利用坛紫菜藻红蛋白制备脯氨酰内肽酶抑制肽
|
摘要
以坛紫菜(Porphyra haitanensis)为原料,通过硫酸铵盐析和DEAE-Sepharose阴离子交换柱层析等方法分离纯化得到一种高纯度藻红蛋白(R-phycoerythrin,R-PE)(A_(565 nm)/A_(280 nm)=5.4)。利用胃、肠液消化酶对R-PE进行酶解制备脯氨酰内肽酶(prolyl endopeptidase,PEP)抑制肽。酶解条件为:1)胃蛋白酶与R-PE比例0.5%(m/m),酶解时间30 min,pH 1.2,温度37℃;2)胰蛋白酶和胰凝乳蛋白酶质量比为1∶1的混合酶,其与R-PE比例0.25%(m/m),酶解时间30 min,pH 7.5,温度37℃。Tricine-十二烷基硫酸钠-聚丙烯酰氨凝胶电泳分析发现,经两步酶解,R-PE被完全降解。采用凝胶过滤色谱法测得R-PE水解液中分子质量在3 kDa以下的小肽含量为86.06%。R-PE水解液对PEP抑制水平IC_(50)为136.35μg/mL。酶抑制动力学研究发现,R-PE水解液对PEP表现为可逆的非竞争性抑制作用,抑制常数Ki为14μg/mL。本研究利用坛紫菜R-PE制备活性高的PEP抑制肽,将为坛紫菜深加工及高值化利用提供一定的理论参考。
In this paper, R-phycoerythrin with relatively high purity(A_(565 nm)/A_(280 nm) = 5.4) was obtained from Porphyra haitanensis by ammonium sulfate fractionation and DEAE-Sepharose anion exchange chromatography. Prolyl endopeptidase(PEP) inhibitory peptides from R-phycoerythrin were prepared by two-step enzymatic digestion with gastrointestinal fluid proteases. Optimal hydrolysis conditions were determined as sequential hydrolysis with 0.5% pepsin at an enzyme-tosubstrate ratio of for 30 min at an initial pH of 1.2 and 37 ℃ followed by a 1:1 mixture of trypsin and chymotrypsin at an enzyme-to-substrate ratio of 0.25% for 30 min at pH 7.5 and 37 ℃. Tricine-SDS-PAGE showed that R-phycoerythrin was completely degraded by two-step enzymatic hydrolysis. Gel filtration chromatography analysis revealed that fractions with molecular mass lower than 3 kDa accounted for 86.06% of the total peptides. The half-maximum inhibitory concentration(IC_(50)) of R-phycoerythrin hydrolysate toward PEP was 136.35 μg/mL. Inhibition kinetic study revealed that the hydrolysate acted as a noncompetitive inhibitor and the constant of inhibition(Ki) was 14 μg/mL. Successful preparation of R-phycoerythrin peptides with potential PEP inhibitory activity provides a theoretical rationale for the processing and utilization of P. haitanensis.
In this paper, R-phycoerythrin with relatively high purity(A_(565 nm)/A_(280 nm) = 5.4) was obtained from Porphyra haitanensis by ammonium sulfate fractionation and DEAE-Sepharose anion exchange chromatography. Prolyl endopeptidase(PEP) inhibitory peptides from R-phycoerythrin were prepared by two-step enzymatic digestion with gastrointestinal fluid proteases. Optimal hydrolysis conditions were determined as sequential hydrolysis with 0.5% pepsin at an enzyme-tosubstrate ratio of for 30 min at an initial pH of 1.2 and 37 ℃ followed by a 1:1 mixture of trypsin and chymotrypsin at an enzyme-to-substrate ratio of 0.25% for 30 min at pH 7.5 and 37 ℃. Tricine-SDS-PAGE showed that R-phycoerythrin was completely degraded by two-step enzymatic hydrolysis. Gel filtration chromatography analysis revealed that fractions with molecular mass lower than 3 kDa accounted for 86.06% of the total peptides. The half-maximum inhibitory concentration(IC_(50)) of R-phycoerythrin hydrolysate toward PEP was 136.35 μg/mL. Inhibition kinetic study revealed that the hydrolysate acted as a noncompetitive inhibitor and the constant of inhibition(Ki) was 14 μg/mL. Successful preparation of R-phycoerythrin peptides with potential PEP inhibitory activity provides a theoretical rationale for the processing and utilization of P. haitanensis.
