Purification and identification of novel angiotensin-I converting enzyme (ACE) inhibitory peptides from cultured marine microalgae (Nannochloropsis oculata) protein hydrolysate

Isolation of bioactive compounds and commercialization of marine microalgae sources are interesting targets in future marine biotechnology. Cultured biomass of the marine microalga, Nannochloropsis oculata, was used to purify angiotensin-I converting enzyme (ACE) inhibitory peptides using proteases including pepsin, trypsin, α-chymotrypsin, papain, alcalase, and neutrase. The pepsin hydrolysate exhibited the highest ACE inhibitory activity, compared to the other hydrolysates and then was separated into three fractions (F1, F2, and F3) using Sephadex G-25 gel filtration column chromatography. First fraction (F1) showed the highest ACE inhibitory activity and it was further purified into two fractions (F1-1 and F1-2) using reverse-phase high-performance liquid chromatography. The IC₅₀ value of purified ACE inhibitory peptides were 123 and 173 μM and identified as novel peptides, Gly-Met-Asn-Asn-Leu-Thr-Pro (GMNNLTP; MW, 728 Da) and Leu-Glu-Gln (LEQ; MW, 369 Da), respectively. In addition, nitric oxide production level (%) was significantly increased by the purified peptide (Gly-Met-Asn-Asn-Leu-Thr-Pro) compared to the purified peptide (Leu-Glu-Gln) and other treated pepsin hydrolysate fractions on human umbilical vein endothelial cells (HUVECs). Cell viability assay showed no cytotoxicity on HUVECs with the treated purified peptides and fractions. These results suggest that the isolated peptides from cultured marine microalga, N. oculata protein sources may have potentiality to use commercially as ACE inhibitory agents in functional food industry.     

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.     

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.     

Direct Resolution of dl-Lysine-3, 5-Dimtrobenzoate

The inhibitory activity of an angiotensin I-converting enzyme (ACE) detected in soy sauce was fractionated into two major fractions of high molecular weight (Hw) and low molecular weight (Lw) by gel filtration chromatography on Bio-gel P-2 after treating with ethanol. The Hw fraction reduced the blood pressure in hypertensive rats after orally administering, while the Lw fraction did not. The ACE inhibitor in the Hw fraction was further purified by Dowex 50W ion-exchange chromatography and four subsequent steps of HPLC. On the basis of the SIMS-mass spectrum, NMR spectrum and other characteristics, the purified ACE inhibitor was identified as nicotianamine (N-[N-(3-amino-3-carboxypropyl)-3-amino-3- carboxypropyl]azetidine-2-carboxylic acid). The IC₅₀ value for this ACE was 0.26 µM.     

Purification and Characterization of an Alkaline Protease from the Marine Yeast <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> for Bioactive Peptide Production from Different Sources

The extracellular alkaline protease in the supernatant of cell culture of the marine yeast Aureobasidium pullulans 10 was purified to homogeneity with a 2.1-fold increase in specific protease activity as compared to that in the supernatant by ammonium sulfate fractionation, gel filtration chromatography (Sephadex™ G-75), and anion-exchange chromatography (DEAE Sepharose Fast Flow). According to the sodium dodecyl sulfate-polyacrylamide gel electrophoresis data, the molecular mass of the purified enzyme was estimated to be 32.0 kDa. The optimal pH and temperature of the purified enzyme were 9.0 and 45°C, respectively. The enzyme was activated by Cu²⁺ (at a concentration of 1.0 mM) and Mn²⁺ and inhibited by Hg²⁺, Fe²⁺, Fe³⁺, Zn²⁺, and Co²⁺. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride, but weakly inhibited by EDTA, 1–10-phenanthroline, and iodoacetic acid. The K _m and V _max values of the purified enzyme for casein were 0.25 mg/ml and 0.0286 μmol/min/mg of protein, respectively. After digestion of shrimp protein, spirulina (Arthospira platensis) protein, proteins of marine yeast strains N3C (Yarrowia lipolytica) and YA03a (Hanseniaspora uvarum), milk protein, and casein with the purified alkaline protease, angiotensin I converting enzyme (ACE) inhibitory activities of the resulting peptides reached 85.3%, 12.1%, 29.8%, 22.8%, 14.1%, and 15.5%, respectively, while the antioxidant activities of these were 52.1%. 54.6%, 25.1%, 35%, 12.5%, and 24.2%, respectively, indicating that ACE inhibitory activity of the resulting peptides from the shrimp protein and antioxidant activity of those produced from the spirulina protein were the highest, respectively. These results suggest that the bioactive peptides produced by digestion of the shrimp protein with the purified alkaline protease have potential applications in the food and pharmaceutical industries.     

