Kinetically Controlled Carboxypeptidase-Catalyzed Synthesis of Novel Antioxidant Dipeptide Precursor BOC-Tyr-Ala
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摘要
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_m~(app) and the apparent maximum reaction rate r_(max)~(app) to be 2.9946 × 10~(-2)mol/L and 2.0406 × 10~(-2)mmol/(mL h), respectively.
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_m~(app) and the apparent maximum reaction rate r_(max)~(app) to be 2.9946 × 10~(-2)mol/L and 2.0406 × 10~(-2)mmol/(mL h), respectively.
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_m~(app) and the apparent maximum reaction rate r_(max)~(app) to be 2.9946 × 10~(-2)mol/L and 2.0406 × 10~(-2)mmol/(mL h), respectively.
引文
1.Kaiser S,Martin M,Lunow D et al(2016)Tryptophan-containing dipeptides are bioavailable and inhibit plasma human angiotensinconverting enzyme in vivo.Int Dairy J 52:107-114
2.Kumar MBA,Gao Y,Shen W et al(2015)Valorisation of protein waste:an enzymatic approach to make commodity chemicals.Front Chem Sci Eng 9(3):295-307
3.Gauthier SF,Pouliot Y,Saint-Sauveur D(2006)Immunomodulatory peptides obtained by the enzymatic hydrolysis of whey proteins.Int Dairy J 16(11):1315-1323
4.Jia J,Ma H,Zhao W et al(2010)The use of ultrasound for enzymatic preparation of ACE-inhibitory peptides from wheat germ protein.Food Chem 119(1):336-342
5.Rahman MS,Choi YH,Choi YS et al(2017)Glycin-rich antimicrobial peptide YD1 from B.amyloliquefaciens,induced morphological alteration in and showed affinity for plasmid DNA of E.coli.AMB Express 7(1):8
6.Yao Z,Che XC,Lu R et al(2007)Inhibition by tyroserleutide(YSL)on the invasion and adhesion of the mouse melanoma cell.Mol Med 13(1-2):14-21
7.Muller FL,Lustgarten MS,Jang Y et al(2007)Trends in oxidative aging theories.Free Radic Bio Med 43(4):477-503
8.White J,Dawson B,Landers G et al(2013)Eff ect of supplemental oxygen on post-exercise inflammatory response and oxidative stress.Eur J Appl Physiol 113(4):1059-1067
9.Chasovnikova LV,Formazyuk VE,Sergienko VI et al(1990)The antioxidative properties of carnosine and other drugs.Biochem Int 20(6):1097-1103
10.Culbertson JY,Kreider RB,Greenwood M et al(2010)Eff ects of beta-alanine on muscle carnosine and exercise performance:a review of the current literature.Nutrients 2(1):75-98
11.Herculano B,Tamura M,Ohba A et al(2013)β-alanyl-L-histidine rescues cognitive deficits caused by feeding a high fat diet in a transgenic mouse model of Alzheimer’s disease.JAlzheimers Dis 33(4):983-997
12.Hisatsune T,Kaneko J,Kurashige H et al(2016)Eff ect of anserine/carnosine supplementation on verbal episodic memory in elderly people.J Alzheimers Dis 50(1):149-159
13.Udenigwe CC,Wu S,Drummond K et al(2014)Revisiting the prospects of plastein:thermal and simulated gastric stability in relation to the antioxidative capacity of casein plastein.J Agric Food Chem 62(1):130-135
14.Je JY,Cho YS,Gong M et al(2015)Dipeptide Phe-Cys derived from in silico thermolysin-hydrolysed RuBisCO large subunit suppresses oxidative stress in cultured human hepatocytes.Food Chem 171:287-291
15.Zhang Z,Zhao Y,Wang X et al(2016)The novel dipeptide TyrAla(TA)significantly enhances the lifespan and healthspan of caenorhabditis elegans.Food Funct 7(4):1975-1984
16.Constable DJC,Dunn PJ,Hayler JD et al(2007)Key green chemistry research areas-a perspective from pharmaceutical manufacturers.Green Chem 9:411-420
17.Yazawa K,Numata K(2014)Recent advances in chemoenzymatic peptide syntheses.Molecules 19(9):13755-13774
18.St-Jacques AD,Rachel NM,Dan RC et al(2016)Specificity of transglutaminase-catalyzed peptide synthesis.J Mol Catal BEnzym 123:53-61
19.Meister A(2006)Proteinase-catalyzed synthesis of peptide bonds.Wiley,New York
