions between peptides and myeloperoxidase (MPO, a mediator and marker of in vivo oxidative stress), were analyzed by in silico methods. Alcalase-produced protein hydrolysates showed 5-35% higher in vitro antioxidant activity than the trypsin-produced ones. UPLC analysis revealed the total amino acid composition in peptide fractions <2 kDa. Alcalase produced 35.4% of peptides with both KCAAA and potential MPO inhibitory activity, compared with only 30.3% for trypsin. A representative peptide sequence TEFHLL generated by alcalase had intense molecular interactions with MPO active site, predicting a capacity to inhibit in vivo oxidative stress.


Assuntos
Antioxidantes/metabolismo , Peptídeos/metabolismo , Pepinos-do-Mar/metabolismo , Subtilisinas/metabolismo , Tripsina/metabolismo , Animais , Cromatografia Líquida , Estresse Oxidativo , Espectrometria de Massas em Tandem
2.
Food Chem ; 311: 125960, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31862569

RESUMO

This study aimed to evaluate the necessity of enzymatic hydrolysis for walnut peptide preparation based on a novel evaluation approach. Defatted walnut meal hydrolysate (DWMH) was prepared by hydrolyzing defatted walnut meal (DWM) with alcalase, and gastrointestinal digestion of DWM and DWMH was simulated in vitro using pepsin and pancreatin. The peptide and free amino acid (FAA) contents, angiotensin-I-converting enzyme (ACE) inhibitory activity, 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity and molecular weight distributions of DWM, DWMH and their gastric and gastrointestinal digestive fluids were compared. Results showed that DWM could be well digested. High peptide content (21.66 mg/mL) with MW < 3000 Da and more FAAs (8.09 mg/mL) were observed in DWM digests. In addition, DWM digests had high ACE inhibitory activity (42.9%) and DPPH radical-scavenging activity (62.58%), which showed no significant difference when compared with DWMH digests. The above results indicate that enzymatic hydrolysis seems unnecessary for the production of walnut peptides; at the least, hydrolysis with alcalase was unnecessary for producing peptides with significant ACE inhibitory and DPPH radical-scavenging activities.


Assuntos
Depuradores de Radicais Livres/química , Juglans/metabolismo , Peptidil Dipeptidase A/metabolismo , Juglans/química , Nozes/química , Nozes/metabolismo , Pancreatina/metabolismo , Pepsina A/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Peptidil Dipeptidase A/química , Proteólise , Solubilidade , Subtilisinas/metabolismo
3.
Nutrients ; 11(9)2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31509959

RESUMO

Being averse to bitter taste is a common phenomenon for humans and other animals, which requires the pharmaceutical and food industries to source compounds that can block bitterness intensity and increase consumer acceptability. In this work, beef protein alcalase hydrolysates (BPAH) and chymotrypsin hydrolysates (BPCH) were reacted with glucose to initiate Maillard reactions that led to the formation of glycated or advanced glycation end products (AGEs), BPAH-AGEs and BPCH-AGEs, respectively. The degree of glycation was higher for the BPAH-AGEs (47-55%) than the BPCH-AGEs (30-38%). Analysis by an electronic tongue instrument showed that BPAH-AGEs and BPCH-AGEs had bitterness scores that were significantly (p < 0.05) less than quinine. The addition of BPAH-AGEs or BPCH-AGEs to quinine led to significant (p < 0.05) reductions (up to 38%) in bitterness intensity of quinine. The use of 3% hydrolysate to react with glucose yielded glycated peptides with a stronger ability to reduce quinine bitterness than when 1% was used. Calcium release from HEK293T cells stably expressing the T2R4 human bitter taste receptor was significantly (p < 0.05) attenuated by BPAH-AGEs (up to 96%) and BPCH-AGEs (up to 92%) when compared to the BPAH (62%) and BPCH (3%) or quinine (0%). We concluded that BPAH-AGEs and BPCH-AGEs may be used as bitter taste blockers to formulate better tasting foods.