Purification and characterization of angiotensin I-converting enzyme inhibitory peptide from enzymatic hydrolysates of Styela clava flesh tissue

Angiotensin I-converting enzyme (ACE) inhibitory peptide was isolated from the Styela clava flesh tissue. Nine proteases (Protamex, Kojizyme, Neutrase, Flavourzyme, Alcalase, pepsin, trypsin, α-chymotrypsin and papain) were used, and their respective enzymatic hydrolysates and an aqueous extract were screened to evaluate their potential ACE inhibitory activity. Among all of the test samples, Protamex hydrolysate possessed the highest ACE inhibitory activity, and the Protamex hydrolysate of flesh tissue showed relatively higher ACE inhibitory activity compared with the Protamex hydrolysate of tunic tissue. We attempted to isolate ACE inhibitory peptide from the Protamex hydrolysate of S. clava flesh tissue using ultrafiltration, gel filtration on a Sephadex G-25 column and high performance liquid chromatography (HPLC) on an ODS column. The purified ACE inhibitory peptide exhibited an IC₅₀ value of 37.1 μM and was identified as non-competitive inhibitor of ACE. Amino acid sequence of the peptide was identified as Ala-His-Ile-Ile-Ile, with a molecular weight 565.3 Da. The results of this study suggested that the peptides derived from enzymes-assisted extracts of S. clava would be useful new antihypertension compounds in functional food resource.     

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.     

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.     

Synergistic Effect of α-Hexachlorocy-clohexane with Pyrethrins

The more potent inhibitory activity against angiotensin-converting enzyme (ACE) was excised from a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) preparation of Bacillus stearothermophilus by heating at 120°C in 1 m AcOH–20mm HCI, as compared with GAPDH preparations of yeast and pig. Sufficient excision of B. stearothermophilus ACE inhibitors required a longer proteolysis time of 60 min. Two inhibitors were then purified by gel-permeation and reverse-phase chromatog-raphies. One of the B. stearothermophilus ACE inhibitors, BG-1, was the GAPDH peptide 68–77 (Gly-Lys-Glu-Ile-Ile-Val-Lys-Ala-Glu-Arg, IC₅₀: 32 μm). Another inhibitor, BG-2 (Gly-Lys-Met-Val-Lys-Val-Val-Ser-Trp-Tyr, IC₅₀: 6 μM), corresponded to GAPDH peptide 304–313. These sequences were quite different from those of vertebrate GAPDH peptides and the venom peptide family with ACE inhibitory activity. BG-2 was found to be a non-competitive type inhibitor, differing from many natural peptide inhibitors. Thus, B. stearothermophilus GAPDH seemed to be a good source of new type ACE inhibitors, in addition to the advantages due to its thermophilic property.     

Critical evaluation of the use of bioinformatics as a theoretical tool to find high-potential sources of ACE inhibitory peptides

A bioinformatics analysis to screen for high-potential sources of angiotensin converting enzyme (ACE) inhibitory peptides was conducted in the area of insect muscle proteins. Vertebrate muscle proteins are reported as good sources of ACE inhibitory peptides, while the research on invertebrate muscle proteins is limited. A phylogenetic tree constructed with actin sequences of both vertebrate and invertebrate species indicated a high homology. Furthermore, a quantitative in silico ACE inhibition analysis suggested that actin proteins of invertebrates have potentials as new sources of ACE inhibitory peptides. On one insect, Bombyx mori, a more detailed in silico analysis was done followed by a small experimental study. The in silico analysis indicated B. mori as a high-potential source of ACE inhibitory peptides and this was supported by the ACE inhibitory activity of the partially purified actin preparation. In conclusion, in food science, in silico analysis can be used as fast initial screening tool to look for high-potential sources of ACE inhibitory peptides and other peptidic bioactivities.     

Structure and ACE-Inhibitory Activity of Peptides Derived from Hen Egg White Lysozyme

Angiotensin I-converting enzyme plays an important role in hypertension and therefore its inhibition is considered to be a useful procedure in the prevention of hypertension. Two novel ACE inhibitory peptides were purified and identified from the papain-trypsin hydrolysate of hen egg white lysozyme using reverse phase-high performance liquid chromatography. The sequences of identified peptides were NTDGSTDYGILQINSR (MW: 1,753.98?±?0.5?Da) and VFGR (MW: 459.26?±?0.5?Da), which were named F2 and F9 peptide, respectively. Analyses of the far-UV CD spectra of ACE in the absence and presence of the F2 peptide revealed ACE secondary structural changes. In the presence of the F2 peptide, a loss of helical content of ACE was observed, which can lead to decrease of the enzymatic activity. Lineweaver?CBurk plots show that the identified peptides both act as non-competitive ACE inhibitors. These findings would be helpful on the understanding of interaction between ACE and its inhibitory peptides.     

