Purification and characterization of a novel angiotensin-I converting enzyme (ACE) inhibitory peptide derived from enzymatic hydrolysate of grass carp protein

Peptides inhibiting angiotensin-I converting enzyme (ACE, EC. 3.4.15.1) are possible cures of hypertension. Food-derived ACE-inhibitory peptides are particularly attractive because of reduced side effects. Previously, we reported ACE-inhibitory activity of grass carp protein hydrolysates. In this work, we report steps for purifying the ACE-inhibitory peptide from the hydrolysate and its biochemical properties. Following steps of ultrafiltration, macroporous adsorption resin, and two steps of reversed phase high performance liquid chromatography (RE-HPLC), a single Val-Ala-Pro (VAP) tripeptide was identified. The tripeptide with excellent ACE-inhibitory activity (IC(50) value of 0.00534 mg/mL) was a competitive ACE inhibitor and stable against both ACE and gastrointestinal enzymes of pepsin and chymotrypsin. This is the first report of food-derived VAP. The identified unique biochemical properties of VAP may enable the application of grass carp protein hydrolysates as a functional food for treatments of hypertension. The developed purification conditions also allow the production of VAP for pharmaceutical applications.     

Angiotensin I converting enzyme (ACE) inhibitory peptides from salmon byproduct protein hydrolysate by Alcalase hydrolysis

Angiotensin I converting enzyme (ACE) inhibitory peptides were produced from salmon byproduct proteins via enzymatic hydrolysis using Alcalase, flavourzyme, neutrase, pepsin, protamex and trypsin. Among them, Alcalase hydrolysate showed the highest ACE inhibitory activity, thus ACE inhibitory peptides were purified using consecutive chromatography. The purified ACE inhibitory peptides were identified to be Val-Trp-Asp-Pro-Pro-Lys-Phe-Asp (P1), Phe-Glu-Asp-Tyr-Val-Pro-Leu-Ser-Cys-Phe (P2), and Phe-Asn-Val-Pro-Leu-Tyr-Glu (P4) by time of flight-mass spectrometry/mass spectrometry (TOF-MS) analysis. The IC₅₀ values against ACE activity were 9.10 μM (P1), 10.77 μM (P2) and 7.72 μM (P4). The inhibition mode of P1, P2 and P4 was analyzed using the Lineweaver–Burk plots, demonstrating P1 to be a non-competitive inhibitor, P2 and P4 having a mixed inhibition mode. Taken together, the salmon byproduct protein hydrolysate and/or its active peptides can be used in foods for its benefits against hypertension and related diseases.     

Free radical scavenging and angiotensin-I converting enzyme inhibitory peptides from Pacific cod (Gadus macrocephalus) skin gelatin

Potent antioxidative peptides were purified from Pacific cod (Gadus macrocephalus) skin gelatin using alcalase, neutrase, papain, trypsin, pepsin, and α-chymotrypsin. Among them, the papain hydrolysate exhibited the highest antioxidant activity. Therefore, it was further purified and obtained two peptides with amino acid sequences of Thr-Cys-Ser-Pro (388 Da) and Thr-Gly-Gly-Gly-Asn-Val (485.5 Da). The antioxidant activity of the purified peptides was performed by electron spin resonance technique. Moreover, their intracellular free radical scavenging activity using 2′,7′-dichlorofluorescin diacetate and the protective effect against oxidation-induced DNA damage were evaluated in mouse macrophages (RAW 264.7 cells). Furthermore, both peptides have shown potential angiotensin-I converting enzyme inhibitory effect. The present study demonstrated that the peptides derived from Pacific cod (G. macrocephalus) skin gelatin could be used in the food industry as functional ingredients with potent antioxidative and antihypertensive benefits.     