Assuntos
Aromatizantes/farmacologia , Produtos Finais de Glicação Avançada/farmacologia , Proteínas de Carne/farmacologia , Hidrolisados de Proteína/farmacologia , Paladar/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Quimotripsina , Nariz Eletrônico , Aromatizantes/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Glicosilação , Células HEK293 , Humanos , Reação de Maillard , Proteínas de Carne/metabolismo , Hidrolisados de Proteína/metabolismo , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/metabolismo , Subtilisinas/metabolismo
4.
J Ind Microbiol Biotechnol ; 46(12): 1745-1755, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31471782

RESUMO

Cell surface engineering was proven as the efficient strategy for enhanced production of target metabolites. In this study, we want to improve the yield of target protein by engineering cell surface in Bacillus licheniformis. First, our results confirmed that deletions of D-alanyl-lipoteichoic acid synthetase gene dltD, cardiolipin synthase gene clsA and CDP-diacylglycerol-serine O-phosphatidyltransferase gene pssA were not conducive to cell growth, and the biomass of gene deletion strains were, respectively, decreased by 10.54 ± 1.43%, 14.17 ± 1.51%, and 17.55 ± 1.28%, while the concentrations of total extracellular proteins were improved, due to the increases of cell surface net negative charge and cell membrane permeability. In addition, the activities of target proteins, nattokinase, and α-amylase were also improved significantly in gene deletion strains. Furthermore, the triplicate gene (dltD, clsA, and pssA) deletion strain was constructed, which further led to the 45.71 ± 2.43% increase of cell surface net negative charge and 26.45 ± 2.31% increase of cell membrane permeability, and the activities of nattokinase and α-amylase reached 37.15 ± 0.89 FU/mL and 305.3 ± 8.4 U/mL, increased by 46.09 ± 3.51% and 96.34 ± 7.24%, respectively. Taken together, our results confirmed that cell surface engineering via deleting dltD, clsA, and pssA is an efficient strategy for enhanced production of target proteins, and this research provided a promising host strain of B. licheniformis for efficient protein expression.


Assuntos
Bacillus licheniformis/enzimologia , Bacillus licheniformis/genética , Membrana Celular/enzimologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Engenharia de Proteínas , Subtilisinas/genética , Subtilisinas/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , alfa-Amilases/genética , alfa-Amilases/metabolismo
5.
Appl Microbiol Biotechnol ; 103(18): 7519-7535, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31378837

RESUMO

Bacillus subtilis natto is a GRAS bacterium. Nattokinase, with fibrinolytic and antithrombotic activities, is one of the major products of this organism. It is being gradually recognized that B. subtilis natto can also be used as a biosynthetic strain for vitamin K2, which has phenomenal benefits, such as effects in the prevention of cardiovascular diseases and osteoporosis along with antitumor effects. Knocking out of the aprN gene by homologous recombination could improve the redox potential and slightly increase the concentration of MK-7. By detecting the change in redox potential during the growth of B. subtilis natto, a good oxygen supply and state of the cell membrane were found to be beneficial to vitamin K2 synthesis. A two-step RSM was used to optimize the operation parameters and substrate concentration in the new residue-free fermentation culture. The optimal conditions for the residue-free medium and control were determined. The optimum concentrations of soybean flour, corn flour, and peptone were 78.9, 72.4, and 24.8 g/L, respectively. The optimum rotational speed and volume of the culture medium using a shaking flask were 117 rpm and 10%, respectively. The state and composition of the cell membranes were more stable when engineered bacteria were cultured in this residue-free fermentation medium. Finally, the concentration of MK-7 increased by 37% to 18.9 mg/L, and the fermentation time was shortened by 24 h.


