Solid-Phase Enzymatic Peptide Synthesis to Produce an Antioxidant Dipeptide
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摘要
Peptide bond synthesis is favorable to the production of bioactive small peptides. However, the abuse of toxic reagents remains an issue for chemical synthesis method, whereas the low product yield and purity limit the widespread use of enzymatic method. In this study, a new solid-phase enzymatic peptide synthesis(SPEPS) strategy was developed to produce an antioxidant tyrosine-alanine dipeptide(Tyr-Ala) by using recombinant carboxypeptidase Y(CPY) as the catalyst. The general SPEPS procedure involves three steps. First, the N-protected acyl donor was covalently attached to solid resin. Second,the peptide bond was condensed between the acyl donor and the nucleophile under the catalysis of CPY. Finally, one-step cleavage was performed to remove the protecting group and cleave the peptides from solid resin. Upon the optimization of reaction conditions, 77.92%(±2.723%) yield of Tyr-Ala with high product purity of 90.971%(±2.695%) was obtained.In addition, the antioxidant activity of Tyr-Ala was determined by ABTS method, indicating that the synthesized Tyr-Ala obtained by SPEPS showed a superior antioxidant capability compared with commercial glutathione.
Peptide bond synthesis is favorable to the production of bioactive small peptides. However, the abuse of toxic reagents remains an issue for chemical synthesis method, whereas the low product yield and purity limit the widespread use of enzymatic method. In this study, a new solid-phase enzymatic peptide synthesis(SPEPS) strategy was developed to produce an antioxidant tyrosine-alanine dipeptide(Tyr-Ala) by using recombinant carboxypeptidase Y(CPY) as the catalyst. The general SPEPS procedure involves three steps. First, the N-protected acyl donor was covalently attached to solid resin. Second,the peptide bond was condensed between the acyl donor and the nucleophile under the catalysis of CPY. Finally, one-step cleavage was performed to remove the protecting group and cleave the peptides from solid resin. Upon the optimization of reaction conditions, 77.92%(±2.723%) yield of Tyr-Ala with high product purity of 90.971%(±2.695%) was obtained.In addition, the antioxidant activity of Tyr-Ala was determined by ABTS method, indicating that the synthesized Tyr-Ala obtained by SPEPS showed a superior antioxidant capability compared with commercial glutathione.
Peptide bond synthesis is favorable to the production of bioactive small peptides. However, the abuse of toxic reagents remains an issue for chemical synthesis method, whereas the low product yield and purity limit the widespread use of enzymatic method. In this study, a new solid-phase enzymatic peptide synthesis(SPEPS) strategy was developed to produce an antioxidant tyrosine-alanine dipeptide(Tyr-Ala) by using recombinant carboxypeptidase Y(CPY) as the catalyst. The general SPEPS procedure involves three steps. First, the N-protected acyl donor was covalently attached to solid resin. Second,the peptide bond was condensed between the acyl donor and the nucleophile under the catalysis of CPY. Finally, one-step cleavage was performed to remove the protecting group and cleave the peptides from solid resin. Upon the optimization of reaction conditions, 77.92%(±2.723%) yield of Tyr-Ala with high product purity of 90.971%(±2.695%) was obtained.In addition, the antioxidant activity of Tyr-Ala was determined by ABTS method, indicating that the synthesized Tyr-Ala obtained by SPEPS showed a superior antioxidant capability compared with commercial glutathione.
引文
1. Li-Chan ECY(2015)Bioactive peptides and protein hydrolysates:research trends and challenges for application as nutraceuticals and functional food ingredients. Curr Opin Food Sci 1:28-37
2. McCann KB, Shiell BJ, Michalski WP et al(2006)Isolation and characterisation of a novel antibacterial peptide from bovineαS1-casein. Int Dairy J 16(4):316-323
3. Mendis E, Rajapakse N, Byun HG et al(2005)Investigation of jumbo squid(Dosidicus gigas)skin gelatin peptides for their in vitro antioxidant effects. Life Sci 77(17):2166-2178
4. 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
5. Garofalo M, Iovine B, Kuryk L et al(2016)Oncolytic adenovirus loaded with L-carnosine as novel strategy to enhance the antitumor activity. Mol Cancer Ther 15(4):651-660
6. 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
