血管縮素轉化酶(Angiotensin-I Converting Enzyme, ACE)在與調節血壓相關之腎素-血管收縮素-醛固酮系統(Renin-Angiotensin-Aldosterone System, RAAS)及激肽釋放酶-激肽系統(kallikrein- kinin system, KKS)中扮演重要的角色。ACE抑制劑被廣泛應用於降血壓。綠藻(又稱小球藻，Chlorella sorokiniana)和藍藻(又稱螺旋藻，Spirulina platensis)是具有高蛋白質含量（乾重40-70％）的食用微藻，由於其多樣的生物活性，長久以來被用作營養補充品或機能性食品。文獻曾報導藍藻蛋白(S. platensis protein, SPP)可被不同酵素(如：鹼性蛋白酶及胃蛋白酶)水解生成降血壓活性胜肽，但綠藻蛋白(C. sorokiniana protein, CSP)水解物之降血壓胜肽尚未被報導。嗜熱菌蛋白酶(thermolysin)具有廣泛特異性及易生成N端疏水性殘基的特性，極具潛力可生成抑制ACE 之活性胜肽。然而，以嗜熱菌蛋白酶水解SPP及CSP生成具抑制ACE活性胜肽之文獻尚未被報導。本研究以不同酵素對上述二種藻類進行水解，發現以嗜熱菌蛋白酶之藍、綠藻水解物具有最佳之ACE抑制活性，在濃度83.3 µg/mL之抑制活性分別為82.60±0.16%及80.54±0.75%。接著，以逆相層析(reversed-phase high performance liquid chromatography, RP-HPLC)及強陽離子交換層析(strong-cation exchange chromatography, SCX)進行藍、綠藻之嗜熱菌蛋白酶水解物之分群，各分液再進行ACE抑制活性評估，並以液相層析-串聯式質譜分析(LC-MS/MS)，分別從CSP及SPP篩選出活性胜肽YR5/ IK15及 IR5 /FY11。其中，IR5半抑制濃度( IC50 = 10.54±1.38 µM)最低。由抑制動力學的結果顯示IR5為非競爭抑制劑，而分子模擬的結果也支持此結果，即IR5與ACE之作用並未落在活性中心。此外，利用類似方法，我們也初步篩選到一些具抗氧化活性之胜肽。本研究結果顯示，藍、綠藻之嗜熱菌蛋白酶水解物存在抑制ACE及抗氧化之活性胜肽，具有防止及治療高血壓之潛力。

Angiotensin-I Converting Enzyme (ACE, EC 3.4.15.1) plays an important role in Renin Angiotensin Aldosterone System (RAAS) and Kallikrein Kinin System (KKS) system of blood pressure regulation. ACE inhibitors (ACEi) are widely used to treat hypertension. Chlorella sorokiniana and Spirulina platensis are edible microalgae with high protein content (40-70% of its dry weight) and have been used as nutraceuticals and functional foods due to its biological activities. Different enzymes have been used for the digestion of S. platensis protein (SPP) containing antihypertensive peptides such as alcalase (Lu et al., 2010) and pepsin (Suetsuna et al., 2001) can be found in the literature, whereas there has no antihypertensive peptide from C. sorokiniana protein (CSP) been reported yet. It has been suggested that thermolysin is the most preferrable due to its broad specificity to generate hydrophobic residues at peptide N-termini. Nevertheless, the use of this enzyme for digesting CSP and SPP peptide has not been elucidated. In this study, thermolysin hydrolysate inhibited 82.60±0.16% and 80.54±0.75% ACE activity at 83.3 µg/mL, which demonstrated potent ACEi activity compared with other enzymes. Subsequently, the thermolysin hydrolysate then was fractionated using two orthogonal bioasssay-guided fractionations using reversed-phase high performance liquid chromatography (RP-HPLC) and strong-cation exchange chromatography (SCX) separation. Peptides YR5 & IK15 from CSP and IR5 & FY11 were characterized and IR5 peptide was further examine its ACE inhibitory activity due to its lowest IC50 (10.54±1.38 µM). This peptide was regarded as non-competitive ACE inhibitor according to inhibition kinetics study. Molecular docking simulation was further conducted to predict the interaction between IR5 and ACE. The result indicates that the preferable interaction is out of ACE active site, which is consistent with the result obtained from inhibition kinetics. Preliminary studies was also conducted to find potential peptide as antioxidant. Our result demonstrate that CSP and SPP are promising material to obtain potential bioactive peptide as ACE inhibitor and antioxidant in the prevention and treatment of hypertension.

