RESUMO
Bromelia serra leaves collected from Corrientes, Argentina, were assessed to analyze and characterize the proteolytic system and to evaluate its potential use as an industrial catalyst. The specific activity of the enzymatic extract (EE), which was prepared using acetone as a precipitating agent of the crude extract (CE), increased 2-3 folds with different substrates (hemoglobin, azocasein and casein). The proteins present in the EE have isoelectric points between 4.55-8.15 and they were significant inhibited by pepstatin A (50%) and E-64 (15%). Proteolytic activity in EE presented high activity in acidic pH (2.7-4), and low activity in neutral alkaline pH (6-11.75). The EE optimum activity was reached at 60ºC, and referring to the thermal stability, it retained over 97% of the proteolytic activity after incubation at a temperature range of 37â60 ºC for 60 min. The effect of reducing agents and ionic strength were also measured, and it showed that the EE had its maximum activity with 5mM of cysteine, and it was inactivated with 2.5 M of NaCl. The chromatography procedures presented two purified enzymes of 21 and 54 KDa with proteolytic activity. The characteristics of the EE suggest that it is a potential candidate as an industrial catalyst.
Assuntos
Bromelia , Bromelia/metabolismo , Concentração de Íons de Hidrogênio , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Folhas de Planta/metabolismo , ProteóliseRESUMO
A crude extract with proteolytic activity was prepared from edible fruits of Bromelia serra, containing cysteine peptidases with molecular masses between 24.1 and 25.9 kDa. The extract presented an optimal pH range of 6.03-9.05, retained more than 80% of activity after thermal pre-treatments at 23, 37, and 45°C (120 min), but it was rapidly inactivated after 10 min at 75°C. These proteases were employed to hydrolyze soybean proteins, bovine casein and bovine whey, achieving degrees of hydrolysis of 18.3 ± 0.6, 29.1 ± 0.7, and 12.6 ± 0.9% (55°C, 180 min), respectively. The casein 180 min-hydrolysate (55°C) presented the maximum value of antioxidant activity (2.89 ± 0.12 mg/mL Trolox), and the whey protein 180 min-hydrolysate (55°C) showed the highest percentage of angiotensin-converting enzyme inhibition (91.9 ± 1.2%). This low-cost enzymatic preparation would be promising for the food industry because it requires mild working conditions and yields hydrolysates with biological activities useful as ingredients for functional food. PRACTICAL APPLICATION: Proteolytic enzymes are employed in the food industry in a wide variety of processes since they modify the properties of proteins causing beneficial effects such as improvement digestibility, diminution of allergenicity, and release of bioactive peptides. Fruits from Bromelia serra possess cysteine peptidases that could be used in food biotechnology because they are capable to hydrolyze soybean and milk proteins by mild working conditions and to provoke the release of bioactive peptides. These hydrolysates containing antioxidative and ACE-inhibitor activities would be useful as ingredients for functional foods or as nutraceuticals, which are nowadays two products highly required by consumers.
Assuntos
Bromelia , Animais , Bromelia/metabolismo , Bovinos , Frutas/metabolismo , Peptídeo Hidrolases , Peptídeos/químicaRESUMO
BACKGROUND: The most common milk-clotting enzymes in the cheese industry are recombinant chymosins. Food naturalness is a factor underpinning consumers' food choice. For consumers who avoid food with ingredients from genetically modified organisms (GMOs), the use of vegetable-based rennet substitute in the cheese formulation may be a suitable solution. Artichokes that deviate from optimal products, when allowed to bloom due to flower protease composition, are excellent as raw material for vegetable rennet preparation. As enzymatic milk clotting exerts a significant impact on the characteristics of the final product, this product should be studied carefully. RESULTS: Mature flowers from unharvested artichokes (Cynara scolymus cv. Francés) that did not meet aesthetic standards for commercialization were collected and used to prepare a flower extract. This extract, as a coagulant preparation, enabled the manufacture of cheeses with distinctive characteristics compared with cheeses prepared with chymosin. Rennet substitution did not affect the actual yield but led to significant changes in dry matter yield, humidity, water activity, protein content, and color, and conferred antioxidant activity to the cheeses. The rennet substitution promoted significant modifications in springiness, and in the microstructure of the cheese, with a more porous protein matrix and an increment in the size of the fat globules. Both formulations showed a similar microbiota evolution pattern with excellent microbiological quality and good sensory acceptance. CONCLUSIONS: The rennet substitute studied here produced a cheese adapted to specific market segments that demand more natural and healthier products made with a commitment to the environment but well accepted by a general cheese consumer. © 2020 Society of Chemical Industry.