Peptidomic Analysis of ACE Inhibitory Peptides Extracted from Fermented Goat Milk

Angiotensin converting enzyme (ACE) is considered as main causative agent in growing hypertension and other cardiovascular disorders. Inhibition of ACE by producing and purifying bioactive peptides of fermented goat milk is aimed in this study. Protein extracted from goat milk was hydrolyzed with proteolytic enzymes of LH (Lactobacillus helveticus-cicc22171). ACE inhibitory peptides were purified from fermented samples of goat milk protein by optimizing incubation time to 8 h (S-8), 16 h (S-16), 24 h (S-24) and 36 h (S-36), via ultrafiltration. S-8 was used as control to compare the ACE inhibition trend. Molecular weight cut-off; 10000 Da (PM-10) and Ultracel 3K membrane was used to perform ultrafiltration. Sample with 24 h incubation time was considered as best hydrolyzed as compared to others, by applying Nin-Hydrin reaction and SDS-PAGE analysis. ACE inhibitory assay validated the authenticity of S-24 in inhibiting ACE, in vitro. Furthermore, Q executive hybrid quadrupole-orbitrap mass spectrometry was used to determine molecular structure and amino acid sequence of ACE inhibitory peptides. Three peptides, VLPVPQKAVPQ, VLPVPQKVVPQ and TQTPVVVPPFLQPEIMGVPKVKE containing functional amino acid structure, has been identified with highest ACE inhibitory activity on the basis of intensity, size and higher concentration of hydrophobic amino acids as shown in figure as graphical abstract. Fermented goat milk containing these novel bioactive peptides, can be used as nutraceuticals to inhibit ACE and control hypertension in future.

Graphical Abstract

     

Preparation and characterization of novel bioactive peptides responsible for angiotensin I‐converting enzyme inhibition from wheat germ

Reported is the preparation of wheat germ (WG) hydrolyzate with potent angiotensin I‐converting enzyme (ACE) inhibitory activity, and the characterization of peptides responsible for ACE inhibition. Successful hydrolyzate with the most potent ACE inhibitory activity was obtained by 0.5 wt.%–8 h Bacillus licheniformis alkaline protease hydrolysis after 3.0 wt.%–3 h α‐amylase treatment of defatted WG (IC₅₀; 0.37 mg protein ml⁻¹). The activity of WG hydrolyzate was markedly increased by ODS and subsequent AG50W purifications (IC₅₀; 0.018 mg protein ml⁻¹). As a result of isolations by high performance liquid chromatographies, 16 peptides with the IC₅₀ value of less than 20 μm , composed of 2–7 amino acid residues were identified from the WG hydrolyzate. Judging from the high content (260 mg in 100 g of AG50W fraction) and powerful ACE inhibitory activity (IC₅₀; 0.48 μm ), Ile‐Val‐Tyr was identified as a main contributor to the ACE inhibition of the hydrolyzate. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Production of novel angiotensin I-converting enzyme inhibitory peptides by fermentation of marine shrimp <Emphasis Type="Italic">Acetes chinensis</Emphasis> with <Emphasis Type="Italic">Lactobacillus fermentum</Emphasis> SM 605

Acetes chinensis is an underutilized shrimp species thriving in Bo Hai Gulf of China. Its hydrolysate digested with protease SM98011 has been previously shown to have high angiotensin I-converting enzyme (ACE) inhibitory activity (He et al., J Pept Sci 12:726-733, 2006). In this article, A. chinensis were fermented by Lactobacillus fermentum SM 605 and the fermented sauce presented high ACE inhibitory activity. The minimum IC(50) value (3.37 +/- 0.04 mg/mL) was achieved by response surface methodology with optimized process parameters such as fermentation time of 24.19 h, incubation temperature at 38.10 degrees C, and pH 6.12. Three ACE inhibitory peptides are purified by ultrafiltration, gel filtration, and reverse-phase high performance liquid chromatography. Identified by mass spectrometry, their amino acid sequences are Asp-Pro, Gly-Thr-Gly, and Ser-Thr, with IC(50) values of 2.15 +/- 0.02, 5.54 +/- 0.09, and 4.03 +/- 0.10 microM, respectively. Also, they are all novel ACE inhibitory peptides. Compared with protease digestion, fermentation is a simpler and cheaper method to produce ACE inhibitory peptides from shrimp A. chinensis.     

