4-HR与抗坏血酸对中华管鞭虾多酚氧化酶的抑制动力学模拟分析
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摘要
研究无硫复合抑制剂主要作用成分4-己基间苯二酚(4-hexylresorcinol,4-HR)、抗坏血酸对中华管鞭虾多酚氧化酶(polyphenoloxidase,PPO)活性的抑制作用,并从抑制剂化学结构及动力学模型出发分析推断其抑制机理。结果表明,4-HR、抗坏血酸均能抑制PPO活力,且对酶促反应有延滞作用,两者半抑制浓度分别为0.072、2.02 mmol/L,对中华管鞭虾PPO的抑制作用都表现为竞争性抑制;化学动力学模拟分析显示4-HR、抗坏血酸均可与PPO双铜活性中心结合,其中4-HR可生成一种高稳定的多酚氧化酶络合物,持续有效地消耗PPO。4-HR、抗坏血酸均能有效抑制中华管鞭虾PPO酶促反应,其中4-HR抑制效果更显著。本实验为无硫复合抑制剂在海水虾捕捞与加工过程中的应用推广提供理论依据。
The purpose of this study was to investigate the inhibitory effects of 4-hexylresorcinol(4-HR) and ascorbic acid(AA) on the activity of purified polyphenoloxidase(PPO) from Solenocera crassicornis. In addition, the inhibitory mechanism was elucidated through kinetic modelling with respect to the structures of the inhibitors. The results showed that both 4-HR and AA could significantly inhibit PPO activity with a half-maximal inhibitory concentration(IC50) of 0.072 and 2.02 mmol/L, respectively and provoke a lag period for the enzymatic reaction. Each of the two compounds exerted competitive inhibition on PPO activity. The kinetic analysis indicated that both 4-HR and AA could be combined with the di-copper binding active center of PPO. 4-HR could consume PPO continuously and effectively by forming a highly stable complex. In conclusion, both 4-HR and AA have been shown to be effective in controlling the enzymatic reaction rate of PPO, 4-HR being stronger than AA. This result provides theoretical support for the application of sulfur-free 4-HR-AA composite preservative in the catching and processing of shrimp.
The purpose of this study was to investigate the inhibitory effects of 4-hexylresorcinol(4-HR) and ascorbic acid(AA) on the activity of purified polyphenoloxidase(PPO) from Solenocera crassicornis. In addition, the inhibitory mechanism was elucidated through kinetic modelling with respect to the structures of the inhibitors. The results showed that both 4-HR and AA could significantly inhibit PPO activity with a half-maximal inhibitory concentration(IC50) of 0.072 and 2.02 mmol/L, respectively and provoke a lag period for the enzymatic reaction. Each of the two compounds exerted competitive inhibition on PPO activity. The kinetic analysis indicated that both 4-HR and AA could be combined with the di-copper binding active center of PPO. 4-HR could consume PPO continuously and effectively by forming a highly stable complex. In conclusion, both 4-HR and AA have been shown to be effective in controlling the enzymatic reaction rate of PPO, 4-HR being stronger than AA. This result provides theoretical support for the application of sulfur-free 4-HR-AA composite preservative in the catching and processing of shrimp.
引文
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[18]赵铮.酶反应机理的多理论层次量子力学/分子力学研究[D].合肥:中国科学技术大学,2008:2-24.
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[20]程林.肽酰转移酶催化肽键形成机理的理论研究[D].武汉:华中师范大学,2006:16-18.
[21]汪敬,顾健德,田安民.5-硝基-1-氢-四唑衍生物热分解机理的从头算分子动力学模拟及密度泛函理论研究[J].四川大学学报(自然科学版),2002,39(2):225-230.DOI:10.3969/j.issn.0490-6756.2002.02.031.
[22]LEEKIM M S,HWANG E S,KIM K H.Inhibition studies on burdock polyphenyl oxidase(PPO)activity[J].Journal of Food Processing&Preservation,2010,21(6):485-494.DOI:10.1111/j.1745-4549.1997.tb00797.x.
[23]KUBO I,KINST-HORI I,ISHIGURO K,et al.Tyrosinase inhibitory flavonoids from Heterotheca inuloides and their structural functions[J].Bioorganic&Medicinal Chemistry Letters,1994,4(12):1443-1446.DOI:10.1016/S0960-894X(01)80510-2.
[24]ARIAS E,GONZáLEZ J,ORIA R,et al.Ascorbic acid and4-hexylresorcinol effects on pear PPO and PPO catalyzed browning reaction[J].Journal of Food Science,2007,72(8):C422-C429.DOI:10.1111/j.1750-3841.2007.00484.x.
[25]ALVAREZ-PARRILLA E,LADELA R,RODRIGO-GARCíA J,et al.Dual effect ofβ-cyclodextrin(β-CD)on the inhibition of apple polyphenol oxidase by 4-hexylresorcinol(HR)and methyl jasmonate(MJ)[J].Food Chemistry,2007,101(4):1346-1356.DOI:10.1016/j.foodchem.2006.03.040.