7. Jemil I, Abdelhedi O, Nasri R et al(2017)Novel bioactive peptides from enzymatic hydrolysate of Sardinelle(Sardinella aurita)muscle proteins hydrolysed by Bacillus subtilis A26 proteases.Food Res Int 100(Pt 1):121-133
8. Ortiz-Martinez M, Otero-Pappatheodorou JT, Serna-Saldivar SO et al(2017)Antioxidant activity and characterization of protein fractions and hydrolysates from normal and quality protein maize kernels. J Cereal Sci 76:85-91
9. Rayaprolu SJ, Hettiarachchy NS, Horax R et al(2017)Purification and characterization of a peptide from soybean with cancer cell proliferation inhibition. J Food Biochem 41(4):e 12374
10. Ozcengiz G, Ogiilur I(2015)Biochemistry, genetics and regulation of bacilysin biosynthesis and its significance more than an antibiotic. New Biotechnol 32(6):612-619
11. Gunes S, Tamburaci S, Dalay MC et al(2017)In vitro evaluation of Spirulina platensis extract incorporated skin cream with its wound healing and antioxidant activities. Pharm Biol55(1):1824-1832
12. Sornwatana T, Bangphoomi K, Roytrakul S et al(2015)Chebulin:Terminalia chebula Retz. fruit-derived peptide with angiotensin-Iconverting enzyme inhibitory activity. Biotechnol Appl Biochem62(6):746-753
13. Ungaro VA, Liria CW, Romagna CD et al(2015)A green route for the synthesis of a bitter-taste dipeptide combining biocatalysis,heterogeneous metal catalysis and magnetic nanoparticles. RSC Adv 5(46):36449-36455
14. Toplak A, Nuijens T, Quaedflieg PJLM et al(2015)Peptide synthesis in neat organic solvents with novel thermostable proteases.Enzyme Microb Tech 73-74:20-28
15. Bordusa F(2002)Proteases in organic synthesis. Chem Rev102(12):4817-4868
16. Manning M(1968)Synthesis by the Merrifield method of a protected nonapeptide amide with the amino acid sequence of oxytocin. J Am Chem Soc 90(5):1348-134917. Hansen PR, Oddo A(2015)Fmoc solid-phase peptide synthesis.
In:Houen G(ed)Peptide antibodies. Humana Press, New York,USA
18. Amblard M, Fehrentz JA, Martinez J et al(2006)Methods and protocols of modern solid phase peptide synthesis. Mol Biotechnol 33(3):239-254
19. Kent SBH(2009)Total chemical synthesis of proteins. Chem Soc Rev 38(2):338-351
20. Zhang M, Yu Q, Liu Z et al(2017)UBX domain-containing proteins are involved in lipid homeostasis and stress responses in Pichia pastoris. Int J Biochem Cell B 90:136-144
21. 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
22. Chiba Y, Midorikawa T, Ichishima E(1995)Cloning and expression of the carboxypeptidase gene from Aspergillus saitoi and determination of the catalytic residues by site-directed mutagenesis. Biochem J 308(2):405-409
23. Dridi F, Marrakchi M, Gargouri M et al(2015)Comparison of carboxypeptidase Y and thermolysin for ochratoxin A electrochemical biosensing. Anal Methods 7(20):8954-8960
24. Mahoney JA, Ntolosi B, DaSilva RP et al(2001)Cloning and characterization of CPVL,a novel serine carboxypeptidase,from human macrophages. Genomics 72(3):243-251
25. Ahmad M, Hirz M, Pichler H et al(2014)Protein expression in Pichia pastoris:recent achievements and perspectives for heterologous protein production. Appl Microbiol Biotechnol98(12):5301-5317
26. Doi E, Shibata D, Matoba T(1981)Modified colorimetric ninhydrin methods for peptidase assay. Anal Biochem 118(1):173-184
27. Wang J, Liu K, Li X et al(2017)Variation of active constituents and antioxidant activity in Scabiosa tschiliensis Grunning from different stages. J Food Sci Technol 54(8):2288-2295
28. Shan Y,Qi W,Wang M et al(2018)Kinetically controlled carboxypeptidase-catalyzed synthesis of novel antioxidantdipeptide precursor BOC-Tyr-Ala. Trans Tianjin Univ. https://doi.org/10.1007/s 12209-018-0166-2
29. Ganesan A(2003)Wang resin. In:Paquette LA(ed)e-EROS encyclopedia of reagents for organic synthesis. Wiley, New York,USA
30. Zhang DY, Luo M, Wang W et al(2013)Variation of active constituents and antioxidant activity in pyrola[P. incarnata Fisch.] from different sites in Northeast China. Food Chem141(3):2213-2219
31. Luo J, Li L, Kong L(2012)Preparative separation of phenylpropenoid glycerides from the bulbs of Lilium lancifolium by highspeed counter-current chromatography and evaluation of their antioxidant activities. Food Chem 131(3):1056-1062
2. McCann KB, Shiell BJ, Michalski WP et al(2006)Isolation and characterisation of a novel antibacterial peptide from bovineαS1-casein. Int Dairy J 16(4):316-323
3. Mendis E, Rajapakse N, Byun HG et al(2005)Investigation of jumbo squid(Dosidicus gigas)skin gelatin peptides for their in vitro antioxidant effects. Life Sci 77(17):2166-2178