Purification and characterization of angiotensin I converting enzyme (ACE) inhibitory peptides from Alaska pollack (Theragra chalcogramma) skin

Proteolytic digestion of gelatin extracts from Alaska Pollack (Theragra chalcogramma) skin brings about a high angiotensin I converting enzyme (ACE) inhibitory activity. Gelatin extracts were hydrolyzed by serial protease-treatments in the order of Alcalase, pronase E, and collagenase using a three-step recycling membrane reactor. Fragments arising from the third step were composed of peptides ranging from 0.9 to 1.9 kDa and responsible for ACE inhibitory activity. Catalytically active two peptides were separated by the consecutive chromatographic methods including gel filtration, ion-exchange chromatography, and reverse-phase high performance liquid chromatography. The isolated peptides were composed of Gly-Pro-Leu and Gly-Pro-Met and showed IC₅₀ values of 2.6 and 17.13 μM, respectively. These results suggested that Gly-Pro-Leu would be useful as a new antihypertensive agent.     

Purification and molecular docking study of angiotensin I-converting enzyme (ACE) inhibitory peptides from hydrolysates of marine sponge Stylotella aurantium

Angioteinsin I-converting enzyme (ACE) inhibitory peptide was isolated from marine sponge (Stylotella aurantium) hydrolysate prepared by various hydrolysis enzymes. The peptic hydrolysate exhibited highest ACE inhibitory activity among them and was fractionated into three ranges of molecular weight. The below 5 kDa fraction showed the highest ACE inhibitory activity and was used for subsequent purification steps. The amino acid sequences of the purified peptides were identified to be Tyr-Arg (337.2 Da), and Ile-Arg (287.2 Da). The purified peptides from marine sponge had an IC₅₀ value of 237.2 μM and 306.4 μM, respectively. The molecular docking study revealed that ACE inhibitory activity of the purified peptides was mainly attributed to the hydrogen bond interactions and Pi interaction between the dipeptides and ACE. The results suggest that marine sponge, S. aurantium would be an attractive raw material for the manufacture of anti-hypertensive nutraceutical ingredients.     

Production of active angiotensin-I converting enzyme inhibitory peptides derived from bovine beta-casein by recombinant DNA technologies

A wide range of anti-hypertensive peptides potentially able to lower blood pressure through the inhibition of vasoactive enzymes such as angiotensin-I converting enzyme (ACE) are known. Currently, ACE-inhibitory peptides can be produced from precursor proteins via enzymatic hydrolysis by proteolytic enzymes, or food fermentation with proteolytic starter cultures. These approaches are neither selective nor easy. In this study a novel procedure has been developed, based on recombinant DNA technologies, for the production of highly purified fractions of three polypeptides derived from bovine beta-casein active as ACE inhibitors in vitro. The procedure includes peptide expression in Escherichia coli cells as recombinant fusion proteins, purification by affinity chromatography, cleavage by proteinase from a selected strain of Lactobacillus helveticus and isolation of bioactive peptides (BPs). The reported concentration of inhibitor needed to reduce at 50% ACE activity (IC(50)) values for single BP calculated in inhibiting the ACE enzyme gave results in agreement with the same parameters available in literature for other milk-derived BPs. This procedure could be used to obtain quantities of pure peptides to determine their interactions with ACE, with the aim of designing peptides that have stronger inhibitory properties and exhibit new pharmacological profiles. Moreover, its scale up would be of commercial interest for the production of functional foods, e.g., milk beverages with blood pressure-lowering effects.     

Purification and identification of antioxidant peptides from egg white protein hydrolysate

Egg white proteins were hydrolysed separately using five different proteases to obtain antioxidant peptides. The antioxidant activity of egg white protein hydrolysates was influenced by the time of hydrolysis and the type of enzyme. Of the various hydrolysates produced, papain hydrolysate obtained by 3-h hydrolysis (PEWPH) displayed the highest DPPH radical scavenging activity. PEWPH could also quench the superoxide anion and hydroxyl radicals, effectively inhibit lipid peroxidation and exhibit reducing power. Then, PEWPH was purified sequentially by ultrafiltration, gel filtration, RP-HPLC and two fractions with relatively strong antioxidant activity were subsequently subjected to LC-MS/MS for peptide sequence identification. The sequences of the two antioxidant peptides were identified to be Tyr-Leu-Gly-Ala-Lys (551.54?Da) and Gly-Gly-Leu-Glu-Pro-Ile-Asn-Phe-Gln (974.55?Da), and they were identified for the first time from food-derived protein hydrolysates. Last, the two purified peptides were synthesized and they showed 7.48- and 6.02-fold higher DPPH radical scavenging activity compared with the crude PEWPH, respectively. These results indicate that PEWPH and/or its isolated peptides may be useful ingredients in food and nutraceutical applications.     