[26]GALV肙J A,YOKOYAMA V A,SAVAY-DA-SILVA L K,et al.Effect of 4-hexylresorcinol and sodium metabisulphite on spoilage and melanosis inhibition in Xiphopenaeus kroyeri shrimps[J].Journal of Food Processing&Preservation,2016,41(3):e12943.DOI:10.1111/jfpp.12943.
[27]SON S M,MOON K D,LEE C Y.Inhibitory effects of various antibrowning agents on apple slices[J].Food Chemistry,2001,73(1):23-30.DOI:10.1016/S0308-8146(00)00274-0.
[28]LIMBO S,PIERGIOVANNI L.Shelf life of minimally processed potatoes:part 1.Effects of high oxygen partial pressures in combination with ascorbic and citric acids on enzymatic browning[J].Postharvest Biology&Technology,2006,39(3):254-264.DOI:10.1016/j.postharvbio.2005.10.016.
[29]蔡燕萍,张建友.虾体多酚氧化酶特性及其抑制技术研究进展[J].食品工业科技,2012,33(13):424-428.DOI:10.13386/j.issn1002-0306.2012.13.053.
[30]ALI H M,ELGIZAWY A M,ELBASSIOUNY R E,et al.Browning inhibition mechanisms by cysteine,ascorbic acid and citric acid,and identifying PPO-catechol-cysteine reaction products[J].Journal of Food Science&Technology,2015,52(6):3651-3659.DOI:10.1007/s13197-014-1437-0.
[31]ARIAS E,GONZáLEZ J,PEIRóJ M,et al.Browning prevention by ascorbic acid and 4-hexylresorcinol:different mechanisms of action on polyphenol oxidase in the presence and in the absence of substrates[J].Journal of Food Science,2007,72(9):464-470.DOI:10.1111/j.1750-3841.2007.00514.x.
[32]ROLFF M,SCHOTTENHEIM J,DECKER H,et al.Copper-O-2reactivity of tyrosinase model towards external monophenolic substrates:molecular mechanism and comparison with the enzyme[J].Chemical Society Reviews,2011,40(7):4077-4098.DOI:10.1039/c0cs00202j.
[2]农业部渔业渔政管理局.2016中国渔业统计年鉴[M].北京:中国农业出版社,2016:28-46.
[3]凌萍华,谢晶.冰温技术结合保鲜剂对南美白对虾品质的影响[J].食品科学,2010,31(14):280-284.
[4]NIRMAL N P,BENJAKUL S,AHMAD M,et al.Undesirable enzymatic browning in crustaceans:causative effects and its inhibition by phenolic compounds[J].Critical Reviews in Food Science and Nutrition,2015,55(14):1992-2003.DOI:10.1080/10408398.2012.755148.
[5]励建荣,李学鹏,周凯,等.我国虾类产品中亚硫酸盐的安全性评价[J].食品安全质量检测学报,2014,5(8):2295-2301.
[6]REIST M,JENNER P,HALLIWELL B.Sulphite enhances peroxynitrite-dependent 1-antiproteinase inactivation:a mechanism of lung injury by sulphur dioxide[J].FEBS Letters,1998,423(2):231-234.DOI:10.1016/S0014-5793(98)00099-4.
[7]XIANG X W,ZHOU Y F,HAO Y B,et al.Purification and characterization of the polyphenoloxidase(PPO)isolated from Solenocera crassicornis(H.Milne Edwards,1837)[J].Crustaceana,2015,88(3):259-272.DOI:10.1163/15685403-00003412.
[8]NURHAYATI T,AMBARSARI L,SUHANDANA M.Partial purification of polyphenoloxidase of black tiger shrimp(Penaeus monodon)from Indonesian waters[J].International Food Research Journal,2015,22(6):2267-2273.
[9]吕艳芳,魏春娇,王娇,等.南美白对虾酚氧化酶的提取及生化特性[J].食品科学,2014,35(7):113-117.DOI:10.7506/spkx1002-6630-201407023.
[10]SAE-LEAW T,BENJAKUL S,SIMPSON B K.Effect of catechin and its derivatives on inhibition of polyphenoloxidase and melanosis of pacific white shrimp[J].Journal of Food Science and Technology,2017,54(5):1098-1107.DOI:10.1007/s13197-017-2556-1.
[11]NIRMAL N P,BENJAKUL S.Effect of ferulic acid on inhibition of polyphenoloxidase and quality changes of pacific white shrimp(Litopenaeus vannamei)during iced storage[J].Food Chemistry,2009,116(1):323-331.DOI:10.1016/j.foodchem.2009.02.054.
[12]陈鲁萍,刘书来,毛泽明,等.不同抑制剂对凡纳滨对虾多酚氧化酶活性的影响[J].水产科学,2010,29(11):639-642.DOI:10.3969/j.issn.1003-1111.2010.11.003.
[13]MONTERO P,AVALOS A,PEREZ-MATEOS M.Characterization of polyphenoloxidase of prawns(Penaeus japonicus).Alternatives to inhibition:additives and high-pressure treatment[J].Food Chemistry,2001,75(3):317-324.DOI:10.1016/S0308-8146(01)00206-0.