4. 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
5. Garofalo M, Iovine B, Kuryk L et al(2016)Oncolytic adenovirus loaded with L-carnosine as novel strategy to enhance the antitumor activity. Mol Cancer Ther 15(4):651-660
6. 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
7. Jemil I, Abdelhedi O, Nasri R et al(2017)Novel bioactive peptides from enzymatic hydrolysate of Sardinelle(Sardinella aurita)muscle proteins hydrolysed by Bacillus subtilis A26 proteases.Food Res Int 100(Pt 1):121-133
8. Ortiz-Martinez M, Otero-Pappatheodorou JT, Serna-Saldivar SO et al(2017)Antioxidant activity and characterization of protein fractions and hydrolysates from normal and quality protein maize kernels. J Cereal Sci 76:85-91
9. Rayaprolu SJ, Hettiarachchy NS, Horax R et al(2017)Purification and characterization of a peptide from soybean with cancer cell proliferation inhibition. J Food Biochem 41(4):e 12374
10. Ozcengiz G, Ogiilur I(2015)Biochemistry, genetics and regulation of bacilysin biosynthesis and its significance more than an antibiotic. New Biotechnol 32(6):612-619
11. Gunes S, Tamburaci S, Dalay MC et al(2017)In vitro evaluation of Spirulina platensis extract incorporated skin cream with its wound healing and antioxidant activities. Pharm Biol55(1):1824-1832
12. Sornwatana T, Bangphoomi K, Roytrakul S et al(2015)Chebulin:Terminalia chebula Retz. fruit-derived peptide with angiotensin-Iconverting enzyme inhibitory activity. Biotechnol Appl Biochem62(6):746-753
13. Ungaro VA, Liria CW, Romagna CD et al(2015)A green route for the synthesis of a bitter-taste dipeptide combining biocatalysis,heterogeneous metal catalysis and magnetic nanoparticles. RSC Adv 5(46):36449-36455
14. Toplak A, Nuijens T, Quaedflieg PJLM et al(2015)Peptide synthesis in neat organic solvents with novel thermostable proteases.Enzyme Microb Tech 73-74:20-28
15. Bordusa F(2002)Proteases in organic synthesis. Chem Rev102(12):4817-4868
16. Manning M(1968)Synthesis by the Merrifield method of a protected nonapeptide amide with the amino acid sequence of oxytocin. J Am Chem Soc 90(5):1348-134917. Hansen PR, Oddo A(2015)Fmoc solid-phase peptide synthesis.
In:Houen G(ed)Peptide antibodies. Humana Press, New York,USA
18. Amblard M, Fehrentz JA, Martinez J et al(2006)Methods and protocols of modern solid phase peptide synthesis. Mol Biotechnol 33(3):239-254
19. Kent SBH(2009)Total chemical synthesis of proteins. Chem Soc Rev 38(2):338-351
20. Zhang M, Yu Q, Liu Z et al(2017)UBX domain-containing proteins are involved in lipid homeostasis and stress responses in Pichia pastoris. Int J Biochem Cell B 90:136-144
21. 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
22. Chiba Y, Midorikawa T, Ichishima E(1995)Cloning and expression of the carboxypeptidase gene from Aspergillus saitoi and determination of the catalytic residues by site-directed mutagenesis. Biochem J 308(2):405-409
23. Dridi F, Marrakchi M, Gargouri M et al(2015)Comparison of carboxypeptidase Y and thermolysin for ochratoxin A electrochemical biosensing. Anal Methods 7(20):8954-8960
24. Mahoney JA, Ntolosi B, DaSilva RP et al(2001)Cloning and characterization of CPVL,a novel serine carboxypeptidase,from human macrophages. Genomics 72(3):243-251
25. Ahmad M, Hirz M, Pichler H et al(2014)Protein expression in Pichia pastoris:recent achievements and perspectives for heterologous protein production. Appl Microbiol Biotechnol98(12):5301-5317
26. Doi E, Shibata D, Matoba T(1981)Modified colorimetric ninhydrin methods for peptidase assay. Anal Biochem 118(1):173-184
27. Wang J, Liu K, Li X et al(2017)Variation of active constituents and antioxidant activity in Scabiosa tschiliensis Grunning from different stages. J Food Sci Technol 54(8):2288-2295
28. Shan Y,Qi W,Wang M et al(2018)Kinetically controlled carboxypeptidase-catalyzed synthesis of novel antioxidantdipeptide precursor BOC-Tyr-Ala. Trans Tianjin Univ. https://doi.org/10.1007/s 12209-018-0166-2
29. Ganesan A(2003)Wang resin. In:Paquette LA(ed)e-EROS encyclopedia of reagents for organic synthesis. Wiley, New York,USA
30. Zhang DY, Luo M, Wang W et al(2013)Variation of active constituents and antioxidant activity in pyrola[P. incarnata Fisch.] from different sites in Northeast China. Food Chem141(3):2213-2219
31. Luo J, Li L, Kong L(2012)Preparative separation of phenylpropenoid glycerides from the bulbs of Lilium lancifolium by highspeed counter-current chromatography and evaluation of their antioxidant activities. Food Chem 131(3):1056-1062