Availability of tyrosine amide for α-chymotrypsin-catalyzed synthesis of oligo-tyrosine peptides

Oligo-tyrosine peptides such as Tyr-Tyr having angiotensin I-converting enzyme (ACE) inhibitory activity could be synthesized by α-chymotrypsin-catalyzed reaction with l-tyrosine ethyl ester in aqueous media. However, peptide yield in the reaction was below 10%. Since l-tyrosine amide showed highly nucleophilic activity for the deacylation of enzyme through which a new peptide bond was made, its application to the enzymatic peptide synthesis was evaluated in this study. Addition of tyrosine amide into the reaction produced Tyr-Tyr-NH₂, of which yield exceeded 130% on the basis of tyrosine ethyl ester. Although purified Tyr-Tyr-NH₂ did not inhibit ACE activity, α-chymotrypsin could act on the dipeptide amide and convert about 40% of it to Tyr-Tyr. The use of both ester and amide forms of tyrosine is expected to be a potent procedure for α-chymotrypsin-catalyzed synthesis of antihypertensive peptides.     

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.     

Isolation and characterization of a novel angiotensin I-converting enzyme inhibitory peptide derived from the edible mushroom Tricholoma giganteum

The fruiting body of Tricholoma giganteum has many pharmaceutical uses and has long been utilized as a home remedy in Asia. This study describes the extraction and characterization of the first angiotensin I-converting enzyme (ACE) inhibitory peptide from T. giganteum. The maximum ACE inhibitory activity (IC50: 0.31 mg) was obtained when the fruiting body of T. giganteum was extracted with distilled water at 30 degrees C for 3 h. After the purification of ACE inhibitory peptides with ultrafiltration, Sephadex G-25 column chromatography, and reverse-phase HPLC, an active fraction with an IC50 of 0.04 mg and a yield of 0.3% was obtained. The ACE inhibitory peptide was a novel tripeptide, showing very low similarity to other ACE inhibitory peptide sequences, and was sequenced as Gly-Glu-Pro. The purified ACE inhibitor from T. giganteum competitively inhibited ACE, and it maintained inhibitory activity even after incubation with proteases. ACE inhibitor from T. giganteum showed a clear antihypertensive effect in spontaneously hypertensive rats (SHR), at a dosage of 1 mg/kg.     

Angiotensin I-converting enzyme inhibitory peptides in red-mold rice made by Monascus purpureus

The ACE inhibitory activity in red-mold rice extracts, prepared from 24 strains of the genus Monascus, was measured. The most effective strain for ACE inhibition was Monascus purpureus IFO 4489 (IC₅₀ = 0.71 mg/ml). Although the antihypertensive substance γ-amino butyric acid was detected in the red-mold rice (85.2 mg/kg), it did not contribute to ACE inhibition. Four ACE inhibitory peptides were isolated from the extract and identified as Ile-Tyr (IC₅₀ = 4.0 μM), Val-Val-Tyr (22.0 μM), Val-Phe (49.7 μM) and Val-Trp (3.1 μM) by protein sequencing. The ACE inhibitory activity of these peptides was almost completely preserved after successive in vitro digestion by pepsin, chymotrypsin and trypsin. These results suggest that red-mold rice made by M. purpureus could be useful in alleviating hypertension.     

Biotechnological production of the angiotensin‐converting enzyme inhibitory dipeptide isoleucine‐tryptophan

Peptides with angiotensin‐converting enzyme (ACE)‐inhibitory and antihypertensive effects are suggested as innovative food additives to prevent or treat hypertension. Currently, these substances are isolated from food proteins following nonselective hydrolysis as a mixture of ACE‐inhibitory peptides and other protein fragments. This study presents an innovative biotechnological method, based on recombinant DNA technology that was established to specifically produce the ACE‐inhibitory dipeptide isoleucine‐tryptophan. In a first step, a repetitive isoleucine‐tryptophan construct fused to the maltose‐binding protein was generated and expressed in Escherichia coli BL21 cells. The chromatographically purified recombinant fusion protein was enzymatically hydrolyzed using α‐chymotrypsin to liberate the dipeptide isoleucine‐tryptophan. The identity of the liberated isoleucine‐tryptophan was confirmed by MS and derivatization of its N‐terminus. The ACE‐inhibitory effect of the recombinant dipeptide on soluble and membrane bound ACE was found to be indistinguishable from the inhibitory potential of the chemically produced commercially available dipeptide. The established experimental strategy represents a promising approach to the biotechnical production of sufficient amounts of recombinant peptide‐based ACE‐inhibitory and antihypertensive substances that are applicable as functional food additives to delay or even prevent hypertension.