[14]曹荣,薛长湖,徐丽敏.复合保鲜剂在对虾保鲜及防黑变中的应用[J].农业工程学报,2009,25(8):294-298.DOI:10.3969/j.issn.1002-6819.2009.08.052.
[15]孟现美,王加磊,张少龙,等.分子动力学研究2-乙酸苯并噻吩在HIV-1蛋白酶与抑制剂结合中的作用[J].化学学报,2013,71(8):1167-1174.DOI:10.6023/A13030327.
[16]潘晓亮.量子力学和分子力学在酶催化研究中的应用[D].长春:吉林大学,2012:4-8.
[17]NIRMAL N P,BENJAKUL S.Inhibition kinetics of catechin and ferulic acid on polyphenoloxidase from cephalothorax of pacific white shrimp(Litopenaeus vannamei)[J].Food Chemistry,2012,131(2):569-573.DOI:10.1016/j.foodchem.2011.09.025.
[18]赵铮.酶反应机理的多理论层次量子力学/分子力学研究[D].合肥:中国科学技术大学,2008:2-24.
[19]熊鹰.四嗪类高氮化合物热分解机理的理论研究[D].绵阳:中国工程物理研究院,2007:7-13.
[20]程林.肽酰转移酶催化肽键形成机理的理论研究[D].武汉:华中师范大学,2006:16-18.
[21]汪敬,顾健德,田安民.5-硝基-1-氢-四唑衍生物热分解机理的从头算分子动力学模拟及密度泛函理论研究[J].四川大学学报(自然科学版),2002,39(2):225-230.DOI:10.3969/j.issn.0490-6756.2002.02.031.
[22]LEEKIM M S,HWANG E S,KIM K H.Inhibition studies on burdock polyphenyl oxidase(PPO)activity[J].Journal of Food Processing&Preservation,2010,21(6):485-494.DOI:10.1111/j.1745-4549.1997.tb00797.x.
[23]KUBO I,KINST-HORI I,ISHIGURO K,et al.Tyrosinase inhibitory flavonoids from Heterotheca inuloides and their structural functions[J].Bioorganic&Medicinal Chemistry Letters,1994,4(12):1443-1446.DOI:10.1016/S0960-894X(01)80510-2.
[24]ARIAS E,GONZáLEZ J,ORIA R,et al.Ascorbic acid and4-hexylresorcinol effects on pear PPO and PPO catalyzed browning reaction[J].Journal of Food Science,2007,72(8):C422-C429.DOI:10.1111/j.1750-3841.2007.00484.x.
[25]ALVAREZ-PARRILLA E,LADELA R,RODRIGO-GARCíA J,et al.Dual effect ofβ-cyclodextrin(β-CD)on the inhibition of apple polyphenol oxidase by 4-hexylresorcinol(HR)and methyl jasmonate(MJ)[J].Food Chemistry,2007,101(4):1346-1356.DOI:10.1016/j.foodchem.2006.03.040.
[26]GALV肙J A,YOKOYAMA V A,SAVAY-DA-SILVA L K,et al.Effect of 4-hexylresorcinol and sodium metabisulphite on spoilage and melanosis inhibition in Xiphopenaeus kroyeri shrimps[J].Journal of Food Processing&Preservation,2016,41(3):e12943.DOI:10.1111/jfpp.12943.
[27]SON S M,MOON K D,LEE C Y.Inhibitory effects of various antibrowning agents on apple slices[J].Food Chemistry,2001,73(1):23-30.DOI:10.1016/S0308-8146(00)00274-0.
[28]LIMBO S,PIERGIOVANNI L.Shelf life of minimally processed potatoes:part 1.Effects of high oxygen partial pressures in combination with ascorbic and citric acids on enzymatic browning[J].Postharvest Biology&Technology,2006,39(3):254-264.DOI:10.1016/j.postharvbio.2005.10.016.
[29]蔡燕萍,张建友.虾体多酚氧化酶特性及其抑制技术研究进展[J].食品工业科技,2012,33(13):424-428.DOI:10.13386/j.issn1002-0306.2012.13.053.
[30]ALI H M,ELGIZAWY A M,ELBASSIOUNY R E,et al.Browning inhibition mechanisms by cysteine,ascorbic acid and citric acid,and identifying PPO-catechol-cysteine reaction products[J].Journal of Food Science&Technology,2015,52(6):3651-3659.DOI:10.1007/s13197-014-1437-0.
[31]ARIAS E,GONZáLEZ J,PEIRóJ M,et al.Browning prevention by ascorbic acid and 4-hexylresorcinol:different mechanisms of action on polyphenol oxidase in the presence and in the absence of substrates[J].Journal of Food Science,2007,72(9):464-470.DOI:10.1111/j.1750-3841.2007.00514.x.
[32]ROLFF M,SCHOTTENHEIM J,DECKER H,et al.Copper-O-2reactivity of tyrosinase model towards external monophenolic substrates:molecular mechanism and comparison with the enzyme[J].Chemical Society Reviews,2011,40(7):4077-4098.DOI:10.1039/c0cs00202j.