引文
[1]CUNNINGHAM D F,O'CONNOR B.Proline specific peptidases[J].Biochimica et Biophysica Acta(BBA)-Protein Structure and Molecular Enzymology,1997,1343(2):160-186.DOI:10.1016/S0167-4838(97)00134-9.
[2]MORENO-BAYLACH M J,PUTTONEN K A,TENORIOLARANGA J,et al.Prolyl endopeptidase is involved in cellular signalling in human neuroblastoma SH-SY5Y cells[J].Neurosignals,2011,19(2):97-109.DOI:10.1159/000326342.
[3]LAFARGA T,O'CONNOR P,HAYES M.In silico,methods to identify meat-derived prolyl endopeptidase inhibitors[J].Food Chemistry,2015,175(15):337-343.DOI:10.10 1 6/j.foodchem.2014.11.150.
[4]JAAKO K,WANIEK A,PARIK K,et al.Prolyl endopeptidase is involved in the degradation of neural cell adhesion molecules in vitro[J].Journal of Cell Science,2016,129(20):3792-3802.DOI:10.1242/jcs.181891.
[5]DA Z,LI B H,JING W,et al.Prolyl oligopeptidase inhibition attenuates steatosis in the L02 human liver cell line[J].PLoS ONE,2016,11(10):1-13.DOI:10.1371/journal.pone.0165224.
[6]BECKER B,NAZIR F H,BRINKMALM G,et al.Alzheimerassociated cerebrospinal fluid f'ragments of neurogranin are generated by Calpain-1 and prolyl endopeptidase[J].Molecular Neurodegeneration,201 8,13(1):47.DOI:10.11 86/s13024-01 8-0279-z.
[7]BABKOVA K,KORABECNY J,SOUKUP O,et al.Prolyl oligopeptidase and its role in the organism:attention to the most promising and clinically relevant inhibitors[J].Future Medicinal Chemistry,2017,9(10):1015-1038.DOI:10.4155/fmc-2017-0030.
[8]FULOP V,BOCSKEI Z,POLGAR L.Prolyl oligopeptidase:an unusual beta-propeller domain regulates proteolysis[J].Cell,1998,94(2):161-170.DOI:10.1016/S0092-8674(00)81416-6.
[9]KUMAR R,BAVI R,MIN G J,et al.New compounds identified through in silico approaches reduce the a-synuclein expression by inhibiting prolyl oligopeptidase in vitro[J].Scientific Report,2017,7(1):1-14.DOI:10.103 8/s41598-017-11302-0.
[10]TOIDE K,SHINODA M,MIYAZAKI A.A novel prolyl endopeptidase inhibitor,JTP-48 19 its behavioral and neurochemical properties for the treatment of Alzheimer's disease[J].Reviews in the Neurosciences,1998,9(1):17-29.DOI:10.15 15/REVNEURO.1998.9.1.17.
[11]MORAIN P,LESTAGE P,DE N G,et al.S 17092:a prolyl endopeptidase inhibitor as a potential therapeutic drug for memory impairment.preclinical and clinical studies[J].CNS Drug Reviews,2002,8(1):31-35.DOI:10.1111/j.1527-3458.2002.tb00214.x.
[12]JALKANEN A J,PUTTONEN K A,VENALAINEN J I,et al.Beneficial effect of prolyl oligopeptidase inhibition on spatial memory in young but not in old scopolamine-treated rats[J].Basic&Clinical Pharmacology&Toxicology,2007,100(2):132-138.DOI:10.1111/j.1742-7843.2006.00021.x.
[13]SHISHIDO Y,FURUSHIRO M,TANABE S,et al.Effects of prolyl endopeptidase inhibitors and neuropeptides on delayed neuronal death in rats[J].European Journal of Pharmacology,1999,372(2):135-142.DOI:10.1016/S0014-2999(99)00185-5.
[14]SILA A,MARTINEZ-ALVAREZ O,HADDAR A,et al.Recovery,viscoelastic and functional properties of Barbel skin gelatine:investigation of anti-DPP-IV and anti-prolyl endopeptidase activities of generated gelatine polypeptides[J].Food Chemistry,2015,168:478-486.DOI:10.1016/j.foodchem.2014.07.086.
