DEAE-琼脂凝胶微球的制备及在R-PE和C-PC提取分离中的应用
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摘要
为了降低天然大分子功能蛋白的分离纯化成本,以琼脂为原料自制弱阴离子交换层析介质DEAE-琼脂凝胶微球。将DEAE-琼脂凝胶微球应用于坛紫菜R-藻红蛋白(R-PE)和螺旋藻C-藻蓝蛋白(C-PC)的提取分离,最佳洗脱条件为:层析柱规格h=35.0 cm,φ=1.0 cm,V=27.5 m L;流速v=0.33 m L/min;洗脱缓冲液为0.01 mol/L磷酸盐溶液(含有0.05~0.5 mol/L Na Cl溶液);0.01 mol/L磷酸盐缓冲液(含0.1 mol/L Na Cl)洗脱出纯度为5.0的坛紫菜R-PE;0.01 mol/L磷酸盐缓冲液(含0.2 mol/L Na Cl)洗脱出纯度为4.4的螺旋藻C-PC。自制层析介质成本低廉、耗能少、可大量制备、分离效果好,可用于坛紫菜R-PE、螺旋藻C-PC的产业化制备。
In order to reduce the separation and purification cost of natural macromolecular functional protein,a kind of weak anion exchange chromatography medium DEAE-agar gel microsphere was made up. Applied DEAE-agar gel microsphere to the separation and purification of the porphyra haitanensis R-phycoerythrin( R- PE) and spirulina C-phycocyanin( C- PC), optimal elution conditions were as follows : chromatography column specifications was h=35.0 cm,φ=1.0 cm,V=27.5 m L,current velocity v=0.33 m L/min,elution buffer solution was 0.01 mol/L phosphate buffer solution(contain 0.05~0.5 mol/L Na Cl). 0.01 mol/L phosphate buffer solution(contain 0.1 mol/L Na Cl) washed porphyra haitanensis R-PE out,its purity was 5.0. 0.01 mol/L phosphate buffer solution(contain 0.2 mol/L Na Cl) washed spirulina C-PC out,its purity was 4.4. The DEAE-agar gel microspheres could be used in the industrial production of R-PE and C-PC because of their low cost,low energy consumption,scale preparation and excellent separation effect.
In order to reduce the separation and purification cost of natural macromolecular functional protein,a kind of weak anion exchange chromatography medium DEAE-agar gel microsphere was made up. Applied DEAE-agar gel microsphere to the separation and purification of the porphyra haitanensis R-phycoerythrin( R- PE) and spirulina C-phycocyanin( C- PC), optimal elution conditions were as follows : chromatography column specifications was h=35.0 cm,φ=1.0 cm,V=27.5 m L,current velocity v=0.33 m L/min,elution buffer solution was 0.01 mol/L phosphate buffer solution(contain 0.05~0.5 mol/L Na Cl). 0.01 mol/L phosphate buffer solution(contain 0.1 mol/L Na Cl) washed porphyra haitanensis R-PE out,its purity was 5.0. 0.01 mol/L phosphate buffer solution(contain 0.2 mol/L Na Cl) washed spirulina C-PC out,its purity was 4.4. The DEAE-agar gel microspheres could be used in the industrial production of R-PE and C-PC because of their low cost,low energy consumption,scale preparation and excellent separation effect.
引文
[1]陈小强.坛紫菜中R-藻红蛋白的分离纯化、理化性质及生物活性的研究[D].杭州:浙江大学,2004.
[2]王广策,曾成奎.藻胆蛋白功能的研究[J].生命科学,1998,10(6):312-315.
[3]顾宁琰,刘宇峰.紫球藻生物活性物质及其利用[J].中国海洋药物,2001(6):43-48.
[4]王璐.多管藻藻胆体中R-藻红蛋白和R-藻蓝蛋白的光谱和结构特性[D].烟台:烟台大学,2010.
[5]GA Basaca-Loya,MA Baldez,EA Enriquez-Guevara,et al.Extraction and purification of B-phycoerythrin from the red microalga Rhodosorus marinus[J].Ciencias Marinas,2009,35(4):359-368.
[6]Anamika Patel,Sandhya Mishra,Richa Pawar,et al.Purification and characterization of C-Phycocyanin from cyanobacterial species of marine and freshwater habitat[J].Protein Expression and Purification,2005,40:248-255.
[7]谭天伟,葛春玲,吕永琴,等.一种琼脂凝胶微球及其制备方法.中国:ZL201410015319.1[P].2014-01-13.
[8]王国祥,聂峰光,苏志国.琼脂糖凝胶偶联DEAE基团反应的影响因素[J].化学反应工程与工艺,2002,18(6):80-85.
[9]高洪峰.不同生长期坛紫菜中藻胆蛋白的含量变化[J].海洋与湖沼,1993,24(6):645-648.
[10]Allen Bennett,Lawrence Bogorad.Complementary chromatic adaptation in a filamentous blue-green alga[J].The Journal of Cell Biology,1973(58):419-435.
[11]郑蔚然.坛紫菜R-藻红蛋白的分离纯化及其稳定性研究[D].杭州:浙江工业大学,2008.
[12]Tripathi,Shivali Kapoor,Alpana Shrivastava.Extraction and purification of an unusual phcoerythrin in a terrestrial desiccation tolerant cyanobacterium Lyngbya arboricola[J].J Appl Phycol,2007,19:441-447.