中文摘要 ii
ABSTRACT iv
ACKNOWLEDGEMENTS vi
TABLE OF CONTENT viii
LIST OF FIGURES xi
LIST OF TABLES xiv
LIST OF APPENDIX xv
I. INTRODUCTION 1
1.1 Background 1
1.2 Future Impact 4
II. LITERATURE REVIEW 5
2.1 The Renin-Angiotensin—Kallikrein-Kinin and Mitochondrial System 5
2.2 Angiotensin Converting Enzyme 7
2.2.1 Structure of Angiotensin Converting Enzyme 7
2.2.2 ACE substrates 9
2.3 Synthetic ACE inhibitors 10
2.4 Bioactive Peptides 11
2.5 Biological Sources Derived from Microalgae 12
2.6 Methods for Separation of Bioactive Peptide 13
2.6.1 RP-HPLC Method 13
2.6.2 Strong Cation Exchange Chromatography Method 14
2.7 Methods for Identification of Bioactive Peptide 14
2.7.1 LC-MS/MS Method 14
2.7.2 Database Search 15
2.7.3 De Novo Sequencing 15
2.8 Measurements of ACEI Inhibition Mode 16
2.9 Gastrointestinal Enzyme Simulation 16
III. Materials and Methods 18
3.1 Experimental Design 18
3.2 Materials and Chemical Reagents 19
3.3 Instruments 20
3.4 Experimental Protocols 20
3.4.1 Cell Disruption 20
3.4.2 Optimization of Protein Purification 21
3.4.3 SDS PAGE-In Gel Digestion 21
3.4.4 Preparation of enzymatic hydrolysates Chlorella sorokiniana Protein (CSP) and Spirulina platensis Protein (SPP) 22
3.4.5 Desalting 22
3.4.6 Separation of Hydrolysate 23
3.4.6.1 Fractionation using RP-HPLC 23
3.4.6.2 Fractionation using Offline SCX 23
3.4.7 ACE and DPPH Inhibitory Studies 23
3.4.7.1 ACE Inhibitory Assay 23
3.4.7.2 DPPH Inhibitory Assay 25
3.4.7.3 IC50 Value Determination 25
3.4.8 Identification of Bioactive Peptides Sequence 25
3.4.8.1 LC-MS/MS Condition 25
3.4.8.2 Mascot Search Protein Database 26
3.4.8.3 De Novo Sequencing 27
3.4.9 Peptide Synthesis 27
3.4.10 Peptide Purification using RP-HPLC 28
3.4.11 MRM Study of Protein Hydrolysates 28
3.4.12 Determination of Stability of Peptides to ACE 29
3.4.13 Determination of the Inhibition Pattern of ACEi Peptide 29
3.4.14 Molecular Docking Study 30
3.4.16 Statistical Analysis 31
IV. RESULTS 32
4.1 Preparation of enzymatic hydrolysate and its ACE inhibitory activities 32
4.3 Identification of ACE Inhibitory Peptide using LC-MS/MS, Database-Assisted Sequencing and De Novo Sequencing 36
4.4 Synthetic Peptide Identification and Purification by RP-HPLC and LC-MS 40
4.4.1 CSP Peptide Characterization 40
4.5 Confirmation of IC50 value ACE inhibition from synthetic peptide 46
4.5.1 IC50 value of ACEi Peptides derived from CSP 46
4.6 Characterization of Peptides Stability to ACE 47
4.7 Inhibitory Kinetics of ACEI Peptides from Microalgae 48
4.7.1 Kinetics Study of ACEi peptide from CSP 48
4.7.2 Kinetics Study of ACEi peptide from SPP 49
4.8 MRM Quantification of Peptide ILLYR on Thermolysin Hydrolysate of S. platensis 50
4.9 Molecular Modelling of Peptides and ACE 51
4.9.1 YDYNR and ACE 51
4.11 Protein Profiling of CSP and SPP by SDS Page 55
4.12 Antioxidative Properties of CSP and SPP 57
V. DISCUSSION 60
VI. CONCLUSION 66
REFERENCES 67
INFORMATION OF AUTHOR 82

Aluko, R. E. 2012. Bioactive Peptides (Chapter 3). In Functional Foods and Nutraceuticals. New York: Springer.
Aissaori, N., Abidi, F., Hardouin, J., Abdelkafi, Z., Marrakchi, N., Jouenne, T., and Marzouki, M. N. 2016. ACE Inhibitory and Antioxidant Activities of Novel Peptides from Scorpaena notata By-product Protein Hydrolysate. International Journal of Peptide Research and Therapeutics. 23(1): 13-23.
Anderson, I. 2008. Catalysis and Regulation in RubisCO. Journal of Experimental Botany. 59(7): 1555-1568.
Anderson, I., and Backlund, A. 2008. Structure and Function of Rubisco. Physiology and Biochemistry. 46: 275-291.
Araujo, M.C., Melo, R.I., Del-Nery, E., Alves, M.F., Juliano, M.A., Casarini, D.E., Juliano, L., and Carmona, A.K. 1999. Internally Quenched Fluorogenic Substrates for Angiotensin I-Converting Enzyme. Journal of Hypertension. 17: 665-672.
Asoodeh, A., Haghighi, L., Chamani, J., Ogholbeyk, M.A.A., Tabatabaei, Z.M., and Lagzian, M. 2014. Potential Angiotensin-I Converting Enzyme Inhibitory Peptides from Gluten Hydrolysate: Biochemical Characterization and Molecular Docking Study. Journal of Cereal Science. 1-7.