[15]MARTINEZ-ALVAREZ O,BATISTA I,RAMOS C,et al.Enhancement of ACE and prolyl oligopeptidase inhibitory potency of protein hydrolysates from sardine and tuna by-products by simulated gastrointestinal digestion[J].Food&Function,2016,7(4):2066-2073.DOI:10.1039/c5fo01603g.
[16]HSIEH C H,WANG T Y,HUNG C C,et al.Isolation of prolyl endopeptidase inhibitory peptides from a sodium caseinate hydrolysate[J].Food&Function,2016,7(1):565-573.DOI:10.1039/c5fo01262g.
[17]HELLINGER R,KOEHBACH J,PUIGPIONS A,et al.Inhibition of human prolyl oligopeptidase activity by the cyclotide psysol 2 isolated from Psychotria solitudinum[J].Journal of Natural Products,2015,78(5):1073-1082.DOI:10.1021/np501061t.
[18]MANZANARES P,MARTINEZ R,GARRIGUES S,et al.Tryptophan-containing dual neuroprotective peptides:prolyl endopeptidase inhibition and caenorhabditis elegans protection fromβ-amyloid peptide toxicity[J].International Journal of Molecular Sciences,2018,19(5):1491-1508.DOI:10.3390/ijms 19051491.
[19]农业部渔业渔政管理局.2017中国渔业统计年鉴[M].北京:中国农业出版社,2017:28-54.
[20]郭雷,王淑军,郝倩,等.紫菜多糖和藻红蛋白生物活性的研究进展[J].食品研究与开发,2010,31(6):182-185.DOI:10.3969/j.issn.1005-6521.2010.06.054.
[21]胡志和,夏磊,孙振刚,等.酪蛋白水解物中ACE抑制肽分离及氨基酸序列分析[J].食品科学,2015,36(24):156-163.DOI:10.7506/spkx 1002-6630-20 1524028.
[22]韩青,周丽杰,李智博,等.酶法制备联合Plastein反应修饰牡蛎ACE抑制肽工艺优化[J].食品科学,2017,38(6):104-110.DOI:10.7506/spkx 1002-6630-201706016.
[23]苗欣宇,王祖浩,王鹏,等.红松仁清蛋白ACE抑制肽的分离纯化与结构鉴定[J].食品科学,2017,38(5):129-133.DOI:10.7506/spkx 1002-6630-201705021.
[24]华鑫,孙乐常,万楚君,等.刺参ACE抑制肽制备及降压功效分析[J].食品科学,2018,39(10):125-130.DOI:10.7506/spkx1002-6630-201810020.
[25]HANAFI M A,HASHIM S M,CHAY S Y,et al.High angiotensin-I converting enzyme(ACE)inhibitory activity of alcalase-digested green soybean(Glycine max)hydrolysates[J].Food Research International,2018,106:589-597.DOI:10.1016/j.foodres.201 8.01.030.
[26]TU M,WANG C,CHEN C,et al.Identification of a novel ACEinhibitory peptide from casein and evaluation of the inhibitory mechanisms[J].Food Chemistry,2018,256:98-104.DOI:10.1016/j.foodchem.201 8.02.107.
[27]伍强.藻红蛋白生物活性肽的分离纯化及其性质分析[D].厦门:集美大学,2015:19-34.
[28]United States Pharmacopeial Convention,Committee of Revision.The United States pharmacopeia USP23:the national formulari NF 18;national formulary supplements 1-9[M].Berlin:Springer Netherlands,1987:279-301.
[29]黄园园,刘光明,周利亘,等.蟹类主要过敏原的模拟肠胃液消化及其对过敏原活性的影响[J].中国食品学报,2009,9(4):15-22.DOI:10.3969/j.issn.1009-7848.2009.04.003.
[30]付晓苹.红毛藻及坛紫菜藻红蛋白的分离纯化与相关性质的研究[D].厦门:集美大学,2007:12-37.
[31]SCHAGGER H.Tricine-SDS-PAGE[J].Nature Protocols,2006,1(1):16-22.
[32]陈清西.酶学及其研究技术[M].厦门:厦门大学出版社,2015:108-118.
