Kinetically Controlled Carboxypeptidase-Catalyzed Synthesis of Novel Antioxidant Dipeptide Precursor BOC-Tyr-Ala

Abstract

Recently, enzymatic peptide synthesis has drawn increasing attention due to its eco-friendly reagents and mild conditions, as compared to traditional chemical peptide synthesis. In this study, we successfully produced an important antioxidant dipeptide precursor, BOC-Tyr-Ala, via a kinetically controlled enzymatic peptide synthesis reaction, catalyzed by the recombinant carboxypeptidase Y (CPY) expressed in P. pastoris GS115. In this reaction, the enzyme activity was 95.043 U/mL, and we used t-butyloxycarbonyl-l-tyrosine-methyl ester (BOC-Tyr-OMe) as the acyl donor and l-alanine (l-Ala) was the amino donor. We optimized the reaction conditions to be: 30 °C, pH 9.5, organic phase (methanol)/aqueous phase = 1:20, BOC-Tyr-OMe 0.05 mol/L, Ala 0.5 mol/L, and a reaction time of 12 h. Under these conditions, the dipeptide yield reached 49.84%. Then, we established the kinetic model of the synthesis reaction in the form of Michaelis–Menten equation according to the concentration–time curve during the process and the transpeptidation mechanism. We calculated the apparent Michaelis constant $K_{\text{m}}^{\text{app}}$ and the apparent maximum reaction rate $r_{\hbox{max} }^{\text{app}}$ to be $2.9946 \times 10^{{^{ - 2} }} {\text{ mol/L}}$ and $2.0406 \times 10^{ - 2} {\text{ mmol/(mL}}\,{\text{h)}}$, respectively.

Keywords

Antioxidant dipeptide precursor / Kinetic control / Reaction kinetic model / Enzymatic peptide synthesis

References

[1] Kaiser S, Martin M, Lunow D, et al. Tryptophan-containing dipeptides are bioavailable and inhibit plasma human angiotensin-converting enzyme in vivo. Int Dairy J, 2016, 52: 107-114.

[2] Kumar MBA, Gao Y, Shen W, et al. Valorisation of protein waste: an enzymatic approach to make commodity chemicals. Front Chem Sci Eng, 2015, 9(3): 295-307.

[3] Gauthier SF, Pouliot Y, Saint-Sauveur D. Immunomodulatory peptides obtained by the enzymatic hydrolysis of whey proteins. Int Dairy J, 2006, 16(11): 1315-1323.

[4] Jia J, Ma H, Zhao W, et al. The use of ultrasound for enzymatic preparation of ACE-inhibitory peptides from wheat germ protein. Food Chem, 2010, 119(1): 336-342.

[5] Rahman MS, Choi YH, Choi YS, et al. Glycin-rich antimicrobial peptide YD1 from B. amyloliquefaciens, induced morphological alteration in and showed affinity for plasmid DNA of E. coli. AMB Express, 2017, 7(1): 8

[6] Yao Z, Che XC, Lu R, et al. Inhibition by tyroserleutide (YSL) on the invasion and adhesion of the mouse melanoma cell. Mol Med, 2007, 13(1–2): 14-21.

[7] Muller FL, Lustgarten MS, Jang Y, et al. Trends in oxidative aging theories. Free Radic Bio Med, 2007, 43(4): 477-503.

[8] White J, Dawson B, Landers G, et al. Effect of supplemental oxygen on post-exercise inflammatory response and oxidative stress. Eur J Appl Physiol, 2013, 113(4): 1059-1067.

[9] Chasovnikova LV, Formazyuk VE, Sergienko VI, et al. The antioxidative properties of carnosine and other drugs. Biochem Int, 1990, 20(6): 1097-1103.

[10] Culbertson JY, Kreider RB, Greenwood M, et al. Effects of beta-alanine on muscle carnosine and exercise performance: a review of the current literature. Nutrients, 2010, 2(1): 75-98.

[11] Herculano B, Tamura M, Ohba A, et al. β-alanyl-l-histidine rescues cognitive deficits caused by feeding a high fat diet in a transgenic mouse model of Alzheimer’s disease. J Alzheimers Dis, 2013, 33(4): 983-997.

[12] Hisatsune T, Kaneko J, Kurashige H, et al. Effect of anserine/carnosine supplementation on verbal episodic memory in elderly people. J Alzheimers Dis, 2016, 50(1): 149-159.

[13] Udenigwe CC, Wu S, Drummond K, et al. Revisiting the prospects of plastein: thermal and simulated gastric stability in relation to the antioxidative capacity of casein plastein. J Agric Food Chem, 2014, 62(1): 130-135.