Atlas, S. A. 2007. The Renin-Angiotensin Aldosterone System: Pathophysiological Role and Pharmacologic Inhibiton. 13(8): S9-S20.
Augenstein, V.A., Heniford, B.T., and Sing, R.F. 2013. Intestinal Angiodema Induced by Angiotensin-Converting Enzyme Inhibitors: An Underrecognized Cause of Abdominal Pain? The Journal of the American Osteopathic Association. 113:221-223.
Barka, A., and Blecker, C. 2016. Microalgae as A Potential Source of Single-Cell Protein. A Review. Biotechnology, Agronomy, Society and Environment. 20(3): 427-436.
Baradaran, A., Nasri, H., and Rafieian-Kopaei, M. 2014. Oxidative stress and hypertension: Possibility of hypertension therapy with antioxidants. Journal of Research in Medical Science. 19(4): 358-367.
Becker, E. W. 2007. Micro-algae as a source of protein. Biotechnology Advances. 25(2): 207-210.
Bersanetti, P. A., Andrade, M.C., Cassarini, D.E., Juliano, M.A., Nchinda, A.T., Sturrock, E.D., Juliano, L., and Carmona, A.K. 2004. Positional-Scanning Combinatorial Libraries of Fluorescence Resonance Energy Transfer Peptides for Defining Substrate Specificity of the Angiotensin-I Converting Enzyme and Development of Selective C-domain Substrates. Biochemistry. 43(50): 15729-15736.
Bryant, D.A. 1991. Cell Culture and Somatic Cell Genetics of Plants, edited by L. Bogorad and I. K. Vasil, Vol. 7B, pp. 255-298. New York: Academic Press.
Burnap, R.L., Hagemann, M., and Kaplan, A. 2015. Regulation of CO2 Concentrating Mechanism in Cyanobacteria. Life. 5:348-371.
Capelli, B., and Cysewski, G. R. 2010. Potential health benefits of Spirulina microalgae. Nutritional Foods. 9: 19-26.
Chia, M.A. 2013. Growth and Biochemical Composition of Chlorella vulgaris in Diffrent Growth Media. Anais da Academia Brasileira de Ciencias. 85(4): 1427-1438.
Cushman, D. W., Cheung, H. S., Sabo, E. F., and Ondetti, M. A. (1977). Design of Potent Competitive Inhibitors of Angiotensin-Converting Enzymes. Carboxyalkanoyl and mercaptoalkanoyl amino acids. Biochemistry. 16: 5484-5491.
Contreras, M.M., Sanchez, D., Sevilla, M.A., Recio, I., and Amigo, L. 2013. Resistance of Casein-Derived Bioactive Peptides to Simulated Gastrointestinal Digestion. International Dairy Joural. 32: 71-78.
Daud, N.A., Babji, A. S., and Yusop, S> M. 2013. Antioxidant Activities of Red Tilapia (Oreochromis niloticus) Protein Hydrolysates as Influenced by Thermolysin and Alcalase. AIP Conference Proceedings. 1571(1): 687-691.
Dauly, C., Perlman, D.H., Costello, C.E, and McComb, M.E. 2006. Protein Separation and Characterization by np-RP-HPLC Followed by Intact MALDI-TOF Mass Spectrometry and Peptide Mass Mapping Analyses. Journal of Proteome Research. 5(7): 1688-1700.
Dikmen, C. D., Yucetepe, A., Guler, F. K., Daskaya, H., and Ozcelik, B. 2017. Angiotensin-I converting enzyme (ACE) Inhibitory Peptides from Plants. Nutrients. 316(9): 1-19.
Duan, X., Wu, F., Li, M., Yang, N., Wu, C., Jin, Y., Yang, J., Jin, Z., and Xu, X. 2014. Naturally occuring angiotensin-I converting enzyme inhibitory peptide from a fertilized egg and its inhibitory mechanism. Journal of Agricultural and Food Chemistry. 62(24): 5500-5506.
Elias, R. J., Kellerby, S. S., and Decker, E. A. 2008. Antioxidant Activity of Proteins and Peptides. Critical Reviewsin Food Science and Nutrition. 48(5): 430-441.
Ellis, R.J., and Vies, S.M.V.D. 1988. The RubisCO Subunit Binding Protein. Photosynthesis Research. 16: 101-115.
Fan, X., Bai, L., Zhu, L., Yang, L., and Zhang, X. 2014. Marine-Algae Derived Bioactive Peptides for Human Nutrition and Health. Journal of Agricultural and Food Chemistry. 62: 9211-9222.
Farvin, K. H. S., Baron, C. P., Nielse, N. S., Otte, J., and Jacobsen, C. 2010. Antioxidant Activity of Yoghurt Peptides: Part 2-Characterisation of peptide Fractions. Food Chemistry. 123: 1090-1097.
Frank, A. M., Savitski, M. M., Nielsen, M. N., Zubarey, R. A., and Pevzner, P. A. 2007. De Novo Peptide Sequencing and Identification with Precision Mass Spectrometry. Journal of Proteome Research. 6(1): 114-123.