Assuntos
Antioxidantes/química , Queijo/análise , Cynara scolymus/química , Extratos Vegetais/química , Animais , Ácido Aspártico Endopeptidases/química , Bactérias/classificação , Bactérias/isolamento & purificação , Bactérias/metabolismo , Bovinos , Queijo/microbiologia , Flores/química , Manipulação de Alimentos , Humanos , Microbiota , Leite/química , PaladarRESUMO
Our objective was to isolate peptidases from the latex of Maclura pomifera fruits and use them to hydrolyze food proteins, as well as to purify and characterize the main peptidase. Two partially purified proteolytic extracts were prepared by ethanol (EE) and acetone (AE) precipitation from an aqueous suspension of exuded fruit latex. EE was used to hydrolyze food proteins with a ratio of 0.19 caseinolytic units (Ucas) per mg of substrate. Different values of hydrolysis degree were observed for hydrolysates of egg white, soy protein isolate, and casein at 180 min (9.3%, 31.1%, and 29.1%, respectively). AE was employed to purify a peptidase which exhibited an isoelectric point (pI) of 8.70 and whose abundance in AE was 28.3%. This enzyme was purified to homogeneity using a single-step procedure by cation-exchange chromatography, achieving an 8.1-fold purification and a yield of 16.7%. The peptidase was named pomiferin I and showed a molecular mass of 63,177.77 Da. Kinetic constants (KM 0.84 mM, Vmax 27.50 uM s-1, kcat 72.37 s-1, and kcat/KM 86.15 mM-1 s-1) were determined employing N-α-carbobenzoxy-L-alanyl-p-nitrophenyl ester as substrate. Analysis by PMF showed only partial homology of pomiferin I with a serine peptidase from a species of the same family.
Assuntos
Proteínas Alimentares/química , Frutas/enzimologia , Maclura/enzimologia , Peptídeo Hidrolases , Proteínas de Plantas , Hidrolisados de Proteína/química , Cromatografia por Troca Iônica , Peptídeo Hidrolases/química , Peptídeo Hidrolases/isolamento & purificação , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificaçãoRESUMO
Binary blends of S. marianum-flower extract and chymosin, as coagulant preparations, enabled the manufacture of miniature cheeses with distinctive characteristics compared to those of chymosin-renneted cheeses. The physicochemical parameters, sensory attributes of the cheeses, and in-vitro water-soluble antioxidant activity were analyzed and compared to those properties obtained from control chymosin-renneted cheeses. The preponderant proteolytic constituent in the flower extract was isolated in a two-step-purification protocol. The thus purified aspartic peptidase was maximally active at acidic pHs and exhibited a preference for peptide bonds between hydrophobic residues. Enzymologic characterization revealed differences in the kinetic parameters and specificity compared to other enzymes employed, such as rennet. S. marianum-flower extract, as a source of peptidase with distinctive characteristics, is a suitable substitute for chymosin in miniature-cheese production. The addition of vegetable rennet contributed to the development of an intense aroma and conferred antioxidant activity to the cheeses and wheys.
Assuntos
Queijo/análise , Quimosina/metabolismo , Manipulação de Alimentos/métodos , Silybum marianum/enzimologia , Animais , Flores/enzimologia , LeiteRESUMO
Pseudocercospora griseola is the causal agent of Angular Leaf Spot (ALS), a disease of common bean. Due to its coevolution with beans, two major groups have been defined, "Andean" (P. griseola f. griseola) and "Mesoamerican" (P. griseola f. mesoamericana). The aim of this study was to characterize the dark pigment, melanin, synthetized by a selected isolate of each genic group of P. griseola when grown on Potato-dextrose broth. P. griseola f. griseola isolate S3b and P. griseola f. mesoamericana isolate T4 produced 1.7⯱â¯0.6 and 4.1⯱â¯0.9â¯mg of melanin per g of dry biomass, respectively. Although both melanins possessed similar UV-visible absorption spectroscopic pattern, P. griseola f. mesoamericana isolate T4 melanin had a lower UV-visible absorption, higher reducing activity and metal chelating ability than melanin from P. griseola f. griseola isolate S3b. However, when the size of the sample was 10â¯mg S3b melanin had a higher content of free phenolic groups. Furthermore, cell wall polysaccharides modified in melanin the availability of active phenolic groups, which was dependent on the fungal isolate and the size of the sample. Therefore, the amount and chemical features of melanin as well as its deposition in mycelium walls within isolates is different, which might explain the different pigmentation and physiological behaviours of these representatives of the two groups of P. griseola.
Assuntos
Ascomicetos/metabolismo , Melaninas/química , Antioxidantes/química , Ascomicetos/crescimento & desenvolvimento , Ascomicetos/isolamento & purificação , Biomassa , Espectroscopia de Ressonância de Spin Eletrônica , Melaninas/metabolismo , Oxirredução , Phaseolus/microbiologia , Doenças das Plantas/microbiologia , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
In this article, we report the cloning of an aspartic protease (AP) from flowers of Arctium minus (Hill) Bernh. (Asteraceae) along with the use of depigmented aqueous flower extracts, as a source of APs, for the hydrolysis of whey proteins. The isolated cDNA encoded a protein product with 509 amino acids called arctiumisin, with the characteristic primary structure organization of typical plant APs. Bovine whey protein hydrolysates, obtained employing the enzyme extracts of A. minus flowers, displayed inhibitory angiotensin-converting enzyme (ACE) and antioxidant activities. Hydrolysates after 3 and 5 h of reaction (degree of hydrolysis 2.4 and 5.6, respectively) and the associated peptide fraction with molecular weight below 3 kDa were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, matrix-assisted laser desorption ionization/time of flight mass spectrometry, and reverse phase-high-performance liquid chromatography. The results obtained in this study demonstrate the viability of using proteases from A. minus to increase the antioxidant and inhibitory ACE capacity of whey proteins.