Assuntos
Bacillus subtilis/enzimologia , Fermentação , Oxirredução , Alimentos de Soja/microbiologia , Vitamina K 2/análogos & derivados , Bacillus subtilis/genética , Biofilmes/crescimento & desenvolvimento , Reatores Biológicos/microbiologia , Meios de Cultura/química , Recombinação Homóloga , Microbiologia Industrial , Subtilisinas/metabolismo , Vitamina K 2/metabolismo
6.
Molecules ; 24(17)2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31438557

RESUMO

Amaranthus hypochondriacus spp. is a commonly grown cereal in Latin America, known for its high protein content. The objective of this study was to separate and identify bioactive peptides found in amaranth seeds through enzymatically-assisted hydrolysis using alcalase and flavourzyme. Hydrolysis was carried out for each enzyme separately and compared to two-step continuous process where both enzymes were combined. The biological activity of the resulting three hydrolysates was analyzed, finding, in general, higher bioactive potential of the hydrolysate obtained in a continuous process (combined enzymes). Its fractions were separated by RP-HPLC, and their bioactivity was analyzed. In particular, two fractions showed the highest biological activity as ACE inhibitors with IC50 at 0.158 and 0.134, thrombin inhibitors with IC50 of 167 and 155, and antioxidants in ABTS assay with SC50 at 1.375 and 0.992 mg/L, respectively. Further sequence analysis of the bioactive peptides was carried out using MALDI-TOF, which identified amino acid chains that have not been reported as bioactive so far. Bibliographic survey allowed identification of similarities between peptides reported in amaranth and other proteins. In conclusion, amaranth proteins are a potential source of peptides with multifunctional activity.


Assuntos
Amaranthus/química , Peptídeos/química , Proteínas de Plantas/química , Sementes/química , Endopeptidases/metabolismo , Subtilisinas/metabolismo
7.
Mar Drugs ; 17(4)2019 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-30987249

RESUMO

Seaweeds, which have been widely used for human consumption, are considered a potential source of biological compounds, where enzyme-assisted extraction can be an efficient method to obtain multifunctional extracts. Chemical characterization of Sargassum muticum and Osmundea pinnatifida extracts obtained by Alcalase and Viscozyme assisted extraction, respectively, showed an increment of macro/micro elements in comparison to the corresponding dry seaweeds, while the ratio of Na/K decreased in both extracts. Galactose, mannose, xylose, fucose, and glucuronic acid were the main monosaccharides (3.2-27.3 mg/glyophilized extract) present in variable molar ratios, whereas low free amino acids content and diversity (1.4-2.7 g/100gprotein) characterized both extracts. FTIR-ATR and 1H NMR spectra confirmed the presence of important polysaccharide structures in the extracts, namely fucoidans from S. muticum or agarans as sulfated polysaccharides from O. pinnatifida. No cytotoxicity against normal mammalian cells was observed from 0 to 4 mglyophilized extract/mL for both extracts. The comprehensive characterization of the composition and safety of these two extracts fulfils an important step towards their authorized application for nutritional and/or nutraceutical purposes.


Assuntos
Suplementos Nutricionais , Extratos Vegetais/química , Rodófitas/química , Sargassum/química , Alga Marinha/química , Animais , Linhagem Celular , Fibroblastos , Camundongos , Estrutura Molecular , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/toxicidade , Polissacarídeos/química , Polissacarídeos/isolamento & purificação , Polissacarídeos/toxicidade , Espectroscopia de Prótons por Ressonância Magnética , Espectroscopia de Infravermelho com Transformada de Fourier , Subtilisinas/metabolismo , Testes de Toxicidade
8.
Nutrients ; 11(4)2019 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-30987324

