1. Please check and comment entries here.
Table of Contents

    Topic review

    Angiotensin-I-converting enzyme

    View times: 10
    Submitted by: Azis Boing Sitanggang


    The Angiotensin-I-converting enzyme (ACE) is a peptidase with a significant role in the regulation of blood pressure. 

    1. Introduction

    Hypertension is a high prevalence disease and is considered one of the major health problems globally [1]. Lim et al. [2] reported that cardiovascular diseases due to complications of hypertension account for 9.4 million deaths every year. It is therefore of importance to take the appropriate mitigations to reduce the mortality rate due to hypertension. Otherwise known as high blood pressure, hypertension is a medical condition where the arterial blood pressure (BP) is abnormally high. According to the 2019 ACC/AHA Guideline on the Primary Prevention of Cardiovascular Disease [3], a normal BP is described as having a systolic and diastolic pressure of less than 120 and 80 mmHg, respectively (BP < 120/80 mmHg). There are two stages of hypertension. Stage 1 is defined with BP 130–139/80–89 mmHg, while stage 2 hypertension is for BP ≥ 140/90 mm Hg [3]. As mentioned above, hypertension could lead to cardiovascular diseases and stroke. Hypertension is usually treated with blood pressure regulating drugs such as angiotensin-I-converting enzyme (ACE) inhibitors (e.g., lisinopril, captopril), vasodilator, etc. Given the side effects of synthetic ACE inhibitors (e.g., taste disturbances, cough, and swelling of the lower layer of human skin or angioneurotic edema) [4], various investigations have been afforded to find potent ACE inhibitors from natural products, especially from food proteins. As a result of the increasing interest regarding functional foods in the past few years, it has been reported that food proteins-derived bioactive peptides have several benevolent effects on human health, including inhibitory activity against ACE [5][6]. Therefore, bioactive peptides can be considered as an alternative for managing hypertension.
    A bioactive peptide is defined as an organic compound with a positive impact on human health (e.g., inhibitory activity against ACE, antioxidant capacity, antimicrobial activity, anti-thrombotic, immunomodulatory, etc.) which consists of 2–20 amino acids joined by covalent bonds called peptide bonds [7]. In the digestive system of the human body, bioactive peptides are liberated by digestive proteases, such as pepsin or microbial enzymes. Additionally, processing food and ripening can release bioactive peptides.
    Nature is an abundant source of bioactive peptides produced by organisms such as plants and animals. Although animal products remain the greatest source of bioactive peptides, this work will mainly discuss plant source bioactive peptides derived specifically from soybean and velvet beans. Soybean nutritional content consists of 35–40% protein, 20% lipids, and 9% dietary fiber based on dry-weight soybean [8][9]. Because of its high protein content, soybean is mostly utilized as a source of bioactive peptides among other plants. Meanwhile, a less well-known type of legumes called velvet bean has a nutritional content of approximately 25% protein and 14% crude fat based on its dry weight [10]. As both beans are considered as potent protein sources in the human diet, their utilization as sources of parent proteins for producing bioactive peptides is promising. However, in the case of velvet bean, studies related to its utilization as a parent protein source are scarce. Thus, it is important to elucidate the technological approach of producing velvet bean-derived peptides especially for inhibiting ACE activity.

    2. Substrate Preparation as Source of ACEi Peptides from Soybean and Velvet Bean

    The preparation of substrates from soybeans is rarely discussed in the literature. Substrates from soybeans as sources of parent proteins can be soy protein concentrate or isolate, soybean flour-rich in protein, and principal soybean storage proteins (i.e., glycinin or β-conglycinin). Gouda et al. [11] prepared the soy protein substrate, glycinin. This method follows a previously described method developed in a study by Rao and Rao [12] with the use of (NH4)2SO4 precipitation and centrifugation. Water containing β-mercaptoethanol (0.1% v/v) is used to extract defatted soybean flour for 4–6 h under constant agitation. The solution is then centrifuged at 6000–8000 rpm for 45 min at 25 °C, followed by the addition of dry MgCl2 until the final MgCl2 concentration in the solution reaches 5 mM. Glycinin is collected by centrifugation, and the precipitate is dried with a freeze drier. Freeze drying is used as a preferred water removal method because it has the advantage to cause less damage to the structure of the protein substrate. Nevertheless, the fractionation of glycinin in most studies involves the precipitation of the alkaline soy protein extract at pH 6.3–7.0 [13][14][15].
    For the preparation of the velvet bean substrate, wet fractionation is the method that is commonly used [16][17][18]. Initially, velvet bean flour is prepared by grounding the grains with a disk mill followed by sieving. The prepared bean flour then undergoes suspension in 3% sodium bisulfite with a 1:6 ratio (w:v) and left to soak for an hour with a constant agitation under alkaline pH (pH = 8). The role of sodium bisulfite is to increase the solubility of the velvet bean protein. Abtahi and Aminlari [19] stated that the modification of protein with a chemical treatment, such as sodium bisulfite, increases the protein dispersibility index (PDI). After fiber solid separation and washing with 3% sodium bisulfite, the protein-starch suspension is then left to sediment for 30 min. The purpose of sedimentation is to recover starch. The pH of protein solution pH is adjusted to an isoelectric point (i.e., pH 4.2) using 1.0 M HCl solution. The precipitate is obtained by centrifuging the solution at 1317× g for 20 min and further dried using a freeze-drier at −47 °C and pressure of 13 × 10−3 mbar [16][17][18]. In another study by Mugendi et al. [20] who characterized the nutritional properties of velvet bean protein isolate, the extraction was conducted with distilled water at pH 9 followed by centrifugation. The pH of the extract was then adjusted to 4.5 to precipitate the protein.