Purification and identification of adipogenesis inhibitory peptide from black soybean protein hydrolysate

Adipogenesis inhibitory peptide was isolated and identified from black soybean (Rhynchosia volubilis Lour.) hydrolysate. An adipogenesis inhibitor was purified using consecutive methods including: ultrafiltration (MWCO; 3 and 10kDa), gel filtration chromatography (Superdex Peptide 10/300 GL column), and reverse-phase high-performance liquid chromatography (microBondapak C(18) column). Also, the adipogenesis inhibition effect of the purified peptide was measured by observation of droplet of 3T3-L1 adipocyte by Oil Red O staining in the highest active fraction in each step. The peptide was shown to inhibit the differentiation of the 3T3-L1 pre-adipocyte, which was confirmed by morphological study. The adipogenesis inhibitory peptide was purified 71.43-fold from black soybean hydrolysate throughout a five-step purification procedure. The adipogenesis inhibitor was identified to be a tripeptide, Ile-Gln-Asn, having an IC(50) value of 0.014 mg protein/ml. Furthermore, the synthetic tripeptide (Ile-Gln-Asn) exhibited the similar adipogenesis effects to the purified peptide. Thus, these results showed the potential anti-obesity effect of the purified peptide through control of adiposity.     

Cryopreservation of a marine microalga, Nannochloropsis oculata (Eustigmatophyceae)

The cryopreservation of algae could prevent genetic drift and minimize labor costs compared to the current method of maintenance and subculturing. Clear, simple protocols for cryopreservation of marine microalga, Nannochloropsis oculata were developed and cryoprotectant choice and concentration optimized. The viability of the microalga was assessed directly after thawing, and algal concentration was measured after 2-30 days of growth. Five cryoprotectants (dimethyl sulphoxide, Me2SO; ethylene glycol, EG; glycerol, Gly; methanol, MeOH; and propylene glycol, PG) at five concentrations (10, 20, 30, 40, and 50%; v/v) were evaluated to determine the toxicity of various cryoprotectants to N. oculata. The toxicity of cryoprotectant (Me2SO, EG, MeOH, and PG) was observed only at higher concentrations of CPAs: > 20% for EG, > 30% for Me2SO and methanol, and > 40% for PG. Direct freezing of algae in liquid nitrogen resulted in a severe loss of viability and a modified cryopreservation protocol proved to be more appropriate for the preservation of N. oculata. Cryopreservation protocols developed and tested in the present study might be applied to cryopreserving other strains, or species, in this genus.     

An angiotensin-I converting enzyme inhibitor from buckwheat (Fagopyrum esculentum Moench) flour

A compound that inhibited angiotensin-I converting enzyme (ACE) activity was isolated from buckwheat powder. This compound is thought to be the hydroxy derivative of nicotianamine and its chemical structure is 2'-hydroxynicotianamine. This compound showed a very high inhibitory activity toward ACE, and the IC(50) was 0.08 microM. Only this hydroxy analog was found in buckwheat powder, at about 30 mg/100g, and no nicotianamine was detected. However, nicotianamine was detected in the buckwheat plant body. 2'-hydroxynicotianamine was also found in other polygonaceous plants.     