Frohlich, H., Nelges, C., Tager, T., Schwenger, V., Cebola, R., Schnorbach, J., Goode, K.M., Kazmi, S., Katus, H.A., Cieland, J.G.F., Clark, A.L., and Frankenstein, L. 2016. Long Term Changes of Renal Function in Relation to ACE inhibitor/Angiotensin Receptor Blocker Dosing in Patients with Heart Failure and Chronic Kidney Disease. Americal Heart Journal. 178: 28-36.
Fujita, H., and Yoshikawa, M. 1999. LKPNM: A Prodrug-type ACE-inhibitory Peptide Derived from Fish Protein. Immunopharmacology. 44: 123-127.
Furuta, T., Miyabe, Y., Yasui, H.., Kinoshita, Y., and Kishimura, H. 2016. Angiotensin-I Converting Enzyme Inhibitory Peptides Derived from Phycobiliproteins of Dulse Palmaria palmata. Marine Drugs. 14(32): 1-10.
Fyhrquist, F., Metsarinne, K., and Tikkanen, I. 1995. Role of Angiotensin II in Blood Pressure Regulation and In The Pathophysiology of Cardiovascular Disorders. Journal of Human Hypertension. 9(5): S19-S24.
Gantenbein, M.H., Bauersfeld, U., Baenziger, O., Frey, B., Neuhaus, T., Sennhauser, F., and Bernet, V. 2008. Side effects of angiotensin converting enzyme inhibitor (captopril) in newborns and young infants. Journal of Perinatal Medicine. 36(5): 448-452.
Garnier, F., Dubacq, J.P., and Thomas, J.C. 1994. Evidence for a Transient Association of New Proteins with the Spirulina maxima Phycobilisome in Relation to Light Intensity. Plant Physiology. 106: 747-754.
Guang, C., Philips, R.D., Jiang, B., and Milani, F. 2012. Three Key Proteases-Angiotensin-I-Converting Enzyme (ACE), ACE2 and Renin – Within and Beyond the Renin-Angiotensin System. Archives of Cardiovascular Diseases. 105(6-7): 373-385.
Guccione, A., Biondi, N., and Tredici, M.R. 2014. Chlorella for Protein and Biofuels: From Strain Selection to Outdoor Cultivation in a Green Wall Panel Photobioreactor. Biotechnology for Biofuels. 7(84): 1-12.
He, H.L., Chen, X.L., Sun, C.Y., Zhang, Y.Z., and Zhou, B.C. 2006. Analysis of novel angiotensin-I-converting enzyme inhibitory peptides from protease-hydrolyzed marine shrimp Acetes chinensis. Journal of Peptide Sciences. 12: 726–733.
Hilgers, K. F., and Mann, J. F. E. 2008.The Choice of Antihypertensive Therapy in Patinets with the Metabolic Syndrome—Time to Change Recommendation? Nephrology Dialysis Transplantation. 23(11): 3389-3391.
Husain, K., Hernandez, W., Ansari, R. A., and Ferder, L. 2015. Inflammation, Oxidative Stress and Renin-Angiotensin System in Atherosclerosis. World Journal of Biological Chemistry. 6(3): 209-217.
Ichimura, M., Kato, S., Tsuneyama, K., Matsutake, S., Kamogawa, M., Hirao, E., Miyata, A., Mori, S., Yamaguchi, N., Suruga, K., and Omagari, K. 2013. Phycocyani Prevents Hypertension and Low Serum Adiponectin Level in A Rat Model of Metabolic Syndrome. Nutrition Research. 33(5): 397-405.
Jao, C.L., Huang, S. L., and Hsu, K. C. 2012. Angiotensin-I Converting Enzyme Inhibitory Peptides: Inhibition Mode, Bioavailabily and Antihypertensive Effects. Biomedicine. 2: 130-136.
Jung, W. K., Mendis, E., Je, J. Y., park, P. J. Son, B. W., and Him, H. C. 2006. Angiotensin-I Converting Enzyme Inhibitory Peptide from yellowfin sole (Limanda aspera) frame protein and its Antihypertensive Effect in Spontaneously Hypertensive Rats. Food Chemistry. 94: 26-32.
Kang, K.H., Qian, Z. J., Ryu, B. M., and Kim, S. K. 2011. Characterization of growth and protein contents from microalgae Navicula incerta with the investigation of antioxidant activity of enzymatic hydrolysates. Food Science and Biotechnology. 20(1): 183-191.
Kaplan, N. M., & Victor, R. G. 2015. Kaplan’s clinical hypertension. (11th ed.). Philadelphia: Wolters Kluwer, (Chapter 7).
Kayashima, Y., Smithies, O., and Kakoki, M. 2013. Kinins—The Kallikrein-Kinin System and Oxidative Stress. CurrentOpinion of Nephrology and Hypertension. 21(1): 92-96.
Keil, B., and Tong, T. N. 1992. LYSIS. New York: Springer-Verlag Berlin Heidelberg.
Kim, D.Y., Vijayan, D., Praveenkumar, R., Han, J.I., Lee, K., Park, J.Y., Chang, W.S., Lee, J.S., and Oh, Y.K. 2016. Cell wall disruption and lipid/astaxanthin extraction from microalgae: Chlorella and Haemotococcus. Bioresource Technology. 199:300-310.