RESUMO

Alcalase- generated potato protein hydrolysate (APPH) is a potential bioactive peptide against diabetes mellitus (DM) and DM-associated secondary effects in animal models. The aim of the present study was to find the efficiency of a deca-peptide DIKTNKPVIF (DF) from APPH against DM. Six-week-old male ICR mice were divided into the following groups: Control, Control+DF (received 50 mg/kg DF), streptozotocin (STZ)-induced DM group, DM+Acarbose group (20 mg/kg of acarbose), DM+DF-L (25 mg/kg of DF), DM+DF-H (50 mg/kg of DF), and DM+APPH (50 mg/kg of APPH). Comparable to APPH, treatment with DF effectively regulated blood glucose level and also controlled plasma total glycerol (TG), total cholesterol (TC), insulin, and HbA1c levels in DM animals. DF treatment also showed evidence of ameliorating DM-associated damages in the pancreatic islets and in the liver, heart, and kidney tissues. Therefore, the results demonstrate that the short synthetic peptide-DF may effectively provide protection against DM-associated damages.


Assuntos
Glicemia/efeitos dos fármacos , Diabetes Mellitus Experimental/prevenção & controle , Hipoglicemiantes/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Oligopeptídeos/farmacologia , Proteínas de Plantas/metabolismo , Hidrolisados de Proteína/metabolismo , Solanum tuberosum/metabolismo , Animais , Biomarcadores/sangue , Glicemia/metabolismo , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/patologia , Hemoglobina A Glicada/metabolismo , Hipoglicemiantes/metabolismo , Insulina/sangue , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Lipídeos/sangue , Masculino , Camundongos Endogâmicos ICR , Oligopeptídeos/metabolismo , Estreptozocina , Subtilisinas/metabolismo
9.
Appl Microbiol Biotechnol ; 103(12): 4789-4799, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31025072

RESUMO

Soybean meal is commonly applied as the raw material in the bio-fermentation industry, and bacitracin is a widely used feed additive in the feed industry. In this study, we investigated the influence of subtilisin enhancement on soybean meal utilization and bacitracin production in Bacillus licheniformis DW2, an industrial strain for bacitracin production. Firstly, blocking sRNA aprA expression benefited bacitracin synthesis, and the bacitracin yield produced by aprA-deficient strain DW2△PaprA reached 931.43 U/mL, 18.92% higher than that of DW2 (783.25 U/mL). The bacitracin yield was reduced by 14.27% in the aprA overexpression strain. Furthermore, our results showed that deficiency of aprA led to a 6.54-fold increase of the aprE transcriptional level and a 1.84-fold increase of subtilisin activity, respectively, which led to the increases of soybean meal utilization rate (28.86%) and precursor amino acid supplies for bacitracin synthesis. Additionally, strengthening the utilization rate of soybean meal also benefited heterologous protein production, and the α-amylase and nattokinase activities were respectively enhanced by 59.81% and 50.53% in aprA-deficient strains. Collectively, this research demonstrated that strengthening subtilisin production could improve the utilization rate of soybean meal and thereby enhance bacitracin and target protein production; also, this strategy would be useful for the improvement of protein/peptide production using soybean meal as the main nitrogen source in the fermentation process.


Assuntos
Bacillus licheniformis/metabolismo , Bacitracina/biossíntese , Fermentação , Soja , Subtilisina/genética , Bacillus licheniformis/genética , Proteínas de Bactérias/genética , Microbiologia Industrial , Interferência de RNA , Subtilisinas/metabolismo , alfa-Amilases/metabolismo
10.
Molecules ; 24(6)2019 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-30897764

RESUMO

To investigate methods for improving the processing of porcine waste, porcine skin was hydrolyzed using different commercially available proteases (Alcalase, Flavorzyme, Neutrase, Bromeline, Protamex, and Papain) under several optimal conditions. Following enzymatic hydrolysis, the collagen hydrolysates (CHs) were fractionated by molecular weight (3 kDa) via membrane ultrafiltration. The CHs were analyzed for physical properties (pH, protein recovery, free amino group content, molecular weight distribution, and amino composition) as well as for functional properties (antioxidant activities and anti-aging activities). Among the CHs, CHs hydrolyzed by Alcalase (CH-Alcalase) exhibited the highest degree of hydrolysis compared to other CHs. Both "CH-Alcalase" and "CH-Alcalase < 3 kDa" fractions showed a considerably high antioxidant activity and collagenase inhibition activity. Therefore, resulting bioactives have potential for development as antioxidants and anti-aging ingredients in the food, cosmetics, and pharmaceuticals, from animal by-products.


