凡纳滨对虾多酚氧化酶的纯化及性质分析
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摘要
通过硫酸铵盐析、DEAE-Cellulose离子交换、Phenyl-Sepharose疏水层析、Hi-Trap Capto-Q强阴离子交换层析等方法,从凡纳滨对虾(Litopenaeus vannamei)虾头中分离纯化出一种多酚氧化酶(polyphenol oxidase,PPO),十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electropheresis,SDS-PAGE)显示其分子质量约为210 ku。肽质量指纹图谱的比对结果表明,纯化得到的PPO中含有187个氨基酸残基的14个片段与NCBI数据库中酚氧化酶原(gi|147724160)序列相似性为100%。该酶的最适温度和最适pH值分别为40℃和6.0,且在温度0～50℃及pH=5.0～8.0可保持相对稳定的活性。圆二色谱分析结果表明,对虾PPO的二级结构主要为反平行结构及无规则卷曲。抗坏血酸、茶多酚和特丁基对苯二酚(tertiary butylhydroquinone,TBHQ)等具有生物活性羟基的抑制剂对其均有较好的抑制效果。
In this study,a polyphenol oxidase(PPO) was purified to homogeneity from Pacific white shrimp(Litopenaeus vannamei) by ammonium sulfate fractionation and column chromatographies including DEAE-Cellulose,Phenyl-Sepharose HP and Hi-Trap Capto-Q. SDS-PAGE showed that the molecular weight of L.vannamei PPO was about 210 ku and its optimal temperature and pH were 40 ℃ and 6.0,respectively.Comparison of sequences between the purified PPO and prophenol oxidase(gi|147724160) of L.vannamei in the database of NCBI showed a similarity of 100% on 187 amino acid residues of 14 peptide fragments.The activity of PPO remained stable at 0-50 ℃ and in the pH range of 5.0-8.0.The secondary structure of PPO was mainly anti-parallel β sheet and random coil as revealed by circular dichrosim(CD).Additives of ascorbic acid,tea-polyphenols,and tertiary butylhydroquinone(TBHQ) inhibited the enzyme activity effectively.
In this study,a polyphenol oxidase(PPO) was purified to homogeneity from Pacific white shrimp(Litopenaeus vannamei) by ammonium sulfate fractionation and column chromatographies including DEAE-Cellulose,Phenyl-Sepharose HP and Hi-Trap Capto-Q. SDS-PAGE showed that the molecular weight of L.vannamei PPO was about 210 ku and its optimal temperature and pH were 40 ℃ and 6.0,respectively.Comparison of sequences between the purified PPO and prophenol oxidase(gi|147724160) of L.vannamei in the database of NCBI showed a similarity of 100% on 187 amino acid residues of 14 peptide fragments.The activity of PPO remained stable at 0-50 ℃ and in the pH range of 5.0-8.0.The secondary structure of PPO was mainly anti-parallel β sheet and random coil as revealed by circular dichrosim(CD).Additives of ascorbic acid,tea-polyphenols,and tertiary butylhydroquinone(TBHQ) inhibited the enzyme activity effectively.
引文
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[13]CHEN B N,XING R,WANG F,et al.Inhibitory effects of α-Na8SiW11CoO40 on tyrosinase and its application in controlling browning of fresh-cut apples[J].Food Chemistry,2015,188:177-183.
[14]蒋经伟.栉孔扇贝(Chlamys farreri)酚氧化酶的分离纯化及特性研究[D].青岛:中国海洋大学,2012.
[15]樊廷俊,汪小锋.中国对虾(Penaeus chinensis)酚氧化酶的分离纯化及其部分生物化学性质[J].生物化学与生物物理学报,2002,34(5):589-594.
[16]林江伟,游洪燕,沈海旺,等.克氏原螯虾原肌球蛋白的纯化及过敏性分析[J].集美大学学报(自然科学版),2012,17(3):167-174.
[17]NIRMAL N P,BENJAKUL S.Use of tea extracts for inhibition of polyphenol oxidase and retardation of quality loss of Pacific white shrimp during iced storage[J].LWT Food Science and Technology,2011,44(4):924-932.
