珍珠层可溶性蛋白质的双水相萃取分离方法研究
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摘要
以合浦珠母贝珍珠层粉为原料,采用双水相萃取法对珍珠层蛋白进行提取,讨论双水相体系中聚合物分子量大小及质量分数、成相盐质量分数、离子强度、粗提液加入量对珍珠层蛋白的提取效果,通过单因素和响应面优化试验确定珍珠层蛋白的最佳提取条件。结果表明:10 g的双水相体系中,PEG 2000、Na_2SO_4、KCl的质量分数分别为30%,6%和1%,蛋白粗提液加入量为2 mL时,蛋白回收率为92.48%,明显高于传统的硫酸铵盐析沉淀法(得率为56.28%)。
A simple aqueous two-phase system(ATPS) was proposed for the purification of proteins from Pinctada fucata(P.fucata) crude extracts. The partition behaviors of nacre proteins were investigated and ATPS composed of Na_2SO_4, and PEG 2000 was chosen. Response surface methodology(RSM) based on Box-Behnken Design was further used to study the effect of the mass fraction of PEG 2000, Na_2SO_4 and KCl on purification of nacre proteins. The optimized ATPS conditions were found at 30% PEG 2000, 6% Na_2SO_4 and 1% KCl, respectively. Under the optimum conditions, the mean recovery of nacre protein was 92.48% when the crude extracts was 2 mL, which is obviously higher than that of precipitation with ammonium sulfate(56.28%).
A simple aqueous two-phase system(ATPS) was proposed for the purification of proteins from Pinctada fucata(P.fucata) crude extracts. The partition behaviors of nacre proteins were investigated and ATPS composed of Na_2SO_4, and PEG 2000 was chosen. Response surface methodology(RSM) based on Box-Behnken Design was further used to study the effect of the mass fraction of PEG 2000, Na_2SO_4 and KCl on purification of nacre proteins. The optimized ATPS conditions were found at 30% PEG 2000, 6% Na_2SO_4 and 1% KCl, respectively. Under the optimum conditions, the mean recovery of nacre protein was 92.48% when the crude extracts was 2 mL, which is obviously higher than that of precipitation with ammonium sulfate(56.28%).
引文
[1]万欣娣,任凤章,刘平,等.贝壳珍珠层的研究现状[J].材料导报,2006,20(10):21-24.
[2]张刚生,童银洪.软体动物贝壳中的有机质研究进展[J].湛江海洋大学学报,2000,20(1):74-78.
[3]BORBAS J E,WHEELER A P,SIKES C S.Molluscan shell matrix phosphoproteins:correlation of degree of phosphorylation to shell mineral microstructure and to in vitro regulation of mineralization[J].Journal of Experimental Zoology,1991,258(1):1-13.
[4]KEITH J,STOCKWELL S,BALL D,et al.Comparative analysis of macromolecules in mollusc shells[J].Comparative Biochemistry and Physiology Part B:Comparative Biochemistry,1993,105(3):487-496.
[5]TAKAHASHI K,YAMAMOTO H,ONODA A,et al.Highly oriented aragonite nanocrystal-biopolymer composites in an aragonite brick of the nacreous layer of Pinctada fucata[J].Chemical Communications,2004(8):996-997.
[6]SUZUKI M,MURAYAMA E,INOUE H,et al.Characterization of Prismalin-14,a novel matrix protein from the prismatic layer of the Japanese pearl oyster(Pinctada fucata)[J].Biochemical Journal,2004,382(1):205-213.
[7]张刚生,李浩璇,陈益兰.珍珠层中的蛋白质及其与碳酸钙相互作用研究进展[J].广西科学,2002,9(4):306-311.
[8]刘茉娥,陈欢林.新型分离技术基础[M].杭州:浙江大学出版社,1999.
[9]王志华,马会民,马泉莉,等.双水相萃取体系达研究[J].应用化学,2001,18(3):173-175.
[10]LI M,KIM J W,PEEPLES T L.Amylase partitioning and extractive bioconversion of starch using thermoseparating aqueous two-phase systems[J].Journal of Biotechnology,2002,93(1):15-26.
[11]RAGHAVARAO K,RANGANATHAN T V,SRINIVAS N D,et al.Aqueous two phase extraction—An environmentally benign technique[J].Clean Technologies and Environmental Policy,2003,5(2):136-141.
[12]范芳.双水相萃取技术的应用进展[J].化学与生物工程,2011,28(7):16-20.
[13]TH?MMES J,ETZEL M.Alternatives to chromatographic separations[J].Biotechnology Progress,2007,23(1):42-45.