Assuntos
Colágeno/metabolismo , Peptídeo Hidrolases/metabolismo , Animais , Antioxidantes , Eletroforese em Gel de Poliacrilamida , Hidrólise , Metaloendopeptidases/metabolismo , Papaína/metabolismo , Subtilisinas/metabolismo , Suínos
11.
Plant Foods Hum Nutr ; 74(2): 225-231, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30912008

RESUMO

Effects of ultrasonication, boiling, steaming, microwaving and autoclaving pretreatments on the production of sweet potato protein hydrolysates (SPPH) by single and combined Alcalase (ALC) and Protease (PRO) were investigated, as well as antioxidant activities of SPPH subjected to in vitro gastrointestinal digestion (GID). All pretreatments significantly increased the degree of hydrolysis (DH) and antioxidant activities of SPPH by ALC, PRO and ALC + PRO in the order of autoclaving > steaming, microwaving, boiling > ultrasonication (P < 0.05). GID significantly enhanced antioxidant activities and increased MW <3 kDa peptide fraction contents of all SPPH. Diverse peptides were identified as sporamin A, A precursor and sporamin B before and after GID from LC-QTOF-MS/MS analysis. Peptides with higher antioxidant amino acids of Trp, Tyr, Met, Cys, His and Phe were found after GID. There is a great potential application of SPPH as a novel food ingredient as a natural antioxidant.


Assuntos
Antioxidantes/metabolismo , Ipomoea batatas/metabolismo , Peptídeos/metabolismo , Proteínas de Plantas/metabolismo , Hidrolisados de Proteína/metabolismo , Precursores de Proteínas/metabolismo , Aminoácidos/metabolismo , Digestão , Hidrólise , Peptídeo Hidrolases/metabolismo , Subtilisinas/metabolismo
12.
Plant Physiol Biochem ; 139: 197-206, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30908971

RESUMO

Proteases play a main role in the mobilization of storage proteins during seed germination. Until today, there is little information about the involvement of serine proteases, particularly subtilases, in the germination of barley grains. The aims of the present work were to study the contribution of serine proteases to the total proteolytic activity induced during germination of barley grains and evaluate the specific involvement of subtilases in this process. Proteolytic activity assayed against azocasein in the presence of specific inhibitors, showed that serine proteases contributed between 10 and 20% of total activity along germination. Subtilase activity increased from day 1 after imbibition with a peak between days 4-5. Moreover, in vivo determination of subtilase activity in germinating grains revealed increasing activity along germination mainly localized in the seed endosperm and developing rootlets. Finally, the expression of 19 barley genes encoding subtilases was measured by real time PCR during germination. Three of the analyzed genes increased their expression along germination, five showed a transient induction, one was down-regulated, nine remained unchanged and one was not expressed. The present work demonstrates the involvement of subtilases in germination of barley grains and describes the positive association of eight subtilase genes to this process.


Assuntos
Germinação , Hordeum/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Plântula/crescimento & desenvolvimento , Subtilisinas/metabolismo , Aminoácidos/metabolismo , Regulação da Expressão Gênica de Plantas , Hordeum/enzimologia , Hordeum/metabolismo , Proteólise , Reação em Cadeia da Polimerase em Tempo Real , Plântula/enzimologia , Plântula/metabolismo
13.
Food Chem ; 283: 637-645, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30722922