Knudzen, A.D., Bennike, H. Kjeldal, Birkelund, S., Otzen, D.E., and Stensballe, A. 2014. Condenser: A Statistical Aggregation Tool for Multi-Sample Quantitative Proteomics Data from Matrix Science Mascot Distiller. Journal of Proteomics. 103: 261-266.
Koa, S.C., Kim, D., and Jeon, Y.J. 2012. Protective Effect of a novel antioxidative peptide purified from a marine Chlorella ellipsoidea protein against free radical-induced oxidative stress. Food and Chemical Toxicology. 50: 2294-2302.
Kob, S.C., Kang, N., Kim, E.A., Kang, M.C., Lee, S.H., Kang, S.M., Lee, J.B., Jeon, B.T., Kim, S.K., Park, S.J., Park, P.J., Jung, W.K., Kim, D., and Jeon, Y.J. 2012. A novel angiotensin I-converting enzyme (ACE) inhibitory peptide from a marine Chlorella ellipsoidea and its antihypertensive effect in spontaneously hyperthensive rats. Process Biochemistry. 47:2005-2011.
Korhonen, H., and Pihlanto, A. 2006. Bioactive Peptides: Production and Functionality. International Dairy Journal. 945-960.
Kroger, W.L. 2009. A Molecular Basis for the C-Domain Selectivity of Angiotensin-Converting Enzyme. Africa: University of Cape Town.
Kumara, K., Dasgupta, C.N., and Das, D. 2014. Cell Growth Kinetics of Chlorella sorokiniana and Nutritional Values of Its Biomass. Bioresource Technology.167: 358-366.
Kumarb, R.R., Rao, P.H, and Arumugam, M. 2015. Lipid Extraction Methods from Microalgae: A Comprehensive Review. Frontiers in Energy Research. 61(2): 1-9.
Lahera, V., Heras, D. L., Farre, L., Manucha, W., and Ferder, L. 2017. Role of Mytochondrial Dysfunction in Hypertension and Obesity. CurrentHypertension Reports. 19(2): 1-9.
Lee, S.H., Qian, Z.J., and Kim, S.K. 2010. A novel angiotensin I converting enzyme inhibitory peptide from tuna frame protein hydrolysate and its antihypertensive effect in spontaneously hypertensive rats. Food Chem. 118: 96–102.
Li, G. H., Lee, G. W., Shi, Y. H., and Shrestha, S. 2004. Angiotensin-I converting enzyme inhibitory peptides derived from food proteins and their physiological and pharmacological effects. iNutrition Research. 24: 469-486.
Li, H., and Aluko, R.E. 2010. Identification and Inhibitory Properties of Multifunctional Peptides from Pea Protein Hydrolysate. Journal of Agricultural and Food Chemistry. 58(21): 11471-11476.
Liu, J., Jin, Y., Lin, S., Jones, G.S., and Chen, F. 2015. Purification and Identification of Novel Antioxidant Peptides from Egg White Protein and Their Antioxidant Activities. Food Chemistry. 175: 258-266.
Lin, K., Zhang, L. W., Han, X., and Cheng, D. Y. 2017. Novel angiotensin-I converting enzyme inhibitory peptides from protease hydrolysates of Qula casein: Quantitative structure-activity relationship modelling and Molecular Docking Study. Journal of Functional Foods. 32: 266-277.
Lu, B., and Chen, T. 2003. A Suffix Tree Approach to the Interpretation of Tandem Mass Spectra: Applications to Peptides of Non-Specific Digestion and Post-Translational Modifications. Bioinformatics. 19(2): 113-121.
Lu, J., Ren, D.F., Xue, Y.L., Sawano, Y., Miyakawa, T., and Tanokura, T. 2010. Isolation of an Antihyperthensive Peptide from Alcalase Digest of Spirulina platensis. Journal of Agricultural and Food Chemistry.m58: 7166-7171.
Mohammed, S., and Heck, A.J.R. 2011. Strong Cation Exchange (SCX) Based Analytical Methods for the Targeted Analysis of Protein Post-Translational Modifications. Current Opinion in Biotechnology. 22(1):9-16.
Mohanty, P., Sruvasta, M., and Krishna, K.B. Edited by Vunshak, A. 2002. The Photosynthetic Apparatus of Spirulina: Electron Transport and Energy Transfer. In Spirulina platensis Arthrospira: Physiology, Cell-Biology and Biotechnology. England: CRC Press.
Mora, L., Escudero, E., Fraser, P. D., Aristoy, M. C., and Toldra, F. 2014. Proteomic Identification of Antioxidant Peptides from 400-2500 Da Generated in Spanish dry-cured Ham Contained in a Size-Exclusion Chromatography Fraction. Food Research International. 56: 68-76.
Natesh, R., Schwager, S.L.U., Sturrock, E.D., and Acharya, K.R. 2003. Crystal Structure of the Human Angiotensin-Converting-Enzyme-Lisinopril Complex. Letters to nature. 421:551-554.
