Protein function analysis of germinated Moringa oleifera seeds, and purification and characterization of their milk-clotting peptidase.

The present study aimed to investigate the biological functions of germinated M. oleifera seed proteins and to identify the identity of milk-clotting proteases. A total of 963 proteins were identified, and those with molecular weights between 10 and 30 kDa were most abundant. The identified proteins were mainly involved in energy-associated catalytic activity and metabolic processes, and carbohydrate and protein metabolisms. The numbers of proteins associated with the hydrolytic and catalytic activities were higher than the matured dry M. oleifera seeds reported previously. Of the identified proteins, proteases were mainly involved in the milk-clotting activity. Especially, a cysteine peptidase with a molecular mass of 17.727 kDa exhibiting hydrolase and peptidase activities was purified and identified. The identified cysteine peptidase was hydrophilic, and its secondary structure consisted of 27.60% alpha helix, 9.20% beta fold, and 63.20% irregular curl; its tertiary structure was also constructed using M. oleifera seed 2S protein as the protein template. The optimal pH and temperature of the purified protease were pH 4.0 and 60 °C, respectively. The protease had high acidic stability and good thermostability, thus could potentially be applied in the dairy industry.

Evaluation of the In Vivo Acute Toxicity and In Vitro Hemolytic and Immunomodulatory Activities of the Moringa oleifera Flower Trypsin Inhibitor (MoFTI).

BACKGROUND: Protease inhibitors have been isolated from plants and present several biological activities, including immunomodulatory action. OBJECTIVE: This work aimed to evaluate a Moringa oleifera flower trypsin inhibitor (MoFTI) for acute toxicity in mice, hemolytic activity on mice erythrocytes and immunomodulatory effects on mice splenocytes. METHODS: The acute toxicity was evaluated using Swiss female mice that received a single dose of the vehicle control or MoFTI (300 mg/kg, i.p.). Behavioral alterations were observed 15-240 min after administration, and survival, weight gain, and water and food consumption were analyzed daily. Organ weights and hematological parameters were analyzed after 14 days. Hemolytic activity of MoFTI was tested using Swiss female mice erythrocytes. Splenocytes obtained from BALB/c mice were cultured in the absence or presence of MoFTI for the evaluation of cell viability and proliferation. Mitochondrial membrane potential (Δψm) and reactive oxygen species (ROS) levels were also determined. Furthermore, the culture supernatants were analyzed for the presence of cytokines and nitric oxide (NO). RESULTS: MoFTI did not cause death or any adverse effects on the mice except for abdominal contortions at 15-30 min after administration. MoFTI did not exhibit a significant hemolytic effect. In addition, MoFTI did not induce apoptosis or necrosis in splenocytes and had no effect on cell proliferation. Increases in cytosolic and mitochondrial ROS release, as well as Δψm reduction, were observed in MoFTI-treated cells. MoFTI was observed to induce TNF-α, IFN-γ, IL-6, IL-10, and NO release. CONCLUSION: These results contribute to the ongoing evaluation of the antitumor potential of MoFTI and its effects on other immunological targets.

Moringa straw as cellulase production inducer and cellulolytic fungi source / Residuo de moringa como inductor de la producción de celulasas y como fuente de hongos celulolíticos

Abstract Currently, the valorization of agroindustrial waste is of great interest. Moringa oleifera is a multipurpose tree whose softwood residues could be used as raw material for low-cost cellulase production. The aim of this study was to isolate, identify, and characterize microorganisms with cellulolytic activity in different carbon sources. We isolated and puri-fied 42 microorganisms from M. oleifera biomass. Fungi presenting the largest hydrolytic halos in carboxymethylcellulose as a substrate were molecularly identified as Penicillium funiculosum (FG1), Fusarium verticillioides (FG3) and Cladosporium cladosporioides (FC2). The ability of these fungal strains to break down cellulose was assessed in a submerged fermentation using either amorphous CMC or crystalline form (Avicel). P. funiculosum and C. cladosporioides displayed similar endoglucanase (606 U/l) and exoglucanase (205 U/l) activities in the Avicel-containing medium, whereas F. verticillioides showed the highest level of p-glucosidase activity (664 U/l) in the carboxymethylcellulose medium. In addition, the effect of three culture media (A, B, and C) on cellulase production was evaluated in P. funiculosum using moringa straw as a carbon source. The results showed a volumetric productivity improvement of cellulases that was 2.77-, 8.26-, and 2.30-fold higher for endoglucanase, exoglucanase and p-glucosidase, respectively when medium C containing moringa straw was used as a carbon source. The enzymatic extracts produced by these fungi have biotechnological potential especially for second-generation bioethanol production (2G) from moringa straw. This is the first report on the use of M. oleifera biomass to induce the production of various cellulases in P. funiculosum. © 2019 Asociación Argentina de Microbiología. Published by Elsevier Espana, S.L.U. This is an open access article under the CC BY-NC-ND license (https://creativecommons.org/licenses/by-nc-nd/4.0/).

