Sergio Medina-Godoy

Modification of the amaranth 11S globulin storage protein to produce an inhibitory peptide of the angiotensin I converting enzyme, and its expression in Escherichia coli.

Amarantin is the predominant seed storage protein from amaranth. It shows a high content of essential amino acids, making this protein important from a nutritional viewpoint. The protein has two disulfide linked subunits: acidic and basic. Acidic subunit has the potential as a functional and nutraceutical protein, and it is structurally a good candidate for modification. In order to improve its functionality, the primary structure was modified in the third variable region of globulins 11S, by inserting four Val-Tyr antihypertensive peptides in tandem. The designed plasmid was expressed in Escherichia coli Origami (DE3) and then the expressed protein was purified. Mass spectrometry analysis was used to corroborate the identity of the protein by peptide mass fingerprinting; also, the modified peptide was fragmented and sequenced by mass spectrometry, corroborating thus the inserted residues. The hydrolyzed protein showed a high inhibitory activity of the angiotensin converting enzyme (IC(50) 0.064 mg ml(-1)); it was nearly eightfold more active than the nonmodified protein. In spite that the nonmodified subunit is less active, its activity is comparable with other hydrolyzed proteins reported as high active inhibitors. The expressed and purified subunit after its engineered modification, may be useful for preventing hypertension and for other medical purposes.

Phenolic Content and Antioxidant and Antimutagenic Activities in Tomato Peel, Seeds, and Byproducts

The phenolic content and antioxidant and antimutagenic activities from the peel and seeds of different tomato types (grape, cherry, bola and saladette type), and simulated tomato industrial byproducts, were studied. Methanolic extracts were used to quantify total phenolic content, groups of phenolic compounds, antioxidant activities, and the profile of phenolic compounds (by HPLC-DAD). Antimutagenic activity was determined by Salmonella typhimurium assay. The total phenolic content and antioxidant activity of tomato and tomato byproducts were comparable or superior to those previously reported for whole fruit and tomato pomace. Phenolic compounds with important biological activities, such as caffeic acid, ferulic acid, chlorogenic acids, quercetin-3-β-O-glycoside, and quercetin, were quantified. Differences in all phenolic determinations due to tomato type and part of the fruit analyzed were observed, peel from grape type showing the best results. Positive antimutagenic results were observed in all samples. All evaluated materials could be used as a source of potential nutraceutical compounds.

Expression and Characterization of a His-Tagged 11S Seed Globulin from Amaranthus hypochondriacus in Escherichia coli

DNA encoding a His‐tagged 11S globulin from Amaranthus hypochondriacus (amarantin) was successfully expressed in Escherichia coli strains BL21 (DE3) and Origami (DE3). The two strains produced different accumulation patterns. Whereas most of the proamarantin expressed in BL21 (DE3) was localized in inclusion bodies, that produced in Origami (DE3) was soluble (76 mg/L). Sucrose density gradient ultracentrifugation analysis of the expressed soluble proamarantin revealed that the protein was assembled into trimers. Treatment of proamarantin trimers in vitro using purified asparaginyl endopeptidase resulted in the appearance of peptides of the sizes expected for acidic and basic chains. Because the proamarantin assembles into trimers with the expected sedimentation characteristics and is cleaved into acidic and basic chains rather than being degraded, the results suggest that the protein folding occurring in E. coliis similar to that taking place in seeds. The His‐tagged proamarantin was purified in a single step by immobilized metal affinity chromatography with a final yield of 48 mg/L. The overexpression of proamarantin in E. coli, together with the one‐step purification will facilitate further investigation of this storage protein through site‐directed mutagenesis.

PvLOX2 silencing in common bean roots impairs arbuscular mycorrhiza-induced resistance without affecting symbiosis establishment

The arbuscular mycorrhizal (AM) symbiosis is an intimate association between specific soil-borne fungi and the roots of most land plants. AM colonisation elicits an enhanced defence resistance against pathogens, known as mycorrhizal-induced resistance (MIR). This mechanism locally and systemically sensitises plant tissues to boost their basal defence response. Although a role for oxylipins in MIR has been proposed, it has not yet been experimentally confirmed. In this study, when the common bean (Phaseolus vulgaris L.) lipoxygenase PvLOX2 was silenced in roots of composite plants, leaves of silenced plants lost their capacity to exhibit MIR against the foliar pathogen Sclerotinia sclerotiorum, even though they were colonised normally. PvLOX6, a LOX gene family member, is involved in JA biosynthesis in the common bean. Downregulation of PvLOX2 and PvLOX6 in leaves of PvLOX2 root-silenced plants coincides with the loss of MIR, suggesting that these genes could be involved in the onset and spreading of the mycorrhiza-induced defence response.

Phenolic profile and antioxidant activity from non-toxic Mexican Jatropha curcas L. shell methanolic extracts

Abstract Jatropha curcas seed shells are the by-product obtained during oil extraction process. Recently, its chemical composition has gained attention since its potential applications. The aim of this study was to identify phenolic compounds profile from a non-toxic J. curcas shell from Mexico, besides, evaluate J. curcas shell methanolic extract (JcSME) antioxidant activity. Free, conjugate and bound phenolics were fractionated and quantified (606.7, 193.32 and 909.59 μg/g shell, respectively) and 13 individual phenolic compounds were detected by HPLC. The radical-scavenging activity of JcSME was similar to Trolox and ascorbic acid by DPPH assay while by ABTS assay it was similar to BHT. Effective antioxidant capacity by ORAC was found (426.44 ± 53.39 μmol Trolox equivalents/g shell). The Mexican non-toxic J. curcas shell is rich in phenolic compounds with high antioxidant activity; hence, it could be considerate as a good source of natural antioxidants.

Antihypertensive Effect of Protein Hydrolysate from Azufrado Beans in Spontaneously Hypertensive Rats

The objective of this study was to evaluate the antihypertensive potential of common bean protein hydrolysate. Protein concentrates were obtained, followed by Alcalase enzymatic hydrolysis, and then ultrafiltrated (3,000 molecular weight cutoff); the lyophilized product was named BP3. The angiotensin converting enzyme (ACE) inhibitory activity was determined as IC50 (3.68 ± 0.07 μg/mL). The antihypertensive effect was evaluated in spontaneously hypertensive rats (SHR) by two assays; Captopril ACE inhibitor was used as a reference compound and water as a control. A short-term assay showed a maximum decrease in mean arterial pressure of –41 ± 5 mmHg in SHR, 3 h after oral administration of 500 mg of BP3/kg of body weight (bw). In a long-term assay, a significant decrease in systolic blood pressure of –24 ± 5 mmHg was observed in SHR, after 45 days of oral administration of 500 mg of BP3/kg of bw/12 h. In both assays, BP3 treatment showed antihypertensive effect over SHR, similar to Captopril treatment. The ...