RESUMO

Significant amount of bran is discarded from sesame processing plants and yet it is seen as waste or animal feed. This study for the first time designed to recover protein and antioxidant compounds from sesame bran. In this respect, effectiveness of four different techniques i.e. viscozyme L, alcalase, ultrasound and ultrasound-assisted enzymatic extractions were tested and compared with standard alkaline method. RSM was used to investigate the effects of extraction parameters and to determine optimum process conditions. All of the independent parameters (enzyme concentrations, pH, ultrasound power, temperature and time) had significant effects on all of the responses. Alcalase exhibited higher recovery efficiency than viscozyme L. The highest protein yield, total phenolic compound and antioxidant capacities were found in ultrasound-assisted enzymatic extraction at 836 W ultrasound power, 43 °C, 98 min, 9.8 pH value and 1.248 AU/100 g enzyme concentration. SDS-PAGE and SEM analyses were also carried out to compare extraction techniques.


Assuntos
Antioxidantes/química , Extratos Vegetais/química , Proteínas de Plantas/química , Sesamum/química , Subtilisinas/metabolismo , Antioxidantes/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Microscopia Eletrônica de Varredura , Fenóis/química , Fenóis/isolamento & purificação , Proteínas de Plantas/isolamento & purificação , Sesamum/metabolismo , Sonicação , Temperatura
14.
Food Chem ; 285: 266-274, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30797344

RESUMO

The present study demonstrated the hydrolysis of peony seed protein isolate (PSPI) by using alcalase and resulted in the generation of an anti-oxidative peptide. In brief, a model was used to illustrate the enzymolysis of PSPI with the determination of kinetic factors as per investigation information. The model proved suitable to explain the PSPI hydrolysis by alcalase. A novel anti-oxidative peptide was obtained successfully by ultrafiltration and a series of chromatography techniques. Subsequently, a purified fragment was identified with the amino acid sequence of SMRKPPG followed by its synthesis and evaluation of its anti-oxidative activities. After hydrolysis, the peony seed protein hydrolysate (PSPH) with the degree of hydrolysis of 18% displayed the most significant antioxidant action which was further used to isolate the anti-oxidative peptide.


Assuntos
Antioxidantes/farmacologia , Paeonia/química , Peptídeos/isolamento & purificação , Hidrolisados de Proteína/farmacologia , Sementes/química , Sequência de Aminoácidos , Antioxidantes/química , Antioxidantes/isolamento & purificação , Hidrólise , Cinética , Modelos Teóricos , Peptídeos/química , Peptídeos/farmacologia , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Hidrolisados de Proteína/análise , Subtilisinas/química , Subtilisinas/metabolismo , Ultrafiltração
15.
Proc Natl Acad Sci U S A ; 116(4): 1279-1288, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30610172

RESUMO

The protein acyl transferase ZDHHC5 was recently proposed to regulate trafficking in the endocytic pathway. Therefore, we explored the function of this enzyme in controlling the action of bacterial toxins. We found that ZDHHC5 activity is required for two very different toxins: the anthrax lethal toxin and the pore-forming toxin aerolysin. Both of these toxins have precursor forms, the protoxins, which can use the proprotein convertases Furin and PC7 for activation. We show that ZDHHC5 indeed affects the processing of the protoxins to their active forms. We found that Furin and PC7 can both be S-palmitoylated and are substrates of ZDHHC5. The impact of ZDHHC5 on Furin/PC7-mediated anthrax toxin cleavage is dual, having an indirect and a direct component. First, ZDHHC5 affects the homeostasis and trafficking of a subset of cellular proteins, including Furin and PC7, presumably by affecting the endocytic/recycling pathway. Second, while not inhibiting the protease activity per se, ZDHHC5-mediated Furin/PC7 palmitoylation is required for the cleavage of the anthrax toxin. Finally, we show that palmitoylation of Furin and PC7 promotes their association with plasma membrane microdomains. Both the receptor-bound toxin and the convertases are of very low abundance at the cell surface. Their encounter is unlikely on reasonable time scales. This work indicates that palmitoylation drives their encounter in specific domains, allowing processing and thereby intoxication of the cell.