[18]SUN J,WANG M,LIU H,et al.Acidic electrolysed water delays browning by destroying conformation of polyphenol oxidase[J].Journal of the Science of Food and Agriculture,2018,98(1):147-153.
[2]符云,麦良彬,钟小庆. 2016年全国南美白对虾养殖渔情报告[J].当代水产,2017(3):43- 45.
[3]农业部渔业渔政管理局.中国渔业统计年鉴2017[M].北京:中国农业出版社,2017:26-34.
[4]翁凌,李腾,阴利华,等.南美白对虾丝氨酸蛋白酶的分离纯化及性质研究[J].集美大学学报(自然科学版),2010,15(4):272-278.
[5]MONTERO P,áVALOS A,PéREZ-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.
[6]BARTOLO I,BIRK E O.Some factors affecting Norway lobster(Nephrops norvegicus) cuticle polyphenol oxidase activity and black spot development[J].International Journal of Food Science & Technology,1998,33(3):329-336.DOI:10.1046/j.1365-2621.1998.00168.x.
[7]SIMPSON B,MARSHALL M R,OTWELL W S.Phenol oxidase from shrimp(Penaeus setiferus):purification and some properties[J].Journal of Agricultural & Food Chemistry,1987,35(6):918-921.DOI:10.1021/jf00078a017.
[8]BENJAKUL S,VISESSANGUAN W,TANAKA M.Properties of phenoloxidase isolated from the cephalothorax of kuruma prawn(Penaeus japonicus)[J].Journal of Food Biochemistry,2005,29(5):470- 485.DOI:10.1111/j.1745-4514.2005.00042.x.
[9]ZAMORANO J P,MARTíNEZ-áLVAREZ O,MONTERO P,et al.Characterisation and tissue distribution of polyphenol oxidase of deepwater pink shrimp(Parapenaeus longirostris)[J].Food Chemistry,2009,112(1):104-111.
[10]LAI C Y,CHENG W,KUO C M.Molecular cloning and characterisation of prophenol oxidase from haemocytes of the white shrimp,Litopenaeus vannamei[J].Fish & Shellfish Immunol,2005,18(5):417- 430.DOI:10.1016/j.fsi.2004.10.004.
[11]NIRMAL N P,BENJAKUL S.Effect of ferulic acid on inhibition of polyphenol oxidase and quality changes of Pacific white shrimp(Litopenaeus vannamei) during iced storage[J].Food Chemistry,2009,116(1):323-331.
[12]NIRMAL N P,BENJAKUL S.Inhibition kinetics of catechin and ferulic acid on polyphenol oxidase from cephalothorax of Pacific white shrimp(Litopenaeus vannamei)[J].Food Chemistry,2012,131(2):569-573.
[13]CHEN B N,XING R,WANG F,et al.Inhibitory effects of α-Na8SiW11CoO40 on tyrosinase and its application in controlling browning of fresh-cut apples[J].Food Chemistry,2015,188:177-183.
[14]蒋经伟.栉孔扇贝(Chlamys farreri)酚氧化酶的分离纯化及特性研究[D].青岛:中国海洋大学,2012.
[15]樊廷俊,汪小锋.中国对虾(Penaeus chinensis)酚氧化酶的分离纯化及其部分生物化学性质[J].生物化学与生物物理学报,2002,34(5):589-594.
[16]林江伟,游洪燕,沈海旺,等.克氏原螯虾原肌球蛋白的纯化及过敏性分析[J].集美大学学报(自然科学版),2012,17(3):167-174.
[17]NIRMAL N P,BENJAKUL S.Use of tea extracts for inhibition of polyphenol oxidase and retardation of quality loss of Pacific white shrimp during iced storage[J].LWT Food Science and Technology,2011,44(4):924-932.
[18]SUN J,WANG M,LIU H,et al.Acidic electrolysed water delays browning by destroying conformation of polyphenol oxidase[J].Journal of the Science of Food and Agriculture,2018,98(1):147-153.