RESUMO
The high content and quality of protein in Andean legumes make them valuable for producing protein hydrolysates using proteases from bacteria isolated from extreme environments. This study aimed to carry out a single-step purification of a haloprotease from Micrococcus sp. PC7 isolated from Peru salterns. In addition, characterize and apply the enzyme for the production of bioactive protein hydrolysates from underutilized Andean legumes. The PC7 protease was fully purified using only tangential flow filtration (TFF) and exhibited maximum activity at pH 7.5 and 40 °C. It was characterized as a serine protease with an estimated molecular weight of 130 kDa. PC7 activity was enhanced by Cu2+ (1.7-fold) and remained active in the presence of most surfactants and acetonitrile. Furthermore, it stayed completely active up to 6% NaCl and kept Ì´ 60% of its activity up to 8%. The protease maintained over 50% of its activity at 25 °C and 40 °C and over 70% at pH from 6 to 10 for up to 24 h. The determined Km and Vmax were 0.1098 mg mL-1 and 273.7 U mL-1, respectively. PC7 protease hydrolyzed 43%, 22% and 11% of the Lupinus mutabilis, Phaseolus lunatus and Erythrina edulis protein concentrates, respectively. Likewise, the hydrolysates from Lupinus mutabilis and Erythrina edulis presented the maximum antioxidant and antihypertensive activities, respectively. Our results demonstrated the feasibility of a simple purification step for the PC7 protease and its potential to be applied in industrial and biotechnological processes. Bioactive protein hydrolysates produced from Andean legumes may lead to the development of nutraceuticals and functional foods contributing to address some United Nations Sustainable Development Goals (SDGs).
Assuntos
Fabaceae , Micrococcus , Hidrolisados de Proteína , Micrococcus/metabolismo , Micrococcus/enzimologia , Concentração de Íons de Hidrogênio , Hidrolisados de Proteína/química , Hidrolisados de Proteína/metabolismo , Peso Molecular , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/isolamento & purificação , Peru , Temperatura , Serina Proteases/metabolismo , Serina Proteases/isolamento & purificação , Serina Proteases/química , Estabilidade Enzimática , Cloreto de Sódio/metabolismo , Cloreto de Sódio/farmacologia , Hidrólise , CinéticaRESUMO
El objetivo del estudio fue realizar la caracterización bioinformática, así como optimizar la producción de L-asparaginasa extracelular de Bacillus sp. M62 aislada de las salinas de Maras (Cusco). Para ello, se verificó la producción de L-asparaginasa mediante el viraje del medio M9 modificado con azul de bromofenol 0.0075%, pH 7.4 a 37 °C por 72 h. A la vez, se extrajo el ADN genómico para amplificar los genes ribosómicos 16S y el gen ansA3. La secuencia aminoacídica codificada por el gen ansA3 se predijo mediante análisis bioinformático. La producción de L-asparaginasa intracelular y extracelular se evaluó a diferentes niveles de glucosa, L-asparagina, NaCl y pH en el medio M9 modificado. Adicionalmente, las actividades enzimáticas de L-asparaginasa y L-glutaminasa se determinaron mediante cuantificación del amonio liberado por el método de Nessler. Así, Bacillus sp. M62 produjo el viraje del medio M9 modificado, obtuvo alta similitud y cercanía evolutiva con Bacillus licheniformis, se encontró que el gen ansA3 amplificado codificaba para 319 aa, dentro de la cual se predijo una secuencia patrón del sitio activo (GFVITHGTDTM ) y 15 sitios inmunogénicos. La producción de L-asparaginasa extracelular fue superior a la intracelular, la que se optimizó de 0.37 U/mL (0.24 U/mg) a 2.15 ± 0.39 U/mL (0.63 U/mg). Finalmente, se encontró que Bacillus sp. M62 presenta L-asparaginasa extracelular con mínima actividad de L-glutaminasa.