[33]PATIL G,CHETHANA S,SRIDEVI A S,et al.Method to obtain C-phycocyanin of high purity[J].Journal of Chromatography A,2006,1 127(1/2):76-81.DOI:10.101 6/j.chroma.2006.05.073.
[2]MORENO-BAYLACH M J,PUTTONEN K A,TENORIOLARANGA J,et al.Prolyl endopeptidase is involved in cellular signalling in human neuroblastoma SH-SY5Y cells[J].Neurosignals,2011,19(2):97-109.DOI:10.1159/000326342.
[3]LAFARGA T,O'CONNOR P,HAYES M.In silico,methods to identify meat-derived prolyl endopeptidase inhibitors[J].Food Chemistry,2015,175(15):337-343.DOI:10.10 1 6/j.foodchem.2014.11.150.
[4]JAAKO K,WANIEK A,PARIK K,et al.Prolyl endopeptidase is involved in the degradation of neural cell adhesion molecules in vitro[J].Journal of Cell Science,2016,129(20):3792-3802.DOI:10.1242/jcs.181891.
[5]DA Z,LI B H,JING W,et al.Prolyl oligopeptidase inhibition attenuates steatosis in the L02 human liver cell line[J].PLoS ONE,2016,11(10):1-13.DOI:10.1371/journal.pone.0165224.
[6]BECKER B,NAZIR F H,BRINKMALM G,et al.Alzheimerassociated cerebrospinal fluid f'ragments of neurogranin are generated by Calpain-1 and prolyl endopeptidase[J].Molecular Neurodegeneration,201 8,13(1):47.DOI:10.11 86/s13024-01 8-0279-z.
[7]BABKOVA K,KORABECNY J,SOUKUP O,et al.Prolyl oligopeptidase and its role in the organism:attention to the most promising and clinically relevant inhibitors[J].Future Medicinal Chemistry,2017,9(10):1015-1038.DOI:10.4155/fmc-2017-0030.
[8]FULOP V,BOCSKEI Z,POLGAR L.Prolyl oligopeptidase:an unusual beta-propeller domain regulates proteolysis[J].Cell,1998,94(2):161-170.DOI:10.1016/S0092-8674(00)81416-6.
[9]KUMAR R,BAVI R,MIN G J,et al.New compounds identified through in silico approaches reduce the a-synuclein expression by inhibiting prolyl oligopeptidase in vitro[J].Scientific Report,2017,7(1):1-14.DOI:10.103 8/s41598-017-11302-0.
[10]TOIDE K,SHINODA M,MIYAZAKI A.A novel prolyl endopeptidase inhibitor,JTP-48 19 its behavioral and neurochemical properties for the treatment of Alzheimer's disease[J].Reviews in the Neurosciences,1998,9(1):17-29.DOI:10.15 15/REVNEURO.1998.9.1.17.
[11]MORAIN P,LESTAGE P,DE N G,et al.S 17092:a prolyl endopeptidase inhibitor as a potential therapeutic drug for memory impairment.preclinical and clinical studies[J].CNS Drug Reviews,2002,8(1):31-35.DOI:10.1111/j.1527-3458.2002.tb00214.x.
[12]JALKANEN A J,PUTTONEN K A,VENALAINEN J I,et al.Beneficial effect of prolyl oligopeptidase inhibition on spatial memory in young but not in old scopolamine-treated rats[J].Basic&Clinical Pharmacology&Toxicology,2007,100(2):132-138.DOI:10.1111/j.1742-7843.2006.00021.x.
[13]SHISHIDO Y,FURUSHIRO M,TANABE S,et al.Effects of prolyl endopeptidase inhibitors and neuropeptides on delayed neuronal death in rats[J].European Journal of Pharmacology,1999,372(2):135-142.DOI:10.1016/S0014-2999(99)00185-5.
[14]SILA A,MARTINEZ-ALVAREZ O,HADDAR A,et al.Recovery,viscoelastic and functional properties of Barbel skin gelatine:investigation of anti-DPP-IV and anti-prolyl endopeptidase activities of generated gelatine polypeptides[J].Food Chemistry,2015,168:478-486.DOI:10.1016/j.foodchem.2014.07.086.