    3. Hydrolytic Conditions for Producing ACEi Peptides from Soybean and Velvet Bean Protein Substrates

    Enzymes for proteolysis are classified as endopeptidases and exopeptidases, based on the site of action on the substrate. Exopeptidases hydrolyze at the N- or C-terminal ends of the peptide, while endopeptidases cleave peptide bonds within and distant from the ends of a polypeptide chain or at the non-terminals of the sequence [21]. The most common enzymes used for producing soybean-based bioactive peptides are pepsin [22][23], papain [6][24], alcalase [25][26][27], proteinase from M. purpureus [28], trypsin, chymotrypsin, ginger protease, and Amano Protease from Aspergillus sp. [11], and protease D3 from E. coli strain JM109 [29]. All of these enzymes are endopeptidases. Endopeptidases, such as alcalase and proteinase K produce short-chain hydrophobic amino acids which are preferred in enhancing ACEi activity [21]. Additionally, prolyl endopeptidases such as Protease P from Aspergillus niger are often used as it can yield in proline-containing bioactive peptides which are favored for their strong affinity to ACE [30]. Hydrolytic conditions of soybean proteins for producing ACEi peptides are shown in Table 1.
    Table 1. Enzymatic hydrolysis conditions of soybean proteins using endopeptidases to produce ACEi peptides.
    Enzyme Substrate Temp. (°C) Time (h) pH Enzyme-to-Substrate Ratio E/S Peptide Sequence Ref.
    Pepsin Protein concentrate 37 24 2 6% IA Chen et al. [22]
    Pepsin Protein isolate 39 12 2 3% - Chen et al. [23]
      Acid-precipitated protein
    Alcalase Protein concentrate 50 12 9 4% - Wu & Ding [25]
    M. purpureus acid proteinase β-conglycinin 37 10 3.3 - LAIPVNKP Kuba et al. [28]
      Glycinin   SPYP
    Bovine trypsin Glycinin 37 18 8.2 2% VLIVP Gouda et al. [11]
    Bovine chymotrypsin 37 18 8.2
    Ginger protease 50 16 6
    Protease P (Amano-P from Aspergillus sp.) 37 18 8.2
    Protease D3 from E. coli strain JM109 Protein isolate 37–40 24–48 4.5 0.2% YVVFK Kodera & Nio [29]
    Pepsin Protein isolate 37 1 5.3 4% - Lo & Li-Chan [31]
    Pancreatin 37 2 7.5
    Alcalase Protein isolate 55 1 8 - - Rayaprolu et al. [26]
    Alcalase Protein isolate 30 0.25 9 6% - Li et al. [27]
    For velvet bean, the proteolytic enzymes reported limitedly in the literature are a combination of pepsin-pancreatin [16][17][18] and alcalase–flavourzyme [16][17]. In contrast to soybean-derived peptides, for velvet bean sourced peptides, the hydrolysis is conducted with a combination of both endopeptidase and exopeptidase. The application of both endo- and exo-peptidase allows it to have a broad cleavage action and produce a shorter chain of peptides. Table 2 shows the enzymatic hydrolysis conditions of velvet bean-derived proteins.
    Table 2. Enzymatic hydrolysis conditions of velvet bean protein concentrate to produce ACEi peptides.
    Enzyme Enzyme Type Hydrolysis Conditions Ref.
    Temp. (°C) Time (h) pH Enzyme-to-Substrate Ratio E/S
    Pepsin Endopeptidase 37 0.75 2 10% Herrera-Chale et al. [17]
    Pancreatin Exopeptidase 37 0.75 7.5
    Alcalase Endopeptidase 50 0.75 8
    Flavourzyme Exopeptidase 50 0.75 7
    Pepsin Endopeptidase 37 0.75 2 10% Tuz & Campos [18]
    Pancreatin Exopeptidase 37 0.75 7
    Pepsin Endopeptidase 37 0.75 2 10% Segura-Campos et al. [16]
    Pancreatin Exopeptidase 37 0.75 7
    Alcalase Endopeptidase 50 0.75 8
    Flavourzyme Exopeptidase 50 0.75 7

