东海中华管鞭虾多酚氧化酶(PPO)的纯化及特性研究
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摘要
本文采用匀浆浸提法提取中华管鞭虾(Solenocera crassicornis,S. crassicornis)多酚氧化酶(polyphenoloxidase,PPO),经硫酸铵质量分级、离子交换、凝胶过滤纯化后,以左旋多巴胺(L-DOPA)为底物研究其底物专一性和动力学特性,并选取不同抑制剂和金属离子探讨对该PPO的抑制影响。结果显示,匀浆浸提法提取PPO的最佳条件为料液比1:2(W/V)、p H 7.5、浸提3 h、温度50℃,从100 g中华管鞭虾中获取的PPO的产量和纯化倍数分别是1.2%和25.9,中华管鞭虾PPO对L-DOPA和儿茶酚具有很显著地催化氧化作用(p≤0.05),Km值为分别是3.8 mM和8.12 mM,说明该酶为儿茶酚家族酶。PPO活性对苯硫脲、抗坏血酸、柠檬酸较为敏感,能显著被Cu~(2+)、Zn~(2+)、乙二胺四乙酸(EDTA)抑制。结果表明,中华管鞭虾PPO可能是活性部位含Cu~(2+)的金属酶,为控制中华管鞭虾黑变提供基础的信息。
In this study, the polyphenoloxidase(PPO) was extracted using the homogenate extraction from Solenocera crassicornis(S. crassicornis) in the East China Sea, and purified through ammonium sulphate precipitation, ion-exchange and gel filtration. The substrate specificity(L-dihydroxyphenylalanine(L-DOPA) as the substrate) and kinetic properties of the purified PPO, along with the effects of different inhibitors and metal ions on the PPO, were determined. The obtained results showed the optimal conditions for the homogenate extraction were: the material to liquid ratio 1:2(W/V), pH 7.5, time 3 h, and temperature 50 ℃. The yield and purification factor were 1.2% and 25.9, respectively. This PPO significantly catalyzed the oxidation of L-DOPA and catechol(p≤0.05), with the Km values being 3.8 mM and 8.12 mM, respectively, indicating that the PPO belongs to the catechol enzyme family. The PPO was sensitive to phenylthiourea, ascorbic acid, and citric acid, and its activity was strongly inhibited by Cu~(2+), Zn~(2+), and ethylenediaminetetraacetic acid(EDTA). Thus, the PPO from S. crassicornis could be a metalloenzyme containing Cu~(2+) in its active center. The results of this study provide the basis for controlling the melanotic blackening of S. crassicornis.
In this study, the polyphenoloxidase(PPO) was extracted using the homogenate extraction from Solenocera crassicornis(S. crassicornis) in the East China Sea, and purified through ammonium sulphate precipitation, ion-exchange and gel filtration. The substrate specificity(L-dihydroxyphenylalanine(L-DOPA) as the substrate) and kinetic properties of the purified PPO, along with the effects of different inhibitors and metal ions on the PPO, were determined. The obtained results showed the optimal conditions for the homogenate extraction were: the material to liquid ratio 1:2(W/V), pH 7.5, time 3 h, and temperature 50 ℃. The yield and purification factor were 1.2% and 25.9, respectively. This PPO significantly catalyzed the oxidation of L-DOPA and catechol(p≤0.05), with the Km values being 3.8 mM and 8.12 mM, respectively, indicating that the PPO belongs to the catechol enzyme family. The PPO was sensitive to phenylthiourea, ascorbic acid, and citric acid, and its activity was strongly inhibited by Cu~(2+), Zn~(2+), and ethylenediaminetetraacetic acid(EDTA). Thus, the PPO from S. crassicornis could be a metalloenzyme containing Cu~(2+) in its active center. The results of this study provide the basis for controlling the melanotic blackening of S. crassicornis.