Using polyethylene glycol as nonionic osmoticum to promote growth and lipid production of marine microalgae Nannochloropsis oculata

To promote the economic feasibility of Nannochloropsis oculata, efficacy of using polyethylene glycol (PEG) to increase microalgal growth and lipid accumulation was investigated. We first examined the effects of PEG concentrations on microalgal growth using 0–5 % (w/v) PEG-6000, and followed by exploring the effects of PEG molecular weights (400, 600, 2,000, 4,000, 6,000, and 20,000) on microalgal growth, size, as well as on yields of biomass, total lipids, and eicosapentaenoic acid. In addition, the capacity of PEG to reduce the effect of oxygen inhibition on microalgal growth was also investigated to evaluate its adaptability for use in large-scale and closed setting. Our results showed that PEG-induced osmotic stress (Π) in the range of 2.465–2.472 MPa can raise microalgal growth. The PEG with higher molecular weight exhibited greater efficacy of growth promotion but less lipid content under equal concentration. In this study, 0.5 % (w/v) PEG-20000 (Π = 2.466 MPa) remarkably enhanced microalgal growth without interference of intracellular lipid productivity and cellular size, yielding >50 % (w/w) increases in biomass, total lipid, and eicosapentaenoic acid amounts after 7 days that provided the optimal condition for microalgal cultivation. These positive effects possibly resulted from the moderate enhancement of osmotic stress in the medium and stronger chaotrope-like behavior from higher molecular weight PEG. With further verification that 0.5 % (w/v) PEG-20000 enabled to reduce the effect of oxygen inhibition on microalgal growth, the PEG-20000-mediated cultivation offers a feasible means for mass culture of N. oculata in closed setting.     

Novel angiotensin I-converting enzyme inhibitory peptides isolated from Alcalase hydrolysate of mung bean protein.

Mung bean protein isolates were hydrolyzed for 2 h by Alcalase. The generated hydrolysate showed angiotensin I-converting enzyme (ACE) inhibitory activity with the IC(50) value of 0.64 mg protein/ml. Three kinds of novel ACE inhibitory peptides were isolated from the hydrolysate by Sephadex G-15 and reverse-phase high performance liquid chromatography (RP-HPLC). These peptides were identified by amino acid composition analysis and matrix assisted-laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF MS/MS), as Lys-Asp-Tyr-Arg-Leu, Val-Thr-Pro-Ala-Leu-Arg and Lys-Leu-Pro-Ala-Gly-Thr-Leu-Phe with the IC(50) values of 26.5 microM, 82.4 microM and 13.4 microM, respectively.     

Identification and molecular docking of novel ACE inhibitory peptides from protein hydrolysates of shrimp waste

The effect of enzymatic hydrolysis by Savinase on the interfacial properties and antihypertensive activity of shrimp waste proteins was evaluated. The physicochemical characterization, interfacial tension, and surface characteristics of shrimp waste protein hydrolysates (SWPH) using different enzyme/substrate (E/S) (SWPH₅ (SWPH using E/S = 5), SWPH₁₅ (SWPH using E/S = 15), and SWPH₄₀ (SWPH using E/S = 40)) were also studied. SWPH₅, SWPH₁₅, and SWPH₄₀ had an isoelectric pH around 2.07, 2.17, and 2.54 respectively. SWPH₅ exhibited the lowest interfacial tension (68.96 mN/m) followed by SWPH₁₅ (69.36 mN/m) and SWPH₄₀ (70.29 mN/m). The in vitro ACE inhibitory activity of shrimp waste protein hydrolysates showed that the most active hydrolysate was obtained using an enzyme/substrate of 15 U/mg (SWPH₁₅). SWPH₁₅ had a lower IC₅₀ value (2.17 mg/mL) than that of SWPH₅ and SWPH₄₀ (3.65 and 5.7 mg/mL, respectively). This hydrolysate was then purified and characterized. Fraction F1 separated by Sephadex G25 column which presents the best ACE inhibition activity was then separated by reversed‐phase high performance liquid chromatography. Four ACE inhibitory peptides were identified and their molecular masses and amino acid sequences were determined using ESI–MS and ESI–MS/MS, respectively. The structures of the most potent peptides were SSSKAKKMP, HGEGGRSTHE, WLGHGGRPDHE, and WRMDIDGDIMISEQEAHQR. The structural modeling of anti‐ACE peptides from shrimp waste through docking simulations results showed that these peptides bound to ACE with high affinity.     