[13]Jian-Feng Niu,Zhang-Fan Chen,Guang-Ce Wang,et al.Purification of phycoerythrin from Porphyra yezoensis Ueda(Bangiales,Rhodophyta)using expanded bed absorption[J].J Appl Phycol,2010,22:25-31.
[14]Caroline Costa Moraes,Susana Juliano Kalil.Strategy for a protein purification design using C-phycocyanin extract[J].Bioresource Technology,2009(100):5312-5317.
[15]廖晓霞,张学武.高效分离纯化藻蓝蛋白新法[J].食品工业科技,2011,32(6):273-275.
[16]Ravi Raghav Sonani,Niraj Kumar Singh,Jitendra Kumar,et al.Concurrent purification and antioxidant activity of phycobiliproteins from Lyngbya sp.A09DM:An antioxidant and anti-aging potential of phycoerythrin in Caenorhabditis elegans[J].Process Biochemistry,2014,49:1757-1766.
[17]Sanjiv K Mishra,Anupama Shrivastav,Sandhya Mishra.Preparation of highly purified C-phycoerythrin from marine cyanobacterium Pseudanabaena sp.[J].Protein Expression and Purification,2011,80:234-238.
[18]Tianfeng Chen,Yum-Shing Wong,Wenjie Zheng.Purification and characterization of selenium-containing phycocyanin from selenium-enriched Spirulina platensis[J].Phytochemistry,2006,67:2424-2430.
[19]陈小强,龚兴国,史锋,等.坛紫菜中R-藻红蛋白的色谱纯化及性质研究[J].分析实验室,2004,23(11):5-9.
[20]章申峰.C-藻蓝蛋白及其亚基的分离纯化和体外抗肿瘤研究[D].杭州:浙江大学,2005.
[2]王广策,曾成奎.藻胆蛋白功能的研究[J].生命科学,1998,10(6):312-315.
[3]顾宁琰,刘宇峰.紫球藻生物活性物质及其利用[J].中国海洋药物,2001(6):43-48.
[4]王璐.多管藻藻胆体中R-藻红蛋白和R-藻蓝蛋白的光谱和结构特性[D].烟台:烟台大学,2010.
[5]GA Basaca-Loya,MA Baldez,EA Enriquez-Guevara,et al.Extraction and purification of B-phycoerythrin from the red microalga Rhodosorus marinus[J].Ciencias Marinas,2009,35(4):359-368.
[6]Anamika Patel,Sandhya Mishra,Richa Pawar,et al.Purification and characterization of C-Phycocyanin from cyanobacterial species of marine and freshwater habitat[J].Protein Expression and Purification,2005,40:248-255.
[7]谭天伟,葛春玲,吕永琴,等.一种琼脂凝胶微球及其制备方法.中国:ZL201410015319.1[P].2014-01-13.
[8]王国祥,聂峰光,苏志国.琼脂糖凝胶偶联DEAE基团反应的影响因素[J].化学反应工程与工艺,2002,18(6):80-85.
[9]高洪峰.不同生长期坛紫菜中藻胆蛋白的含量变化[J].海洋与湖沼,1993,24(6):645-648.
[10]Allen Bennett,Lawrence Bogorad.Complementary chromatic adaptation in a filamentous blue-green alga[J].The Journal of Cell Biology,1973(58):419-435.
[11]郑蔚然.坛紫菜R-藻红蛋白的分离纯化及其稳定性研究[D].杭州:浙江工业大学,2008.
[12]Tripathi,Shivali Kapoor,Alpana Shrivastava.Extraction and purification of an unusual phcoerythrin in a terrestrial desiccation tolerant cyanobacterium Lyngbya arboricola[J].J Appl Phycol,2007,19:441-447.
[13]Jian-Feng Niu,Zhang-Fan Chen,Guang-Ce Wang,et al.Purification of phycoerythrin from Porphyra yezoensis Ueda(Bangiales,Rhodophyta)using expanded bed absorption[J].J Appl Phycol,2010,22:25-31.
[14]Caroline Costa Moraes,Susana Juliano Kalil.Strategy for a protein purification design using C-phycocyanin extract[J].Bioresource Technology,2009(100):5312-5317.
[15]廖晓霞,张学武.高效分离纯化藻蓝蛋白新法[J].食品工业科技,2011,32(6):273-275.
[16]Ravi Raghav Sonani,Niraj Kumar Singh,Jitendra Kumar,et al.Concurrent purification and antioxidant activity of phycobiliproteins from Lyngbya sp.A09DM:An antioxidant and anti-aging potential of phycoerythrin in Caenorhabditis elegans[J].Process Biochemistry,2014,49:1757-1766.
[17]Sanjiv K Mishra,Anupama Shrivastav,Sandhya Mishra.Preparation of highly purified C-phycoerythrin from marine cyanobacterium Pseudanabaena sp.[J].Protein Expression and Purification,2011,80:234-238.
[18]Tianfeng Chen,Yum-Shing Wong,Wenjie Zheng.Purification and characterization of selenium-containing phycocyanin from selenium-enriched Spirulina platensis[J].Phytochemistry,2006,67:2424-2430.
[19]陈小强,龚兴国,史锋,等.坛紫菜中R-藻红蛋白的色谱纯化及性质研究[J].分析实验室,2004,23(11):5-9.
[20]章申峰.C-藻蓝蛋白及其亚基的分离纯化和体外抗肿瘤研究[D].杭州:浙江大学,2005.