引文
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[3]Barjinder P K,Rao P S.Kinetic modeling of polyphenoloxidase inactivation during thermal-assisted high pressure processing in black tiger shrimp(Penaeus monodon)[J].Food Control,2017,80:388-394
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[6]Zotos A,Taylorr K D A.Partial purification and characterization of proteases from Norway lobster(Nephrops norvegicus)and their role in the phenolase activation process[J].Food Chemistry,1996,56:61-68
[7]Encarnasion A B,Fagutao F,Hiryama J,et al.Edible mushroom(Flammulina velutipes)extract inhibits melanosis in Kuruma shrimp(Marsupenaeus japonicus)[J].Journal of Food Science,2011,76(1):52-58
[8]Manheem K,Benjakul S,Kijroongrojana K,et al.The effect of heating conditions on polyphenol oxidase,proteases and melanosis in precooked Pacific white shrimp during refrigerated storage[J].Food Chemistry,2012,131(4):1370-1375
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[11]郑斌,郝云彬,杨会成,等.中华管鞭虾PPO生化特性研究[J].浙江海洋学院:自然科学版,2010,29(6):526-530ZHENG Bin,HAO Yun-bin,YANG Hui-cheng,et al.Study of biochemical characteristics of polyphenoloxidase in deepwater pink shrimp(Solenocera crassicornis)[J].Journal of Zhejiang Ocean University(Natural Science),2010,29(36):526-530
[12]杨会成,郝云彬,郑斌,等.中华管鞭虾PPO的分离制备[J].食品科技,2011,36(7):24-28YANG Hui-cheng,HAO Yun-bin,ZHENG Bin,et al.Extraction of polyphenoloxidase in deepwater pink shrimp(Solenocera crassicornis)[J].Food Science and Technology,2011,36(7):24-28
[13]吴亮亮,杨会成,廖妙飞,等.不同对虾中PPO的提取比较及在虾体的分布研究[J].食品工业科技,2012,33(7):5-62WU Liang-liang,YANG Hui-cheng,LIAO Miao-fei,et al.Study on extraction of polyphenoloxidase and enzyme distribution on different shrimps[J].Science and Technology of Food Industry,2012,33(7):55-62
[14]Bibiuti B,Mishr A,Satyendra G,et al.Purification and characterization of polyphenol oxidase(OPP)from eggplant(Solanum melongena)[J].Food Chemistry,2012,13(4):1855-1861
[15]Zamorano J P,Oscar M A,Pilar M,et al.Characterisation and tissue distribution of polyphenol oxidase of deepwater pink shrimp(Parapenaeus longirostris)[J].Food Chemistry,2009,112:104-111
[16]Benjakul S,Visessanguan W,Tanaka M.Inhibitory effect of cysteine and glutathione on phenoloxidase from kuruma prawn(Penaeus japonicus)[J].Food Chemistry,2006,98:158-163
[17]Liu G,Yang L,Fan T,et al.Purification and characterization of phenoloxidase from crab Charybdis japonica[J].Fish and Shellfish Immunology,2006,20:47-57
[18]Chakraborty A,Raro P S,Mishra H N.Kinetic modeling of polyphenoloxidase and peroxidase inactivation in pineapple(Ananas comosus L.)puree during high-pressure and thermal treatments[J].Innovative Food Science and Emerging Technologies,2015,27:57-68
[19]Nirmal N P,Benjakul S.Inhibitory effect of mimosine on polyphenoloxidase from cephalothoraxes of pacific white shrimp(Litopenaeus vannamei)[J].Journal of Agricultural and Food Chemistry,2011b,59(18):10256-10260
[2]Simpson B K,Mashall M R,Otwell W S.Phenoloxidases from pink and white shrimp:Kinetic and other properties[J].Journal of Food Biochemistry,1988,12:205-217
[3]Barjinder P K,Rao P S.Kinetic modeling of polyphenoloxidase inactivation during thermal-assisted high pressure processing in black tiger shrimp(Penaeus monodon)[J].Food Control,2017,80:388-394