Resumen Actualmente, la valorización de los residuos agroindustriales es de gran interés. En este trabajo se emplearon residuos de madera blanda de Moringa oleifera para la producción de celulasas de bajo costo. El objetivo fue aislar, identificar y caracterizar microorganismos con actividad celulolítica en diferentes fuentes de carbono. A partir de la biomasa de M. oleifera, se aislaron e identificaron 42 microorganismos productores de celulasas. Los hongos que presentaron los mayores halos de hidrólisis en carboximetilcelulosa como sustrato fueron identificados molecularmente como Penicillium funiculosum (FG1), Fusarium verticillioides (FG3) y Cladosporium cladosporioides (FC2). Mediante fermentación sumergida, se evaluó la capacidad de estas cepas en la producción de celulasas utilizando celulosa cristalina (Avicel) y amorfa (CMC) como fuentes de carbono. P. funiculosum y C. cladosporioides presentaron las mayores actividades de endoglucanasa (606 U/l) y exoglucanasa (205 U/l) en medio Avicel, mientras que F. verticillioides mostró la mayor actividad de p-glucosidasa (664 U/l) en medio CMC. Además, se evaluó el efecto de tres medios de cultivo (A, B y C) sobre la producción de celulasas en P. funiculosum empleando residuos de moringa como fuente de carbono. Los resultados mostraron que en el medio C, la productividad volumétrica de celulasas se incrementó en 2,77; 8,26 y 2,30 veces para las actividades de endoglucanasa, exoglucanasa y p-glucosidasa, respectivamente. Los extractos enzimáticos producidos tienen gran potencial para su utilización biotecnológica, especialmente en la sacarificación de residuos de moringa y la producción de bioetanol de segunda generación. Este es el primer estudio del uso de la biomasa de M. oleifera para inducir la producción de diversas celulasas en P. funiculosum.

Moringa straw as cellulase production inducer and cellulolytic fungi source.

Currently, the valorization of agroindustrial waste is of great interest. Moringa oleifera is a multipurpose tree whose softwood residues could be used as raw material for low-cost cellulase production. The aim of this study was to isolate, identify, and characterize microorganisms with cellulolytic activity in different carbon sources. We isolated and purified 42 microorganisms from M. oleifera biomass. Fungi presenting the largest hydrolytic halos in carboxymethylcellulose as a substrate were molecularly identified as Penicillium funiculosum (FG1), Fusarium verticillioides (FG3) and Cladosporium cladosporioides (FC2). The ability of these fungal strains to break down cellulose was assessed in a submerged fermentation using either amorphous CMC or crystalline form (Avicel). P. funiculosum and C. cladosporioides displayed similar endoglucanase (606U/l) and exoglucanase (205U/l) activities in the Avicel-containing medium, whereas F. verticillioides showed the highest level of ß-glucosidase activity (664U/l) in the carboxymethylcellulose medium. In addition, the effect of three culture media (A, B, and C) on cellulase production was evaluated in P. funiculosum using moringa straw as a carbon source. The results showed a volumetric productivity improvement of cellulases that was 2.77-, 8.26-, and 2.30-fold higher for endoglucanase, exoglucanase and ß-glucosidase, respectively when medium C containing moringa straw was used as a carbon source. The enzymatic extracts produced by these fungi have biotechnological potential especially for second-generation bioethanol production (2G) from moringa straw. This is the first report on the use of M. oleifera biomass to induce the production of various cellulases in P. funiculosum.

Label-free quantitative proteomic analysis of the biological functions of Moringa oleifera seed proteins provides insights regarding the milk-clotting proteases.