20.Schellenberger V,Jakubke HD(1991)Protease-catalyzed kinetically controlled peptide synthesis.Angew Chem(Int Ed Engl)30(11):1437-1449
21.Chen GH,Yao LU(2007)Progress about short peptide synthesis by enzyme in reverse micelle.J Sichuan Univ Sci Eng20(1):104-106(in Chinese)
22.Noritomi H,Suzuki K,Kikuta M et al(2009)Catalytic activity ofα-chymotrypsin in enzymatic peptide synthesis in ionic liquids.Biochem Eng J 47(1-3):27-30
23.Kuhn RW,Walsh KA,Neurath H(1974)Isolation and partial characterization of an acid carboxypeptidase from yeast.Biochem 13(19):3871-3877
24.Zhang QH,Yang GP(2014)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 1325(2):83-91
25.Breddam K,Johansen JT(1984)Semisynthesis of human insulin utilizing chemically modified carboxypeptidase Y.Carlsberg Res Commun 49(4):463-472
26.Peschke B,Bak S(2009)Controlled coupling of peptides at their C-termini.Peptides 30(4):689-698
27.Duan W,Zhang Y,Xu G(2016)Optimization and application of protein C-terminal labeling by carboxypeptidase Y.Chin JBiotechnol 32(1):135-148
28.Doi E,Shibata D,Matoba T(1981)Modified colorimetric ninhydrin methods for peptidase assay.Anal Biochem118(1):173-184
29.Guzmán Barberis F,Illanes S et al(2007)Peptide synthesis:chemical or enzymatic.Electron J Biotechn 10(2):279-314
30.Rawal VH,Cava MP(1985)Thermolytic removal of t-butyloxycarbonyl(BOC)protecting group on indoles and pyrroles.Tetrahedron Lett 26(50):6141-6142
31.Ogino H,Yamada M,Watanabe F et al(1999)Peptide synthesis catalyzed by organic solvent-stable protease from Pseudomonas aeruginosa PST-01 in monophasic aqueous-organic solvent systems.J Biosci Bioeng 88(5):513-518
32.Widmer F,Breddam K,Johansen JT(1981)Influence of the structure of amine components on carboxypeptidase Y catalyzed amide bond formation.Carlsberg Res Commun 46(1):97-106
33.Wang M,Wei Q,Yu Q et al(2011)Kinetically controlled enzymatic synthesis of dipeptide precursor of L-alanyl-L-glutamine.Biotechnol Appl Biochem 58(6):449-455
34.Breddam K,Widmer F,Meldal M(1991)Amidation of growth hormone releasing factor(1-29)by serine carboxypeptidase catalysed transpeptidation.Int J Pept Prot Res 37(2):153-160
35.Yu X,Liu F,Zou Y et al(2016)Biosynthesis of strained piperazine alkaloids:uncovering the concise pathway of herquline A.JAm Chem Soc 138(41):13529-13532
36.Marangoni AG(2003)Enzyme kinetics:a modern approach.Wiley,New York
2.Kumar MBA,Gao Y,Shen W et al(2015)Valorisation of protein waste:an enzymatic approach to make commodity chemicals.Front Chem Sci Eng 9(3):295-307
3.Gauthier SF,Pouliot Y,Saint-Sauveur D(2006)Immunomodulatory peptides obtained by the enzymatic hydrolysis of whey proteins.Int Dairy J 16(11):1315-1323
4.Jia J,Ma H,Zhao W et al(2010)The use of ultrasound for enzymatic preparation of ACE-inhibitory peptides from wheat germ protein.Food Chem 119(1):336-342
5.Rahman MS,Choi YH,Choi YS et al(2017)Glycin-rich antimicrobial peptide YD1 from B.amyloliquefaciens,induced morphological alteration in and showed affinity for plasmid DNA of E.coli.AMB Express 7(1):8
6.Yao Z,Che XC,Lu R et al(2007)Inhibition by tyroserleutide(YSL)on the invasion and adhesion of the mouse melanoma cell.Mol Med 13(1-2):14-21
7.Muller FL,Lustgarten MS,Jang Y et al(2007)Trends in oxidative aging theories.Free Radic Bio Med 43(4):477-503
8.White J,Dawson B,Landers G et al(2013)Eff ect of supplemental oxygen on post-exercise inflammatory response and oxidative stress.Eur J Appl Physiol 113(4):1059-1067
9.Chasovnikova LV,Formazyuk VE,Sergienko VI et al(1990)The antioxidative properties of carnosine and other drugs.Biochem Int 20(6):1097-1103
10.Culbertson JY,Kreider RB,Greenwood M et al(2010)Eff ects of beta-alanine on muscle carnosine and exercise performance:a review of the current literature.Nutrients 2(1):75-98