    The entry is from 10.3390/molecules26133822


    1. Mills, K.T.; Bundy, J.D.; Kelly, T.N.; Reed, J.E.; Kearney, P.M.; Reynolds, K.; Chen, J.; He, J. Global disparities of hypertension prevalence and control. Circulation 2016, 134, 441–450.
    2. Lim, S.S.; Vos, T.; Flaxman, A.D.; Danaei, G.; Shibuya, K.; Adair-Rohani, H.; Amann, M.; Anderson, H.R.; Andrews, K.G.; Aryee, M.; et al. A comparative risk assessment of burden of disease and injury attributable to 67 risk factors and risk factor clusters in 21 regions, 1990–2010: A systematic analysis for the global burden of disease study 2010. Lancet 2012, 380, 2224–2260.
    3. Arnett, D.K.; Blumenthal, R.S.; Albert, M.A.; Buroker, A.B.; Goldberger, Z.D.; Hahn, E.J.; Himmelfarb, C.D.; Khera, A.; Lloyd-Jones, D.; McEvoy, J.W.; et al. 2019 ACC/AHA Guideline on the primary prevention of cardiovascular disease: A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines. Circulation 2019, 140, e596–e646.
    4. Atkinson, A.; Robertson, J.I. Captopril in the treatment of clinical hypertension and cardiac failure. Lancet 1979, 314, 836–839.
    5. Sánchez, A.; Vázquez, A. Bioactive peptides: A review. Food Qual. Saf. 2017, 1, 29–46.
    6. Sitanggang, A.B.; Sumitra, J.; Budijanto, S. Continuous production of tempe-based bioactive peptides using an automated enzymatic membrane reactor. Innov. Food Sci. Emerg. Technol. 2021, 68, 102639.
    7. Chalamaiah, M.; Keskin Ulug, S.; Hong, H.; Wu, J. Regulatory requirements of bioactive peptides (protein hydrolysates) from food proteins. J. Funct. Foods 2019, 58, 123–129.
    8. Chatterjee, C.; Gleddie, S.; Xiao, C.-W. Soybean bioactive peptides and their functional properties. Nutrients 2018, 10, 1211.
    9. Syah, D.; Sitanggang, A.B.; Faradilla, R.F.; Trisna, V.; Karsono, Y.; Septianita, D.A. The influences of coagulation conditions and storage proteins on the textural properties of soy-curd (tofu). CYTA J. Food 2015, 13.
    10. Balogun, I.O.; Olatidoye, O.P. Chemical composition and nutritional evaluation of velvet bean seeds (Mucuna utilis) for domestic consumption and industrial utilization in Nigeria. Pak. J. Nutr. 2012, 11, 116–122.
    11. Mallikarjun Gouda, K.G.; Gowda, L.R.; Rao, A.G.A.; Prakash, V. Angiotensin I-converting enzyme inhibitory peptide derived from glycinin, the 11S globulin of soybean (Glycine max). J. Agric. Food Chem. 2006, 54, 4568–4573.
    12. Rao, A.G.A.; Narasinga Rao, M.S. A method for isolation of 2S, 7S and 11S proteins of soybean. Prep. Biochem. 1977, 7, 89–101.
    13. Deng, K.; Huang, Y.; Hua, Y. Isolation of glycinin (11S) from lipid-reduced soybean flour: Effect of processing conditions on yields and purity. Molecules 2012, 17, 2968–2979.
    14. Deak, N.A.; Murphy, P.A.; Johnson, L.A. Effects of NaCl concentration on salting-in and dilution during salting-out on soy protein fractionation. J. Food Sci. 2006, 71, C247–C254.
    15. Wu, S.; Murphy, P.A.; Johnson, L.A.; Fratzke, A.R.; Reuber, M.A. Pilot-plant fractionation of soybean glycinin and β-conglycinin. J. Am. Oil Chem. Soc. 1999, 76, 285–293.
    16. Segura-Campos, M.; CP, E.-A.; Chel-Guerrero, L.; Betancur-Ancona, D. ACE-I inhibitory peptide fractions from enzymatic hydrolysates of velvet bean (Mucuna pruriens). Agric. Sci. 2013, 4, 767–773.