The aim of this study was to perform bioinformatics characterization and optimize the production of extracellular L-asparaginase from Bacillus sp. M62, isolated from the Maras salt ponds (Cusco). To achieve this, the production of L-asparaginase was verified by the change in color of modified M9 medium, containing 0.0075% bromophenol blue, at pH 7.4 and 37°C for 72 hours. Genomic DNA was extracted to amplify the 16S ribosomal genes and the ansA3 gene. The amino acid sequence encoded by the ansA3 gene was predicted using bioinformatic analysis. The production of intracellular and extracellular L-asparaginase was evaluated at different levels of glucose, L-asparagine, NaCl, and pH in modified M9 medium. Additionally, the enzymatic activities of L-asparaginase and L-glutaminase were determined by quantifying the released ammonium using the Nessler method. Bacillus sp. M62 showed the change in color of the modified M9 medium, high similarity, and evolutionary closeness to Bacillus licheniformis. The amplified ansA3 gene was found to encode for 319 amino acids, with a predicted active site pattern (GFVITHGTDTM) and 15 immunogenic sites. The production of extracellular L-asparaginase was found to be higher than intracellular L-asparaginase and was optimized from 0.37 U/mL (0.24 U/mg) to 2.15 ± 0.39 U/mL (0.63 U/mg). Finally, it was found that Bacillus sp. M62 presents extracellular L-asparaginase with minimal L-glutaminase activity.
RESUMO
The ability of multifunctional food-derived peptides to act on different body targets make them promising alternatives in the prevention/management of chronic disorders. The potential of Erythrina edulis (pajuro) protein as a source of multifunctional peptides was proven. Fourteen selected synthetic peptides identified in an alcalase hydrolyzate from pajuro protein showed in vitro antioxidant, anti-hypertensive, anti-diabetic, and/or anti-obesity effects. The radical scavenging properties of the peptides could be responsible for the potent protective effects observed against the oxidative damage caused by FeSO4 in neuroblastoma cells. Moreover, their affinity towards the binding cavity of angiotensin-converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV) were predicted by molecular modeling. The results demonstrated that some peptides such as YPSY exhibited promising binding at both enzymes, supporting the role of pajuro protein as a novel ingredient of functional foods or nutraceuticals for prevention/management of oxidative stress, hypertension, and metabolic-alteration-associated chronic diseases.
RESUMO
Multifunctional peptides, capable of acting on different body systems through multiple mechanisms of action, offer many advantages over monofunctional peptides, including lower adverse side effects and costs. Erythrina edulis (pajuro) is a legume with a large number of high-quality proteins, of which their potential as a source of antioxidant peptides has been recently reported. In this study, the behavior of these proteins under a sequential enzymatic hydrolysis with digestive and microbial enzymes was investigated by evaluating the multi-functionality of the hydrolyzates. The albumin hydrolyzates obtained after the action of pepsin, pancreatin, and Alcalase showed antioxidant, angiotensin-converting enzyme (ACE), α-amylase, α-glucosidase, and dipeptidyl peptidase (DPP)-IV inhibitory activities. The radical scavenging properties of the hydrolyzate could be responsible for the potent protective effects observed in FeSO4-induced neuroblastoma cells. The findings support the role of pajuro protein as an ingredient of functional foods or nutraceuticals for health promotion and the prevention of oxidative stress, hypertension, and metabolic alteration-associated chronic diseases.
RESUMO
BACKGROUND: Oxidative reactions are responsible for the changes in quality during food processing and storage. Oxidative stress is also involved in multiple chronic diseases, such as cardiovascular and neurodegenerative disorders, diabetes, cancer, and aging. The consumption of dietary antioxidants has been demonstrated to help to reduce the oxidative damage in both the human body and food systems. In this study, the potential of Erythrina edulis (pajuro) protein as source of antioxidant peptides was evaluated. RESULTS: Pajuro protein concentrate hydrolyzed by alcalase for 120 min showed potent ABTS·+ and peroxyl radical scavenging activity with Trolox equivalent antioxidant capacity (TEAC) and oxygen radical absorbance capacity (ORAC) values of 1.37 ± 0.09 µmol TE mg-1 peptide and 2.83 ± 0.07 µmol TE mg-1 peptide, respectively. Fractionation of the hydrolyzate to small peptides resulted in increased antioxidant activity. De novo sequencing of most active fractions collected by chromatographic analysis enabled 30 novel peptides to be identified. Of these, ten were synthesized and their radical activity evaluated, demonstrating their relevant contribution to the antioxidant effects observed for pajuro protein hydrolyzate. CONCLUSIONS: The sequences identified represent an important advance in the molecular characterization of the pajuro protein, demonstrating its potential as a source of antioxidant peptides for food and nutraceutical applications. © 2018 Society of Chemical Industry.