Assuntos
Acetiltransferases/metabolismo , Antígenos de Bactérias/metabolismo , Bacillus anthracis/metabolismo , Toxinas Bacterianas/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Transporte Proteico/fisiologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Endocitose/fisiologia , Furina/metabolismo , Células HeLa , Humanos , Microdomínios da Membrana/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Pró-Proteína Convertases/metabolismo , Subtilisinas/metabolismo
16.
J Biol Chem ; 294(13): 4806-4814, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30692196

RESUMO

Mycobacteria use type VII secretion systems to secrete proteins across their highly hydrophobic diderm cell envelope. Pathogenic mycobacteria, such as Mycobacterium tuberculosis and Mycobacterium marinum, have up to five of these systems, named ESX-1 to ESX-5. Most of these systems contain a set of five conserved membrane components, of which the four Ecc proteins form the core membrane-embedded secretion complex. The fifth conserved membrane protein, mycosin protease (MycP), is not part of the core complex but is essential for secretion, as it stabilizes this membrane complex. Here we investigated which MycP domains are required for this stabilization by producing hybrid constructs between MycP1 and MycP5 in M. marinum and analyzed their effect on ESX-1 and ESX-5 secretion. We found that both the protease and transmembrane domain are required for the ESX system-specific function of mycosins. In addition, we observed that the transmembrane domain strongly affects MycP protein levels. We also show that the extended loops 1 and 2 in the protease domain are probably primarily involved in MycP stability, whereas loop 3 and the MycP5-specific loop 5 are dispensable. The atypical propeptide, or N-terminal extension, is required only for MycP stability. Finally, we show that the protease domain of MycPP1, encoded by the esx-P1 locus on the pRAW plasmid, is functionally redundant to the protease domain of MycP5 These results provide the first insight into the regions of mycosins involved in interaction with and stabilization of their respective ESX complexes.


Assuntos
Proteínas de Bactérias , Mycobacterium marinum , Mycobacterium tuberculosis , Subtilisinas , Sistemas de Secreção Tipo IV , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Mycobacterium marinum/enzimologia , Mycobacterium marinum/genética , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Domínios Proteicos , Estrutura Secundária de Proteína , Subtilisinas/química , Subtilisinas/genética , Subtilisinas/metabolismo , Sistemas de Secreção Tipo IV/química , Sistemas de Secreção Tipo IV/genética , Sistemas de Secreção Tipo IV/metabolismo
17.
J Ind Microbiol Biotechnol ; 46(3-4): 537-549, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30484123

RESUMO

CylA is a subtilisin-like protein belonging to a recently expanded serine protease family related to class II lanthipeptide biosynthesis. As a leader peptidase, CylA is responsible for maturation of the enterococcal cytolysin, a lantibiotic important for Enterococcus faecalis virulence. In vitro reconstitution of CylA reveals that it accepts both linear and modified cytolysin peptides with a preference for cyclized peptides. Further characterization indicates that CylA activates itself by removing its N-terminal 95 amino acids. CylA achieves sequence-specific traceless cleavage of non-cognate peptides even if they are post-translationally modified, which makes the peptidase a powerful tool for mining novel lanthipeptides by providing a general strategy for leader peptide removal. Knowledge about the substrate specificity of CylA may also facilitate the development of protease inhibitors targeting cytolysin biosynthesis as a potential therapeutic approach for enterococcal infections.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Membrana/genética , Serina Endopeptidases/genética , Subtilisinas/genética , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Vias Biossintéticas/genética , Enterococcus/enzimologia , Enterococcus/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Proteínas de Membrana/metabolismo , Peptídeos/química , Perforina/metabolismo , Serina Endopeptidases/metabolismo , Especificidade por Substrato , Subtilisinas/metabolismo
18.
Food Chem ; 277: 314-322, 2019 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-30502151