Ni, H., Li, N., Liu, G., and Hu, S.Q. 2012. Inhibition Mechanism and Model of an Angiotensin-I Converting Enzyme (ACE)-Inhibitory Hexapeptide from Yeast (Saccharomyces cerevisiae). 7(5): 1-7.
Ngo, D. H., Ryu, B. M., Vo, T. S., Himaya, S. W. A., Wijesekara, I., and Kim, S. K. 2011. Free Radical Scavenging and Angiotensin-I Converting Enzyme Inhibitory Peptides from Pacific cod (Gadus macrocephalus) skin gelatin. International Journal of Biological Macromolecules.49: 1110-1116.
O’Neill, H. G., Redelinghuys, P., Schwager, S.L., and Sturrock, E.D. 2008. The Role of Glycosylation and Domain Interaction in the Thermal Stability of Human Angiotensin-Converting Enzyme. Biological Chemistry. 16(1):351-359.
Pacurari, M., Kafoury, R., Tchounwou, P.B., and Debele, K.N. 2014. The Renin-Angiotensin-Aldosterone System in Vascular Inflammation and Remodeling. International Journal of Inflammation. 2014: 1-13.
Paiva, L., Lima, L., Neto, A.I., and Baptista, J. 2016. Isolation and Characterization of Angiotensin-I Converting Enzyme (ACE) Inhibitory Peptides from Ulva rigida C. Agardh Protein Hydrolysate. Journal of Functional Foods. 26: 65-76.
Pan, S., Wang, S., Jing, L., and Yao, D. 2016. Purification and Characterization of a Novel Angiotensin-I Converting Enzyme (ACE)-Inhibitory Peptide Derived from the Enzymatic Hydrolysate of Enteromorpha clathrata protein. Food Chemistry. 211: 423-430.
Parish, R.C., and Miller, L.J. 1992. Adverse Effects of Angiotensin Converting Enzyme (ACE) Inhibitors. An Update. Drug Safety. 7(1): 14-31.
Patchett, A. A., Harris, E., Tristam, E. W., Wyvratt, M. J., Wu, M. T., Taub, D., Peterson, E. R., Ikeler, T. J., ten-Brooke, J., Payne, L. G., Ondeyka, D. L., Thorsett, E> D., Greenlee, W. J., Lohr, N. S., et al. 1980. A New Class of Angiotensin-Converting Enzyme Inhibitors. Nature. 288: 280-283.
Picariello, G., Ferranti, P., Fierro, O., Mamone, G., Caira, S., Luccia, A.D., Monica, S., and Addeo, F. 2010. Peptides Surviving the Simulated Gastrointestinal Digestion of Milk Proteins: Biological and Toxicological Implications. Journal of Chromatography B. 878 (293-308).
Pripp, A.H. 2007. Docking and Virtual Screening of ACE Inhibitory Dipeptides. European Food Research and Technology. 225: 589-592.
Priyanto, A.D., Doerksen, R.J., Chang, C.I., Sung, W.C., Widjanarko, S.B., Kusnadi, J., Lin, Y.C., Wang, T.C., and Hsu, J.L. 2015. Screening, Discovery, and Characterization of Angiotensin-I Converting Enzyme Inhibitory Peptides Derived from Proteolytic Hydrolysate of Bitter Melon Seed Proteins. Journal of Proteomics. 128: 424-435.
Puddu, P., Puddu, G. M., Cravero, E., Pascalis, S. D., and Muscari, A. 2007. The Putative Role of Mytochondrial Dysfunction in Hypertension. 29: 427-434.
Pujiastuti, D.Y., Shih, Y.H., Chen, W.L., Sukoso., and Hsu, J.L. 2016. Screening of Angiotensin-I Converting Enzyme Inhibitory Peptides Derived from Soft-Shelled Turtle Yolk Using Two Orthogonal Bioassay-Guided Fractionations. Journal of Functional Foods. 28: 36-47.
Qian, Z.J., Je, J.Y., and Kim, S.K. 2007. Antihypertensive effect of angiotensin I converting enzyme-inhibitory peptide from hydrolysates of bigeye tuna dark muscle, Thunnus obesus. Journal of Agricultural and Food Chemistry. 55: 8398–8403.
Quiros, A., Contreras, M.D.M., Ramos, M., Amigo, L., and Recio, I. 2009. Stability to Gastrointestinal Enzymes and Structure-Activity Relationship of β-Casein-Peptides with Antihypertensive Properties. Peptides. 1848-1853.
Rajalingam, D., Loftis, C., Xu, J.J., and Kumar, T.K.S. 2009. Trichloroacetic acid-Induced Protein Precipitation Involves the Reversible Association of a Stable Partially Structured Intermediate. Protein Science. 18(5): 980-993.
Ramalingam, L., Menikdiwela, K., LeMieux, M., Dufour, J.M., Kaur, G., Kalupahana, N., and Moussa, N.M. 2016. The Renin Angiotensin System, Oxidative Stress and Mitochondrial Function in Obesity and Insulin Resistance. Biochimica et Biophysica Acta. 1-9.