[15]MARTINEZ-ALVAREZ O,BATISTA I,RAMOS C,et al.Enhancement of ACE and prolyl oligopeptidase inhibitory potency of protein hydrolysates from sardine and tuna by-products by simulated gastrointestinal digestion[J].Food&Function,2016,7(4):2066-2073.DOI:10.1039/c5fo01603g.
[16]HSIEH C H,WANG T Y,HUNG C C,et al.Isolation of prolyl endopeptidase inhibitory peptides from a sodium caseinate hydrolysate[J].Food&Function,2016,7(1):565-573.DOI:10.1039/c5fo01262g.
[17]HELLINGER R,KOEHBACH J,PUIGPIONS A,et al.Inhibition of human prolyl oligopeptidase activity by the cyclotide psysol 2 isolated from Psychotria solitudinum[J].Journal of Natural Products,2015,78(5):1073-1082.DOI:10.1021/np501061t.
[18]MANZANARES P,MARTINEZ R,GARRIGUES S,et al.Tryptophan-containing dual neuroprotective peptides:prolyl endopeptidase inhibition and caenorhabditis elegans protection fromβ-amyloid peptide toxicity[J].International Journal of Molecular Sciences,2018,19(5):1491-1508.DOI:10.3390/ijms 19051491.
[19]农业部渔业渔政管理局.2017中国渔业统计年鉴[M].北京:中国农业出版社,2017:28-54.
[20]郭雷,王淑军,郝倩,等.紫菜多糖和藻红蛋白生物活性的研究进展[J].食品研究与开发,2010,31(6):182-185.DOI:10.3969/j.issn.1005-6521.2010.06.054.
[21]胡志和,夏磊,孙振刚,等.酪蛋白水解物中ACE抑制肽分离及氨基酸序列分析[J].食品科学,2015,36(24):156-163.DOI:10.7506/spkx 1002-6630-20 1524028.
[22]韩青,周丽杰,李智博,等.酶法制备联合Plastein反应修饰牡蛎ACE抑制肽工艺优化[J].食品科学,2017,38(6):104-110.DOI:10.7506/spkx 1002-6630-201706016.
[23]苗欣宇,王祖浩,王鹏,等.红松仁清蛋白ACE抑制肽的分离纯化与结构鉴定[J].食品科学,2017,38(5):129-133.DOI:10.7506/spkx 1002-6630-201705021.
[24]华鑫,孙乐常,万楚君,等.刺参ACE抑制肽制备及降压功效分析[J].食品科学,2018,39(10):125-130.DOI:10.7506/spkx1002-6630-201810020.
[25]HANAFI M A,HASHIM S M,CHAY S Y,et al.High angiotensin-I converting enzyme(ACE)inhibitory activity of alcalase-digested green soybean(Glycine max)hydrolysates[J].Food Research International,2018,106:589-597.DOI:10.1016/j.foodres.201 8.01.030.
[26]TU M,WANG C,CHEN C,et al.Identification of a novel ACEinhibitory peptide from casein and evaluation of the inhibitory mechanisms[J].Food Chemistry,2018,256:98-104.DOI:10.1016/j.foodchem.201 8.02.107.
[27]伍强.藻红蛋白生物活性肽的分离纯化及其性质分析[D].厦门:集美大学,2015:19-34.
[28]United States Pharmacopeial Convention,Committee of Revision.The United States pharmacopeia USP23:the national formulari NF 18;national formulary supplements 1-9[M].Berlin:Springer Netherlands,1987:279-301.
[29]黄园园,刘光明,周利亘,等.蟹类主要过敏原的模拟肠胃液消化及其对过敏原活性的影响[J].中国食品学报,2009,9(4):15-22.DOI:10.3969/j.issn.1009-7848.2009.04.003.
[30]付晓苹.红毛藻及坛紫菜藻红蛋白的分离纯化与相关性质的研究[D].厦门:集美大学,2007:12-37.
[31]SCHAGGER H.Tricine-SDS-PAGE[J].Nature Protocols,2006,1(1):16-22.
[32]陈清西.酶学及其研究技术[M].厦门:厦门大学出版社,2015:108-118.
[33]PATIL G,CHETHANA S,SRIDEVI A S,et al.Method to obtain C-phycocyanin of high purity[J].Journal of Chromatography A,2006,1 127(1/2):76-81.DOI:10.101 6/j.chroma.2006.05.073.