RESUMO

The enzymatic hydrolysis (at 2.5, 5.0, 10.0, and 15.0% degree of hydrolysis, DH) of kilka fish (Clupeonella cultriventris caspia) was investigated using a crude melon extract (CME) as well as a commercial serine protease (Alcalase). Hydrolysates from both enzymatic treatments were analyzed for their antioxidant and functional properties. The hydrolysis resulted in increased antioxidant activities of kilka fish protein hydrolysates and the highest antioxidant activity was obtained for the CME hydrolysates with 5.0% DH level. Both treatments improved the protein solubility level to >65% within a pH range of 2.0-10.0. At a given DH level, CME hydrolysates showed higher oil and water holding capacities than Alcalase hydrolysates. Hydrolysates from CME exhibited better emulsifying properties compared to those prepared by Alcalase, particularly at low DH (2.5 and 5.0%). According to the results of this study, CME can be suggested as a new source of proteolytic enzymes for fish protein hydrolysis.


Assuntos
Cucurbitaceae/enzimologia , Proteínas de Peixes/metabolismo , Peptídeo Hidrolases/metabolismo , Subtilisinas/metabolismo , Animais , Antioxidantes/análise , Peixes , Hidrólise , Modelos Teóricos , Peso Molecular , Hidrolisados de Proteína/metabolismo , Solubilidade
19.
Food Chem ; 277: 655-663, 2019 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-30502200

RESUMO

The low solubility of wheat gluten (WG) considerably limits its application. Owing to its high hydrolytic efficiency, alcalase was the protease selected for the enzymatic hydrolysis of WG. The functional properties of WG hydrolysate prepared by alcalase (AHWG) with a hydrolysis degree (DH) of 10% were better than those with DH 5% and DH 15%. The application of AHWG was hindered by its bitterness. To mask the bitterness of AHWG, WG was respectively deamidated with acetic acid, tartaric acid, and citric acid, followed by being hydrolyzed by alcalase to DH 10%. The citric acid deamidation-alcalase hydrolysis WG hydrolysate (CDAH) exhibited the best functional properties. Partial least squares regression analysis results indicated that CDAH exhibited an enhanced bitter-masking property attributable to a high content of umami taste amino acids (glutamic acid and aspartic acid). Thus, CDAH showed the greatest potential as a modified WG product to expand the application of WG.


Assuntos
Ácido Acético/química , Proteínas de Bactérias/metabolismo , Glutens/metabolismo , Subtilisinas/metabolismo , Paladar , Triticum/metabolismo , Ácido Aspártico/química , Proteínas de Bactérias/química , Ácido Glutâmico/química , Glutens/química , Hidrólise , Análise dos Mínimos Quadrados , Solubilidade , Subtilisinas/química , Tartaratos/química
20.
Biochim Biophys Acta Proteins Proteom ; 1867(2): 152-162, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30502512

RESUMO

Cloning into a pET 11a vector, followed by high-level expression of the cold adapted subtilase, VPR, utilizing the rhamnose titratable T7 system of Lemo21, resulted in a dramatic increase of soluble protein compared to the older system used. Expression optimization clearly shows the importance of calcium in the medium after induction, both for stability of the proteinase and cell health. Characterization of the purified enzyme obtained in a redesigned purification protocol which removed apparent RNA contaminants, resulted in a significantly higher value for kcat than previously reported. The new recombinant protein exhibited slightly lower stability against thermal denaturation and thermal inactivation. Our results also indicate that two of the calcium binding sites have apparent binding constants in the mM range. Binding of calcium to the weaker of those two sites only affects resistance of the enzyme against irreversible thermal inactivation. Differential scanning calorimetry revealed a non-two-state denaturation process, with indication of presence of intermediates caused by unfolding of calcium binding motifs.


Assuntos
Engenharia de Proteínas/métodos , Subtilisinas/genética , Subtilisinas/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Endopeptidase K , Cinética , Proteínas Recombinantes/metabolismo , Serina Proteases/metabolismo
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