Rawendra, R.D.S., Aisha., Chang, C.I., Aulanni’am., Chen, H.H., Huang, T.C., and Hsu, J.L. 2013. A Novel Angiotensin Converting Enzyme Inhibitory Peptide Derived from Proteolytic Digest of Chinese Soft-Shelled Turtle Egg White Proteins. Journal of Proteomics. 54: 359-369.
Reboldi, G., Gentile, G., and Verdecchia, P. 2009. Choice of ACE Inhibitor Combinations in Hypertensive Patients with Type 2 Diabetes: Update after recent Clinical Trials. Vascular Health and Risk Management. 5:411-427.
Remuzzi, G., Perico, N., Macia, M., and Ruggenenti, P. 2005. The Role of Renin-Angiotensin-Aldosterone System in the Progression of Chronic Kidney Disease. Kidney International. 68 (99): S57-S65.
Safi, C., Ursu, A.V., Laroche, C., Zebib, B., Merah, O., Pontalier, P.Y., and Garcia, C.V. 2014. Aqueous Extraction of Proteins from Microalgae: Effect of Different Cell Disruption Methods. Algal Research. 3: 61-65.
Samarakoon, K.W., Nam, K.O., Ko, J.Y., Lee, J.H., Kang, M.C., Kim, D., Lee, J.B., Lee, J.S., and Jeon, Y.J. 2013. Purification and identification of novel angiotensin-I converting enzyme (ACE) inhibitory peptides from cultured marine microalgae (Nannochloropsis oculata) protein hydrolysate. Journal of Applied Phycology. 1-12.
Sato, M., Hosokawa, T., Yamaguchi, T., Nakano, T., Muramoto, K., and Kahara, T. 2002. Angiotensin-I Converting Enzyme Inhibitory Peptides Derived from Wakame (Undaria pinnatifida) and Their Antihypertensive Effect in Spontaneously Hypertensive Rats. Journal of Agricultural and Food Chemistry. 50: 6245-6252.
Saveliev, S., Engel, L., Strauss, E., Jones, R., and Rosenblatt, M. 2012. The Advantages to Using Arg-C, Elastase, Thermolysin, and Protein Analysis. Promega Corporation Website. https://worldwide.promega.com/resources/pubhub/the-advantages-to-using-arg-c-elastase-thermolysin-and-pepsin-for-protein-analysis/. Accessed October 21, 2016.
Sharma, R. (2012). Enzyme Inhibition: Mechanisms and Scope, Enzyme Inhibition and Bioapplications. InTech, DOI: 10.5772/39273. Available from: https://www.intechopen.com/ books/enzyme-inhibition-and-bioapplications/enzyme-inhibition-mechanisms-and-scope
Shamloo, M., Eck, P., and Beta, T. 2014. Angiotensin Converting Enzyme Inhibitory Peptides Derived from Cereals. Journal of Human Nutrition & Food Science. 3(1): 1-10.
Silverman, R.B., and Holladay, M.W. 2014. The Organic Chemistry of Drug Design Third Edition. Academic Press: USA.
Siragy, H. M., and Carey, R. M. 2010. Role of the Intrarenal Renin-Angiotensin Aldosterone System in Chronic Kidney Disease. American Journal of Nephrology. 31(6): 541-550.
Spyroulias, G. A., Galanis, A. S., Pairas, G., Zoupa, E. M., and Cordopatis, P. 2004. Structural Features of Angiotensin-I Converting Enzyme Catalytic Sites: Conformational Studies in Solution, Homology Models and Comparison with Other Zinc Metallopeptidases. Current Topics in Medicinal Chemistry. 4: 403-429.
Spreitzer, R.J. 2003. Role of the Small Subunit in Ribulose-1.5-Biphospate Carboxylase/Oxygenase. Archives of Biochemistry and Biophysics. 414(2): 141-149.
Sturrock, E.D., Natesh, R., Van-Rooyen, J.M., and Acharya, K.R. 2004. Structure of Angiotensin I-Converting Enzyme. Cellular and Molecular Life Science. 61:2677-2686.
Suetsuna, K. 1998. Isolation and Characterization of Angiotensin-I Converting Enzyme Inhibitor Dipeptides Derived from Allium sativum L (garlic). Journal of Nutrition and Biochemistry. 9: 415-419.
Suetsuna, K., and Nakano, T. 2000. Identification of an Antihypertensive Peptide from Peptic Digest of Wakame (Undaria pinnatifida). Journal of Nutritional Biochemistry. 11(9): 450-454.
Suetsuna, K., and Chen, J.R. 2001. Identification of Antihyperthensive Peptides from Peptic Digest of Two Microalgae, Chlorella vulgaris and Spirulina platensis. Marine Biotechnology. 3: 305-309.
Suetsuna, K., Maekawa, K., and Chen, J. R. 2004. Antihypertensive Effects of Undaria pinnatifida (wakame) peptide on blood pressure in Spontaneously Hypertensive Rats. The Journal of Nutritional Biochemistry. 15: 262-267.