[14] Je JY, Cho YS, Gong M, et al. Dipeptide Phe-Cys derived from in silico thermolysin-hydrolysed RuBisCO large subunit suppresses oxidative stress in cultured human hepatocytes. Food Chem, 2015, 171: 287-291.

[15] Zhang Z, Zhao Y, Wang X, et al. The novel dipeptide Tyr-Ala (TA) significantly enhances the lifespan and healthspan of caenorhabditis elegans. Food Funct, 2016, 7(4): 1975-1984.

[16] Constable DJC, Dunn PJ, Hayler JD, et al. Key green chemistry research areas—a perspective from pharmaceutical manufacturers. Green Chem, 2007, 9: 411-420.

[17] Yazawa K, Numata K. Recent advances in chemoenzymatic peptide syntheses. Molecules, 2014, 19(9): 13755-13774.

[18] St-Jacques AD, Rachel NM, Dan RC, et al. Specificity of transglutaminase-catalyzed peptide synthesis. J Mol Catal B Enzym, 2016, 123: 53-61.

[19] Meister A. Proteinase-catalyzed synthesis of peptide bonds, 2006, New York: Wiley.

[20] Schellenberger V, Jakubke HD. Protease-catalyzed kinetically controlled peptide synthesis. Angew Chem (Int Ed Engl), 1991, 30(11): 1437-1449.

[21] Chen GH, Yao LU. Progress about short peptide synthesis by enzyme in reverse micelle. J Sichuan Univ Sci Eng, 2007, 20(1): 104-106 (in Chinese)

[22] Noritomi H, Suzuki K, Kikuta M, et al. Catalytic activity of α-chymotrypsin in enzymatic peptide synthesis in ionic liquids. Biochem Eng J, 2009, 47(1–3): 27-30.

[23] Kuhn RW, Walsh KA, Neurath H. Isolation and partial characterization of an acid carboxypeptidase from yeast. Biochem, 1974, 13(19): 3871-3877.

[24] Zhang QH, Yang GP. Selenium speciation in bay scallops by high performance liquid chromatography separation and inductively coupled plasma mass spectrometry detection after complete enzymatic extraction. J Chromatogr A, 2014, 1325(2): 83-91.

[25] Breddam K, Johansen JT. Semisynthesis of human insulin utilizing chemically modified carboxypeptidase Y. Carlsberg Res Commun, 1984, 49(4): 463-472.

[26] Peschke B, Bak S. Controlled coupling of peptides at their C-termini. Peptides, 2009, 30(4): 689-698.

[27] Duan W, Zhang Y, Xu G. Optimization and application of protein C-terminal labeling by carboxypeptidase Y. Chin J Biotechnol, 2016, 32(1): 135-148.

[28] Doi E, Shibata D, Matoba T. Modified colorimetric ninhydrin methods for peptidase assay. Anal Biochem, 1981, 118(1): 173-184.

[29] Guzmán Barberis F, Illanes S, et al. Peptide synthesis: chemical or enzymatic. Electron J Biotechn, 2007, 10(2): 279-314.

[30] Rawal VH, Cava MP. Thermolytic removal of t-butyloxycarbonyl (BOC) protecting group on indoles and pyrroles. Tetrahedron Lett, 1985, 26(50): 6141-6142.

[31] Ogino H, Yamada M, Watanabe F, et al. Peptide synthesis catalyzed by organic solvent-stable protease from Pseudomonas aeruginosa PST-01 in monophasic aqueous-organic solvent systems. J Biosci Bioeng, 1999, 88(5): 513-518.

[32] Widmer F, Breddam K, Johansen JT. Influence of the structure of amine components on carboxypeptidase Y catalyzed amide bond formation. Carlsberg Res Commun, 1981, 46(1): 97-106.

[33] Wang M, Wei Q, Yu Q, et al. Kinetically controlled enzymatic synthesis of dipeptide precursor of l-alanyl-l-glutamine. Biotechnol Appl Biochem, 2011, 58(6): 449-455.

[34] Breddam K, Widmer F, Meldal M. Amidation of growth hormone releasing factor (1–29) by serine carboxypeptidase catalysed transpeptidation. Int J Pept Prot Res, 1991, 37(2): 153-160.

[35] Yu X, Liu F, Zou Y, et al. Biosynthesis of strained piperazine alkaloids: uncovering the concise pathway of herquline A. J Am Chem Soc, 2016, 138(41): 13529-13532.

[36] Marangoni AG. Enzyme kinetics: a modern approach, 2003, New York: Wiley .