Purification,modification and inhibition mechanism of angiotensin I-converting enzyme inhibitory peptide from silkworm pupa (Bombyx mori) protein hydrolysate

Angiotensin I-converting enzyme (ACE) inhibitory peptide from silkworm pupa (Bombyx mori) was purified, modified, as well as inhibition mechanism by using molecular docking analysis. Silkworm pupa protein was hydrolyzed by neutral protease and the obtained hydrolysate was subjected to various types of chromatography to acquire peptide isolate. Then the molecular mass and amino acid sequence of the peptide was determined by MALDI-TOF/TOF MS. Subsequently, thermal and digestive stability of the peptide were explored through a high temperature processing and a simulated gastrointestinal digestion. Finally, the peptide was modified to smaller peptides and investigated their potentiate activities. Results showed that the peptide from silkworm pupa was determined to be Gly-Asn-Pro-Trp-Met (603.7 Da) with IC₅₀ 21.70 μM. Stability testing showed that ACE inhibitory activities were not significantly changed at temperature from 40 to 80 °C as well as during in vitro gastrointestinal digestion. The inhibitory activity of four modified peptides were Trp-Trp > Gly-Asn-Pro-Trp-Trp > Asn-Pro-Trp-Trp > Pro-Trp-Trp, and the IC₅₀ of Trp-Trp was 10.76 μM Docking simulation revealed that the inhibitory activity was closely related to the spatial structure of peptide and zinc ions. The purified peptide and four modified peptides may be beneficial as functional food or drug for treating hypertension.     

Isolation of angiotensin converting enzyme (ACE) binding protein from human serum with an ACE affinity column.

Immobilized angiotensin-converting enzyme (ACE) was utilized as an affinity ligand to isolate a naturally occurring ACE binding protein from normal human serum. The enzyme was isolated from solubilized bovine lung membrane preparations by lisinopril affinity chromatography. It had an estimated molecular weight of 180 000 and was recognized by the anti-ACE antibody for the rabbit testicular ACE in immunoblots. ACE was immobilized onto epoxy Sepharose as well as Affi-Gel 15. Immobilized ACE on Affi-Gel 15 had higher catalytic activity (0.176 U/mL) compared with the enzyme immobilized on epoxy Sepharose (0.00005 U/mL). Immobilized ACE served as the affinity ligand for the identification of the ACE binding protein in human serum with an estimated molecular weight of 14 000 as observed by SDS polyacrylamide gel electrophoresis. The identification and further characterization of ACE binding proteins in serum and tissues may facilitate the greater understanding of the endogenous regulation of this key enzyme, which is involved in blood pressure homeostasis.     

Analysis of novel angiotensin-I-converting enzyme inhibitory peptides from protease-hydrolyzed marine shrimp Acetes chinensis.

Acetes chinensis is an underutilized shrimp species thriving in the Bo Hai Gulf of China. In a previous study, we had used the protease from Bacillus sp. SM98011 to digest this kind of shrimp and found that the oligopeptide-enriched hydrolysate possessed antioxidant activity and high angiotensin I-converting enzyme (ACE) inhibitory activity with an IC50 value of 0.97 mg/ml. In this paper, by ultrafiltration, gel permeation chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC), five peptides with high ACE inhibitory activity were purified from the shrimp hydrolysates and their sequences were identified by amino acid composition analysis and molecular weight (MW) analysis. Three of them, FCVLRP (a), IFVPAF (f) and KPPETV (j), were novel ACE inhibitory peptides. Their IC50 values were 12.3 microM, 3.4 microM and 24.1 microM, respectively, and their recoveries were 30 mg/100 g (solid basis of shrimp), 19 mg/100 g and 33 mg/100 g, respectively. Lineweaver-Burk plots for the three novel peptides showed that they are all competitive inhibitors. To test the ACE inhibitory activity of peptide a, f, j after they were digested by digestive enzymes in vivo, 12 derived peptides from FCVLRP and IFVPAF were synthesized based on their amino acid sequences and the cleavage sites of digestive enzymes. No digestive enzyme cleavage site was found in KPPETV. The IC50 values of the derived peptides were determined and the result showed that except for VPAF, FC and FCVL, the ACE inhibitory activity of the other nine derived peptides did not significantly change when compared with their original peptides. Surprisingly, five peptides had lower IC50 values than their original peptides, particularly for RP (IC50 value = 0.39 microM), which is about 30 times lower than its original peptide and almost the lowest IC50 value for ACE inhibitory peptides reported. Therefore, the novel peptides identified from A. chinensis hydrolysates probably still maintain a high ACE inhibitory activity even if they are digested in vivo. This is the first report about novel ACE inhibitory peptides from hydrolysates of marine shrimp A. chinensis. The novel peptides from hydrolysate of A. chinensis and some of their derived peptides with high ACE inhibitory activity probably have potential in the treatment of hypertension or in clinical nutrition.     