In this study, label-free quantitative proteomics was used to investigate the biological functions of M. oleifera seed proteins, which resulted in the identification of milk-clotting proteases. In total, 921 proteins were identified, and proteins within the molecular weight range of 30-50 kDa were abundant. The identified proteins were mainly involved in catalytic activity and metabolic processes associated with carbohydrate and protein metabolism, among which, proteases in the observed molecular weight range could possibly be responsible for the previously reported milk-clotting activity. An aspartic-type endopeptidase with molecular mass of 45,517 Da was purified from M. oleifera seeds using ammonium sulfate precipitation, ultrafiltration, and preparative high performance liquid chromatography, and was characterized using liquid chromatography-mass spectrometry (LC-MS)/MS. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the purified protease exhibited hydrolase activity and was involved in several metabolic pathways, which further confirmed that proteomic analysis can assist in the purification of the milk-clotting protease. The optimal temperature and pH required for protease activity were 60 °C and 5.0, respectively. The high thermal stability and good pH stability of the protease indicated that it can be used in the dairy industry.

Extraction, purification, and activity of protease from the leaves of Moringa oleifera.

Background: Proteases cleave proteins, thereby providing essential amino acids for protein synthesis, and degrade misfolded and damaged proteins to maintain homeostasis. Proteases also serve as signaling molecules, therapeutic agents and find wide applications in biotechnology and pharmaceutical industry.  Plant-derived proteases are suitable for many biomedical applications due to their easy availability and activity over a wide range of pH, temperature, and substrates. Moringa oleifera Lam (Moringaceae) is a very common food plant with medicinal property and geographically distributed in tropical countries. Here, we isolate proteases from the leaves of Moringa oleifera and characterize its enzymatic activity. Methods: Proteases were isolated from the aqueous leaf extract of Moringa oleifera by ammonium sulfate precipitation and purified by ion exchange chromatography. Subsequently, the enzyme kinetics was determined using casein as a substrate and calibrated over different pH and temperature range for maximal activity. Results: We obtained purified fraction of the protease having a molecular weight of 51 kDa. We observed that for the maximal caseinolytic activity of the protease, a pH of 8 and temperature of 37ºC was found to be most effective. Conclusion: The plant-derived proteolytic enzymes are finding increasing clinical and industrial applications. We could extract, purify and characterize the enzymatic activity of proteases from the leaves of Moringa oleifera. Further molecular characterization, substrate specificity and activity of the extracted protease are required for determining its suitability as a proteolytic enzyme for various applications.

Aqueous enzymatic extraction of Moringa oleifera oil.

This paper reports on the extraction of Moringa oleifera (MO) oil by using aqueous enzymatic extraction (AEE) method. The effect of different process parameters on the oil recovery was discovered by using statistical optimization, besides the effect of selected parameters on the formation of its oil-in-water cream emulsions. Within the pre-determined ranges, the use of pH 4.5, moisture/kernel ratio of 8:1 (w/w), and 300stroke/min shaking speed at 40°C for 1h incubation time resulted in highest oil recovery of approximately 70% (goil/g solvent-extracted oil). These optimized parameters also result in a very thin emulsion layer, indicating minute amount of emulsion formed. Zero oil recovery with thick emulsion were observed when the used aqueous phase was re-utilized for another AEE process. The findings suggest that the critical selection of AEE parameters is key to high oil recovery with minimum emulsion formation thereby lowering the load on the de-emulsification step.

Purification and characterization of a class II α-Mannosidase from Moringa oleifera seed kernels.

α-Mannosidase (EC. 3.2.1.114) belonging to class II glycosyl hydrolase family 38 was purified from Moringa oleifera seeds to apparent homogeneity by conventional protein purification methods followed by affinity chromatography on Con A Sepharose and size exclusion chromatography. The purified enzyme is a glycoprotein with 9.3 % carbohydrate and exhibited a native molecular mass of 240 kDa, comprising two heterogeneous subunits with molecular masses of 66 kDa (α-larger subunit) and 55 kDa (ß-smaller subunit). Among both the subunits only larger subunit stained for carbohydrate with periodic acid Schiff's staining. The optimum temperature and pH for purified enzyme was 50 °C and pH 5.0, respectively. The enzyme was stable within the pH range of 3.0-7.0. The enzyme was inhibited by EDTA, Hg(2+), Ag(2+), and Cu(2+). The activity lost by EDTA was completely regained by addition of Zn(2+). The purified enzyme was characterized in terms of the kinetic parameters K m (1.6 mM) and V max (2.2 U/mg) using para-nitrophenyl-α-D-mannopyranoside as substrate. The enzyme was very strongly inhibited by swainsonine (SW) at 1 µM concentration a class II α-Mannosidase inhibitor, but not by deoxymannojirimycin (DMNJ). Chemical modification studies revealed involvement of tryptophan at active site. The inhibition by SW and requirement of the Zn(2+) as a metal ion suggested that the enzyme belongs to class II α-Mannosidase.