[4]Gimenez B,Martinez A O,Monetero P,et al.Characterization of phenoloxidase activity of carapace and viscera from cephalothorax of Norway lobster(Nephrops norvegicus)[J].LWT-Food Science and Technology,2008,43(8):1240-1245
[5]Vazquez J A,Amado I R,Montemayor M I,et al.Chondroitin sulfate,hyaluronic acid and chitin/chitosan production using marine waste sources:Characteristics,applications and eco-friendly processes:A review[J].Marine Drugs,2013,11,747-774
[6]Zotos A,Taylorr K D A.Partial purification and characterization of proteases from Norway lobster(Nephrops norvegicus)and their role in the phenolase activation process[J].Food Chemistry,1996,56:61-68
[7]Encarnasion A B,Fagutao F,Hiryama J,et al.Edible mushroom(Flammulina velutipes)extract inhibits melanosis in Kuruma shrimp(Marsupenaeus japonicus)[J].Journal of Food Science,2011,76(1):52-58
[8]Manheem K,Benjakul S,Kijroongrojana K,et al.The effect of heating conditions on polyphenol oxidase,proteases and melanosis in precooked Pacific white shrimp during refrigerated storage[J].Food Chemistry,2012,131(4):1370-1375
[9]Ali M T,Gleeson R A,Wei C I,et al.Activation mechanisms of prophenoloxidase on melanosis development in Florida spiny lobster(Panulirus argus)cuticle[J].Journal of Food Science,1994,59:1024-1030
[10]Nirmal N P,Benjakul S.Biochemical properties of polyphenoloxidase from the cephalothorax of Pacific white shrimp(Litopenaeus vannamei)[J].International Aquatic Research,2011a,4(6):1-13
[11]郑斌,郝云彬,杨会成,等.中华管鞭虾PPO生化特性研究[J].浙江海洋学院:自然科学版,2010,29(6):526-530ZHENG Bin,HAO Yun-bin,YANG Hui-cheng,et al.Study of biochemical characteristics of polyphenoloxidase in deepwater pink shrimp(Solenocera crassicornis)[J].Journal of Zhejiang Ocean University(Natural Science),2010,29(36):526-530
[12]杨会成,郝云彬,郑斌,等.中华管鞭虾PPO的分离制备[J].食品科技,2011,36(7):24-28YANG Hui-cheng,HAO Yun-bin,ZHENG Bin,et al.Extraction of polyphenoloxidase in deepwater pink shrimp(Solenocera crassicornis)[J].Food Science and Technology,2011,36(7):24-28
[13]吴亮亮,杨会成,廖妙飞,等.不同对虾中PPO的提取比较及在虾体的分布研究[J].食品工业科技,2012,33(7):5-62WU Liang-liang,YANG Hui-cheng,LIAO Miao-fei,et al.Study on extraction of polyphenoloxidase and enzyme distribution on different shrimps[J].Science and Technology of Food Industry,2012,33(7):55-62
[14]Bibiuti B,Mishr A,Satyendra G,et al.Purification and characterization of polyphenol oxidase(OPP)from eggplant(Solanum melongena)[J].Food Chemistry,2012,13(4):1855-1861
[15]Zamorano J P,Oscar M A,Pilar M,et al.Characterisation and tissue distribution of polyphenol oxidase of deepwater pink shrimp(Parapenaeus longirostris)[J].Food Chemistry,2009,112:104-111
[16]Benjakul S,Visessanguan W,Tanaka M.Inhibitory effect of cysteine and glutathione on phenoloxidase from kuruma prawn(Penaeus japonicus)[J].Food Chemistry,2006,98:158-163
[17]Liu G,Yang L,Fan T,et al.Purification and characterization of phenoloxidase from crab Charybdis japonica[J].Fish and Shellfish Immunology,2006,20:47-57
[18]Chakraborty A,Raro P S,Mishra H N.Kinetic modeling of polyphenoloxidase and peroxidase inactivation in pineapple(Ananas comosus L.)puree during high-pressure and thermal treatments[J].Innovative Food Science and Emerging Technologies,2015,27:57-68
[19]Nirmal N P,Benjakul S.Inhibitory effect of mimosine on polyphenoloxidase from cephalothoraxes of pacific white shrimp(Litopenaeus vannamei)[J].Journal of Agricultural and Food Chemistry,2011b,59(18):10256-10260