Sheih, I.C., Wu, T.K., and Fang, T.J. 2009. Isolation and Characterization of A Novel Angiotensin I-Converting Enzyme (ACE) Inhibitory Peptide from the Algae Protein Waste. Food Chemistry. 115: 279-284.
Su, J.B. 2014. Different Cross-Talk Sites Between the Renin-Angiotensin and the Kallikrein-Kinin System. Journal of the Renin-Angiotensin Aldosterone System. 15(4): 319-328.
Tsai, J.S., Chen, J.L., and Pan, B.S. 2008. ACE-inhibitory peptides identified from the muscle protein hydrolysate of hard clam (Meretrix lusoria). Process Biochemistry. 43: 743–747.
Tumanan-Mendoza, B. A., Dans, A. L., Villacin, L. L., Mendoza, V. L., Rellama-Black, S., Bartolome, M., et al. 2007. Dechallenge and rechallenge method showed different incidences of cough among four ACE-Is. Journal of Clinical Epidemiology. 60: 547-553.
Vaz, B.D.S., Moreira, J.B., Morais, M.G., and Costa, J.AV. 2016. Microalgae As A New Source of Bioactive Compounds in Food Supplements. Current Opinion in Food Science. 7: 73-77.
Vermeirssen, V., Camp, J.V., and Verstraete, W. 2004. Bioavailability of Angiotensin-I Converting Enzyme Inhibitory Peptides. British Journal of Nutrition. 92: 357-366.
Vo, T. S., Ryu, B. M., and Kim, S> K. 2013. Purification of Novel Anti-Inflammatory Peptides from Enzymatic Hydrolysate of the Edible Microalgal Spirulina maxima. Journal of Functional Foods. 1336-1346.
Wanga, Y.K., He, H.L., Chen, X.L., Sun, C.Y., Zhang, Y.Z., and Zhou, B.C. 2008. Production of novel angiotensin I-converting enzyme inhibitory peptides by fermentation of marine shrimp Acetes chinensis with Lactobacillus fermentum SM 605. Applied Microbiology and Biotechnology. 79: 785–791.
Wangb, X., Wu, S., Xu, D., Xie, D., Guo, H. 2011. Inhibitor and Substrate Binding by Angiotensin - converting Enzyme: Quantum Mechanical/Molecular Mechanical Molecular Dynamics Studies. Journal of chemical information and modeling. 51(5):1074-1082.
Weller, M.G. 2012. A Unifying Review of Bioassay-Guided Fractionation, Effect-Directed Analysis and Related Techniques. Sensors. 12(7): 9181-9209.
WHO. (World Health Organization). 2013. A Global Brief on Hypertension. Silent killer, global public health crisis. Switzerland: WHO.
Wu, Z.Y., Qu, C.B., Shi, X.M. 2009. Biochemical System Analysis of Lutein Production by Heterotrophic Chlorella pyrenoidosa in a fermentor. Food Technology and Biotechnology. 47: 450-455.
Wu, R., and Jiafeng, H. 2015. Overview of Antioxidant Peptides Derived from Marine Resources: The Sources, Characteristics, Purification, and Evaluation Methods. Application of Biochemistry and Biotechnology. 176: 1815-1833.
Yokota, A., and Canvin, D.T. 1985. Ribulose Biphosphate Carboxylase/Oxygenase Content Determined with [14C] Carboxypentitol Biphosphate in Plants and Algae. Plant Physiology. 77(3): 735-739.
Yu, J., Hu, Y., Xue, M., Dun, Y., Li, S., Peng, N., Liang, Y., and Zhao, S. 2016. Purification and Identification of Antioxidant Peptides from Enzymatic Hydrolysate of Spirulina platensis. Journal of Microbiology and Biotechnology. 26(7): 1216-1223.
Zhanga, X., Xie, Y.W., Nasjletti, A., Xu, X., Wolin, M.S., and Hintze, T.H. 1997. ACE Inhibitors Promote Nitric Oxide Accumulation to Modulate Myocardial Oxygen Consumption. Circulation. 95: 176-182.
Zhangb, Li., Liu, Y., Lu, D., Han, J., Lu, X., and Tian, Z. 2015. Angiotensin Converting Enzyme Inhibitory, Antioxidant Activities, and Antihyperlipidaemic Activities of Protein Hydrolysates From Scallop Mantle (Chlamys farreri). International Journal of Food Properties. 18(1): 33-42.
Zhao, Y., and Xu, C. 2008. Structure and Function of Angiotensin Converting Enzyme and Its Inhibition. Chinese Journal of Biotechnology. 24(2): 171-176.
Zhao, Y., Bafang, L., Dong, S., Liu, Z., Zhao, X., Wang, J., and Zeng, M. 2009. A novel ACE inhibitory peptide isolated from Acaudina molpadioidea hydrolysate. Peptides. 30: 1028–1033.
Zou, T. B., He, T. P., Li, H. B., Tang, H. W., and Xia, E. Q. 2016. The Structure-Activity Relationship of the Antioxidant Peptides from Natural Proteins. Molecules. 21(72): 1-14.