三相法分离番薯过氧化物酶
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摘要
采用三相法(TPP)分离纯化番薯过氧化物酶(SPP),以番薯过氧化物酶的酶活回收率和纯化倍数为优化目标,分别利用单因素法和响应面分析法对其影响因素进行考察。建立了最佳提取条件:以叔丁醇作溶剂、硫酸铵饱和度为66%、粗提物与叔丁醇体积比为1. 0∶1. 13、温度为26℃、pH=5. 0,在最优提取条件下,其酶活回收率和纯化倍数分别达133. 5%和3. 5。SDS-PAGE电泳分析结果显示SPP酶蛋白主要为一条带,其分子质量约为40 kDa左右。三相法操作简便、耗时短、成本低,此外分离过程增强了SPP酶与底物的亲和力,提高了酶活回收率,有利于工业化应用。
Sweet potato peroxidase( SPP) is separated and purified by three-phase partitioning( TPP) method.The recovery rate and purification ratio of sweet potato peroxidase are optimized by single factor method and response surface analysis.The optimal extraction conditions are established as follows: t-butyl alcohol is used as solvent,the saturation of ammonium sulfate is 66%,the volumetric ratio of crude solution to t-butyl alcohol is 1. 0 ∶1. 13,the temperature is set at 26°C and p H is 5. 0.Under these optimal conditions,the purification efficiency of sweet potato peroxidase can reach 3. 5 times and the activity recovery can be 133. 5%.It is shown by SDS-PAGE that SPP enzyme protein is a single band with a molecular weight of around 40 k Da.TPP is a simple,quick,low cost and very attractive process for primary purification of SPP and has a big potential use for industrial applications.
Sweet potato peroxidase( SPP) is separated and purified by three-phase partitioning( TPP) method.The recovery rate and purification ratio of sweet potato peroxidase are optimized by single factor method and response surface analysis.The optimal extraction conditions are established as follows: t-butyl alcohol is used as solvent,the saturation of ammonium sulfate is 66%,the volumetric ratio of crude solution to t-butyl alcohol is 1. 0 ∶1. 13,the temperature is set at 26°C and p H is 5. 0.Under these optimal conditions,the purification efficiency of sweet potato peroxidase can reach 3. 5 times and the activity recovery can be 133. 5%.It is shown by SDS-PAGE that SPP enzyme protein is a single band with a molecular weight of around 40 k Da.TPP is a simple,quick,low cost and very attractive process for primary purification of SPP and has a big potential use for industrial applications.
引文
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[12]Wang S,Wu P,Han Z. Random conjugated polybenzazole copolymers:Synthesis,characterization,and exciton confinement effects in photophysical properties[J].Journal of Materials Science,2004,39(8):2717-2726.
[13]Dennison C,Lovrien R.Three phase partitioning:Concentration and purification of proteins[J]. Protein Expression&Purification,1997,11(2):149.
[14]Zhang Z,Lai J,Wu K.Peroxidase-catalyzed chemiluminescence system and its application in immunoassay[J]. Talanta,2018,180:260-270.
[15]Vetal M,Rathod V.Three phase partitioning a novel technique for purification of peroxidase from orange peels(citrus sinenses)[J].Food and Bioproducts Processing,2014,94:284-289.
[16]Duman Y,Kaya E. Purification,recovery,and characterization of chick pea(cicer arietinum)β-galactosidase in single step by three phase partitioning as a rapid and easy technique[J]. Protein Expression and Purification,2013,91:155-160.
[2]Leon J,Alpeeva I,Chubar T,et al.Purification and substrate specificity of Peroxidase from sweet potato tubers[J]. Plant Science,2002,163(5):1011-1019.
[3]Kosman J,Juskowiak B. Peroxidase-mimicking DNAzymes for biosensing applications:A review[J]. Analytica Chimica Acta,2011,707(1-2):7-17.
[4]Patil P,Yadav G. Application of microwave assisted three phase partitioning method for purification of laccase from Trametes hissuta[J].Process Biochemistry,2018,65:220-227.
[5]Mangesh D,Virendra K.Three phase partitioning:A novel technique for purification of peroxidase from orange peels[J]. Food and Bioproducts Processing,2015,94:284-289.
[6]Yan J,Wang Y,Qiu W,et al. Ultrasound synergized with threephase partitioning for extraction and separation of corbicula fluminea polysaccharides and possible relevant mechanisms[J].Ultrasonics Sonochemistry,2018,40:128-134.
[7]Yadav N,Gupta M,Khare S.Three phase partitioning and spectroscopic characterization of bioactive constituent from halophilic Bacillus subtilis EMB M15[J]. Bioresource Technology,2017,242:283-286.
[8]Li D,Zheng D,Cao Z.Response surface methods for slope reliability analysis:Review and comparison[J]. Engineering Geology,2016,203(25):3-14.
[9]Yusri I,Majeed A,Mamat R,et al.A review on the application of response surface method and artificial neural network in engine performance and exhaust emissions characteristics in alternative fuel[J].Renewable and Sustainable Energy Reviews,2018,90:665-686.
[10]Bradford M.A rapid and sensitive method for the quantification of microgram quantities of protein using the principle of protein-dye binding[J].Analytical Biochemistry,1976,72(s 1-2):248-254.
[11]Laemmlli U.Cleavage of structural proteins during assembly of head of bacteriophage-T4[J].Nature,1970,227:680-685.
[12]Wang S,Wu P,Han Z. Random conjugated polybenzazole copolymers:Synthesis,characterization,and exciton confinement effects in photophysical properties[J].Journal of Materials Science,2004,39(8):2717-2726.
[13]Dennison C,Lovrien R.Three phase partitioning:Concentration and purification of proteins[J]. Protein Expression&Purification,1997,11(2):149.
[14]Zhang Z,Lai J,Wu K.Peroxidase-catalyzed chemiluminescence system and its application in immunoassay[J]. Talanta,2018,180:260-270.
[15]Vetal M,Rathod V.Three phase partitioning a novel technique for purification of peroxidase from orange peels(citrus sinenses)[J].Food and Bioproducts Processing,2014,94:284-289.
[16]Duman Y,Kaya E. Purification,recovery,and characterization of chick pea(cicer arietinum)β-galactosidase in single step by three phase partitioning as a rapid and easy technique[J]. Protein Expression and Purification,2013,91:155-160.