    17. Chalé, F.G.H.; Ruiz, J.C.R.; Fernández, J.J.A.; Ancona, D.A.B.; Campos, M.R.S. ACE inhibitory, hypotensive and antioxidant peptide fractions from Mucuna pruriens proteins. Proc. Biochem. 2014, 39, 1691–1698.
    18. Tuz, M.A.O.; Campos, M.R.S. Purification of Mucuna pruriens (L) peptide fractions and evaluation of their ACE inhibitory effect. Biocatal. Agric. Biotechnol. 2017, 10, 390–395.
    19. Abtahi, S.; Aminlari, M. Effect of sodium sulfite, sodium bisulfite, cysteine, and pH on protein solubility and sodium dodecyl sulfate−polyacrylamide gel electrophoresis of soybean milk base. J. Agric. Food Chem. 1997, 45, 4768–4772.
    20. Mugendi, J.B.W.; Njagi, E.N.M.; Kuria, E.N.; Mwasaru, M.A.; Mureithi, J.G.; Apostolides, Z. Nutritional quality and physicochemical properties of Mucuna bean (Mucuna pruriens L.) protein isolates. Int. Food Res. J. 2010, 17, 357–366.
    21. Mótyán, J.; Tóth, F.; Tőzsér, J. Research applications of proteolytic enzymes in molecular biology. Biomolecules 2013, 3, 923–942.
    22. Chen, J.-R.; Okada, T.; Muramoto, K.; Suetsuna, K.; Yang, S.-C. Identification of angiotensin I-converting enzyme inhibitory peptides derived from the peptic digest of soybean protein. J. Food Biochem. 2002, 26, 543–554.
    23. Chen, J.R.; Yang, S.C.; Suetsuna, K.; Chao, J.C.J. Soybean protein-derived hydrolysate affects blood pressure in spontaneously hypertensive rats. J. Food Biochem. 2004, 28, 61–73.
    24. Sitanggang, A.B.; Lesmana, M.; Budijanto, S. Membrane-based preparative methods and bioactivities mapping of tempe-based peptides. Food Chem. 2020, 329, 127193.
    25. Wu, J.; Ding, X. Hypotensive and physiological effect of angiotensin converting enzyme inhibitory peptides derived from soy protein on spontaneously hypertensive rats. J. Agric. Food Chem. 2001, 49, 501–506.
    26. Rayaprolu, S.; Hettiarachchy, N.; Horax, R.; Satchithanandam, E.; Chen, P.; Mauromoustakos, A. Amino acid profiles of 44 soybean lines and ACE-I inhibitory activities of peptide fractions from selected lines. J. Am. Oil Chem. Soc. 2015, 92, 1023–1033.
    27. Li, M.; Xia, S.; Zhang, Y.; Li, X. Optimization of ACE inhibitory peptides from black soybean by microwave-assisted enzymatic method and study on its stability. LWT 2018, 98, 358–365.
    28. Kuba, M.; Tana, C.; Tawata, S.; Yasuda, M. Production of angiotensin I-converting enzyme inhibitory peptides from soybean protein with Monascus purpureus acid proteinase. Process Biochem. 2005, 40, 2191–2196.
    29. Kodera, T.; Nio, N. Identification of an angiotensin I-converting enzyme inhibitory peptides from protein hydrolysates by a soybean protease and the antihypertensive effects of hydrolysates in 4 spontaneously hypertensive model rats. J. Food Sci. 2006, 71, C164–C173.
    30. Martínez-Medina, G.A.; Barragán, A.P.; Ruiz, H.A.; Ilyina, A.; Martínez Hernández, J.L.; Rodríguez-Jasso, R.M.; Hoyos-Concha, J.L.; Aguilar-González, C.N. Fungal proteases and production of bioactive peptides for the food industry. In Enzymes in Food Biotechnology; Elsevier: Amsterdam, The Netherlands, 2019; pp. 221–246. ISBN 9780128132807.
    31. Lo, W.M.Y.; Li-Chan, E.C.Y. Angiotensin I converting enzyme inhibitory peptides from in vitro pepsin–pancreatin digestion of soy protein. J. Agric. Food Chem. 2005, 53, 3369–3376.