Purification and characterization of angiotensin I converting enzyme inhibitory peptides from the rotifer, Brachionus rotundiformis

Angiotensin I converting enzyme (ACE) inhibitory peptide was isolated from the marine rotifer, Brachionus rotundiformis. ACE inhibitory peptides were separated from rotifer hydrolysate prepared by Alcalase, α-chymotrypsin, Neutrase, papain, and trypsin. The Alcalase hydrolysate had the highest ACE inhibitory activity compared to the other hydrolysates. The IC₅₀ value of Alcalase hydrolysate for ACE inhibitory activity was 0.63 mg/ml. We attempted to isolate ACE inhibitory peptides from Alcalase prepared rotifer hydrolysate using gel filtration on a Sephadex G-25 column and high performance liquid chromatography on an ODS column. The IC₅₀ value of purified ACE inhibitory peptide was 9.64 μM, and Lineweaver–Burk plots suggest that the peptide purified from rotifer protein acts as a competitive inhibitor against ACE. Amino acid sequence of the peptide was identified as Asp-Asp-Thr-Gly-His-Asp-Phe-Glu-Asp-Thr-Gly-Glu-Ala-Met, with a molecular weight 1538 Da. The results of this study suggest that peptides derived from rotifers may be beneficial as anti-hypertension compounds in functional foods resource.     

Angiotensin-I-converting enzyme inhibitory peptides from tryptic hydrolysate of bovine alphaS2-casein

Angiotensin-I-converting enzyme (ACE) inhibitory activity of a tryptic digest of bovine alpha(S2)-casein (alpha(S2)-CN) was extensively investigated. Forty-three peptide peaks were isolated and tested. Seven casokinins (i.e. CN-derived ACE inhibitory peptides) were identified and their IC50 values were determined. Four peptides exhibited an IC50 value lower than 20 microM. Peptides alpha(S2)-CN (f174-181) and alpha(S2)-CN (f174-179) had IC50 values of 4 microM. Surprisingly, deletion of the C-terminal dipeptide of two of these casokinins did not significantly alter their inhibitory activity.     

The utilization of bicarbonate ions by the marine microalga Nannochloropsis oculata (Droop) Hibberd

HCO₃^? utilization by the marine microalga Nannochloropsis oculata was investigated using a pH drift technique in a closed system. Light-dependent alkalization of the medium resulted in a final pH of 10.5, confirming substantial HCO₃^? use by this alga. Alkalinity remained constant throughout the pH drift. Measurement of dissolved inorganic carbon (DIC) or the uptake of H¹⁴CO₃^? showed that nearly 50% of the total DIC remained external to the plasma membrane on completion of a pH drift. The rate of light-driven alkalization was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and thus was dependent on photosynthesis. Light-driven alkalization was not inhibited by a membrane-impermeable inhibitor of carbonic anhydrase (CA), dcxtran-bound sulphonamide (DBS), indicating that external CA was not involved in HCO₃^? utilization. The anion-cxchangc inhibitor 4′,4′-diisothiocyanostilbene-2,2-disulphonic acid (DIDS) completely inhibited light-driven alkalization of the medium and H¹⁴CO₃^? uptake, providing unequivocal support for a direct uptake of H¹⁴CO₃^?. Chloride ions were essential for DIC-dependent photosynthetic oxygen evolution, suggesting that bicarbonate transport occurs by HCO₃^?/CI^? exchange.