Assuntos
Arctium/química , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/farmacologia , Ácido Aspártico Proteases/química , Hidrólise/efeitos dos fármacos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/farmacologia , Proteínas do Soro do Leite/química , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Antioxidantes/química , Antioxidantes/farmacologia , Bovinos , Cromatografia Líquida de Alta Pressão/métodos , DNA Complementar , Flores/química , Espectrometria de Massas/métodos , Fragmentos de Peptídeos/genética , Extratos Vegetais/químicaRESUMO
Fruits of Bromelia hieronymi, a tropical South American plant, possess a high content of peptidases with potential biotechnological uses. Total RNA was extracted from unripe fruits and peptidase cDNA was obtained by 3'RACE-PCR. The consensus sequence of the cysteine peptidase cDNA contained 875 bp, the 690 first ones codifying for a hypothetical polypeptide chain of the mature peptidase, named Bh-CP1 (molecular mass 24.773 kDa, pI 8.6, extinction molar coefficient 58,705 M(-1) cm(-1)). Bh-CP1 sequence shows a high percentage of identity with those of other cysteine plant proteases. The presence of highly preserved residues is observed, like those forming the catalytic site (Gln19, Cys25, His159, and Asn175, papain numbering), as well as other six Cys residues, involved in the formation of disulfide bounds. Molecular modeling results suggest the enzyme belongs to the α + ß class of proteins, with two disulfide bridges (Cys23-Cys63 and Cys57-Cys96) in the α domain, while the ß domain is stabilized by another disulfide bridge (Cys153-Cys203). Additionally, peptide mass fingerprints (PMFs) of the three peptidases previously isolated from B. hieronymi fruits (namely hieronymain I, II, and III) were performed and compared with the theoretical fingerprint of PMF of Bh-CP1, showing a partial matching between the in silico-translated protein and hieronymain II.
Assuntos
Bromelia/enzimologia , Sequência Conservada/genética , Cisteína Endopeptidases/química , Proteínas de Plantas/química , Proteômica/métodos , Proteínas Recombinantes/química , Sequência de Aminoácidos , Sequência de Bases , Domínio Catalítico , Clonagem Molecular , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/isolamento & purificação , Escherichia coli , Frutas/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Plasmídeos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transformação BacterianaRESUMO
A new proteolytic enzyme, named hieronymain III, has been purified by ion-exchange chromatography from unripe fruits of Bromelia hieronymi Mez. The new peptidase belongs to the cysteine catalytic type, as well as hieronymain I and II, the other two peptidases previously isolated from this species. Hieronymain III showed optimum alkaline pH range (8.6-9.3) and the molecular mass (MALDI-TOF) was 23713 Da. The N-terminal sequence (AVPQSIDWRRYGAVTTSRNQG) exhibited a higher percentage identity with hieronymain II (93%) than with hieronymain I (71%). The three peptidases showed notable differences on synthetic substrates degradation: whereas hieronymain III was the only one able to hidrolyze Z-Arg-Arg-p-nitroanilide, hieronymain I and II could degrade Z-Phe-Arg-p-nitroanilide; on the other hand, PFLNA was only split by hieronymain I. Finally, the three proteases showed different preferences on N-alpha-CBZ-p-nitrophenyl aminoacid ester substrates. From a biotechnological point of view, cleavage specificity differences are significant enough to use these enzymes as potential tools in that area.
Assuntos
Amidoidrolases/isolamento & purificação , Bromelia/enzimologia , Cisteína Endopeptidases/isolamento & purificação , Esterases/isolamento & purificação , Proteínas de Plantas/isolamento & purificação , Amidoidrolases/química , Amidoidrolases/metabolismo , Sequência de Aminoácidos , Cromatografia por Troca Iônica , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Eletroforese em Gel de Poliacrilamida , Esterases/química , Esterases/metabolismo , Frutas/enzimologia , Focalização Isoelétrica , Dados de Sequência Molecular , Proteínas de Plantas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
A new cysteine peptidase (Granulosain I) was isolated from ripe fruits of Solanum granuloso-leprosum Dunal (Solanaceae) by means of precipitation with organic solvent and cation exchange chromatography. The enzyme showed a single band by SDS-PAGE, its molecular mass was 24,746 Da (MALDI-TOF/MS) and its isoelectric point was higher than 9.3. It showed maximum activity (more than 90%) in the pH range 7-8.6. Granulosain I was completely inhibited by E-64 and activated by the addition of cysteine or 2-mercaptoethanol, confirming its cysteinic nature. The kinetic studies carried out with PFLNA as substrate, showed an affinity (Km 0.6 mM) slightly lower than those of other known plant cysteine proteases (papain and bromelain). The N-terminal sequence of granulosain I (DRLPASVDWRGKGVLVLVKNQGQC) exhibited a close homology with other cysteine proteases belonging to the C1A family.