11.Herculano B,Tamura M,Ohba A et al(2013)β-alanyl-L-histidine rescues cognitive deficits caused by feeding a high fat diet in a transgenic mouse model of Alzheimer’s disease.JAlzheimers Dis 33(4):983-997
12.Hisatsune T,Kaneko J,Kurashige H et al(2016)Eff ect of anserine/carnosine supplementation on verbal episodic memory in elderly people.J Alzheimers Dis 50(1):149-159
13.Udenigwe CC,Wu S,Drummond K et al(2014)Revisiting the prospects of plastein:thermal and simulated gastric stability in relation to the antioxidative capacity of casein plastein.J Agric Food Chem 62(1):130-135
14.Je JY,Cho YS,Gong M et al(2015)Dipeptide Phe-Cys derived from in silico thermolysin-hydrolysed RuBisCO large subunit suppresses oxidative stress in cultured human hepatocytes.Food Chem 171:287-291
15.Zhang Z,Zhao Y,Wang X et al(2016)The novel dipeptide TyrAla(TA)significantly enhances the lifespan and healthspan of caenorhabditis elegans.Food Funct 7(4):1975-1984
16.Constable DJC,Dunn PJ,Hayler JD et al(2007)Key green chemistry research areas-a perspective from pharmaceutical manufacturers.Green Chem 9:411-420
17.Yazawa K,Numata K(2014)Recent advances in chemoenzymatic peptide syntheses.Molecules 19(9):13755-13774
18.St-Jacques AD,Rachel NM,Dan RC et al(2016)Specificity of transglutaminase-catalyzed peptide synthesis.J Mol Catal BEnzym 123:53-61
19.Meister A(2006)Proteinase-catalyzed synthesis of peptide bonds.Wiley,New York
20.Schellenberger V,Jakubke HD(1991)Protease-catalyzed kinetically controlled peptide synthesis.Angew Chem(Int Ed Engl)30(11):1437-1449
21.Chen GH,Yao LU(2007)Progress about short peptide synthesis by enzyme in reverse micelle.J Sichuan Univ Sci Eng20(1):104-106(in Chinese)
22.Noritomi H,Suzuki K,Kikuta M et al(2009)Catalytic activity ofα-chymotrypsin in enzymatic peptide synthesis in ionic liquids.Biochem Eng J 47(1-3):27-30
23.Kuhn RW,Walsh KA,Neurath H(1974)Isolation and partial characterization of an acid carboxypeptidase from yeast.Biochem 13(19):3871-3877
24.Zhang QH,Yang GP(2014)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 1325(2):83-91
25.Breddam K,Johansen JT(1984)Semisynthesis of human insulin utilizing chemically modified carboxypeptidase Y.Carlsberg Res Commun 49(4):463-472
26.Peschke B,Bak S(2009)Controlled coupling of peptides at their C-termini.Peptides 30(4):689-698
27.Duan W,Zhang Y,Xu G(2016)Optimization and application of protein C-terminal labeling by carboxypeptidase Y.Chin JBiotechnol 32(1):135-148
28.Doi E,Shibata D,Matoba T(1981)Modified colorimetric ninhydrin methods for peptidase assay.Anal Biochem118(1):173-184
29.Guzmán Barberis F,Illanes S et al(2007)Peptide synthesis:chemical or enzymatic.Electron J Biotechn 10(2):279-314
30.Rawal VH,Cava MP(1985)Thermolytic removal of t-butyloxycarbonyl(BOC)protecting group on indoles and pyrroles.Tetrahedron Lett 26(50):6141-6142
31.Ogino H,Yamada M,Watanabe F et al(1999)Peptide synthesis catalyzed by organic solvent-stable protease from Pseudomonas aeruginosa PST-01 in monophasic aqueous-organic solvent systems.J Biosci Bioeng 88(5):513-518
32.Widmer F,Breddam K,Johansen JT(1981)Influence of the structure of amine components on carboxypeptidase Y catalyzed amide bond formation.Carlsberg Res Commun 46(1):97-106
33.Wang M,Wei Q,Yu Q et al(2011)Kinetically controlled enzymatic synthesis of dipeptide precursor of L-alanyl-L-glutamine.Biotechnol Appl Biochem 58(6):449-455
34.Breddam K,Widmer F,Meldal M(1991)Amidation of growth hormone releasing factor(1-29)by serine carboxypeptidase catalysed transpeptidation.Int J Pept Prot Res 37(2):153-160
35.Yu X,Liu F,Zou Y et al(2016)Biosynthesis of strained piperazine alkaloids:uncovering the concise pathway of herquline A.JAm Chem Soc 138(41):13529-13532
36.Marangoni AG(2003)Enzyme kinetics:a modern approach.Wiley,New York