[14]PRZYBYCIEN T M,PUJAR N S,STEELE L M.Alternative bioseparation operations:life beyond packed-bed chromatography[J].Current Opinion in Biotechnology,2004,15(5):469-478.
[15]马春宏,朱红,王良,等.双水相萃取技术的应用研究进展[J].光谱实验室,2010,27(5):1906-1914.
[16]ALBERTSSON P?.Partition of cell particles and macromolecules:separation and purification of biomolecules,cell organelles,membranes,and cells in aqueous polymer two-phase systems and their use in biochemical analysis and biotechnology[M].New York:Wiley,1986.
[17]丙潇潇.藻胆蛋白的双水相绿色提取技术研究[D].哈尔滨:哈尔滨工业大学,2010.
[18]刘箭.生物化学实验教程[M].北京:科学出版社,2010:21.
[19]ZASLAVSKY B Y.Aqueous two-phase partitioning:physical chemistry and bioanalytical applications[M].CRC Press,1994.
[20]CAO Q,LI S,HE C,et al.Extraction and determination of papaverin in pericarpium papaveris using aqueous two-phase system of poly(ethylene glycol)-(NH4)2SO4 coupled with high-performance liquid chromatography[J].Analytica Chimica Acta,2007,590(2):187-194.
[21]谢云飞,常雅宁,谢志镭,等.用双水相体系分离血红蛋白(HB)[J].华东理工大学学报(自然科学版),2010,36(5):685-689.
[22]王倩,贾长虹,常丽新,等.双水相萃取法分离纯化狗枣猕猴桃叶总黄酮[J].食品科学,2011,32(16):167-171.
[2]张刚生,童银洪.软体动物贝壳中的有机质研究进展[J].湛江海洋大学学报,2000,20(1):74-78.
[3]BORBAS J E,WHEELER A P,SIKES C S.Molluscan shell matrix phosphoproteins:correlation of degree of phosphorylation to shell mineral microstructure and to in vitro regulation of mineralization[J].Journal of Experimental Zoology,1991,258(1):1-13.
[4]KEITH J,STOCKWELL S,BALL D,et al.Comparative analysis of macromolecules in mollusc shells[J].Comparative Biochemistry and Physiology Part B:Comparative Biochemistry,1993,105(3):487-496.
[5]TAKAHASHI K,YAMAMOTO H,ONODA A,et al.Highly oriented aragonite nanocrystal-biopolymer composites in an aragonite brick of the nacreous layer of Pinctada fucata[J].Chemical Communications,2004(8):996-997.
[6]SUZUKI M,MURAYAMA E,INOUE H,et al.Characterization of Prismalin-14,a novel matrix protein from the prismatic layer of the Japanese pearl oyster(Pinctada fucata)[J].Biochemical Journal,2004,382(1):205-213.
[7]张刚生,李浩璇,陈益兰.珍珠层中的蛋白质及其与碳酸钙相互作用研究进展[J].广西科学,2002,9(4):306-311.
[8]刘茉娥,陈欢林.新型分离技术基础[M].杭州:浙江大学出版社,1999.
[9]王志华,马会民,马泉莉,等.双水相萃取体系达研究[J].应用化学,2001,18(3):173-175.
[10]LI M,KIM J W,PEEPLES T L.Amylase partitioning and extractive bioconversion of starch using thermoseparating aqueous two-phase systems[J].Journal of Biotechnology,2002,93(1):15-26.
[11]RAGHAVARAO K,RANGANATHAN T V,SRINIVAS N D,et al.Aqueous two phase extraction—An environmentally benign technique[J].Clean Technologies and Environmental Policy,2003,5(2):136-141.
[12]范芳.双水相萃取技术的应用进展[J].化学与生物工程,2011,28(7):16-20.
[13]TH?MMES J,ETZEL M.Alternatives to chromatographic separations[J].Biotechnology Progress,2007,23(1):42-45.
[14]PRZYBYCIEN T M,PUJAR N S,STEELE L M.Alternative bioseparation operations:life beyond packed-bed chromatography[J].Current Opinion in Biotechnology,2004,15(5):469-478.
[15]马春宏,朱红,王良,等.双水相萃取技术的应用研究进展[J].光谱实验室,2010,27(5):1906-1914.
[16]ALBERTSSON P?.Partition of cell particles and macromolecules:separation and purification of biomolecules,cell organelles,membranes,and cells in aqueous polymer two-phase systems and their use in biochemical analysis and biotechnology[M].New York:Wiley,1986.
[17]丙潇潇.藻胆蛋白的双水相绿色提取技术研究[D].哈尔滨:哈尔滨工业大学,2010.
[18]刘箭.生物化学实验教程[M].北京:科学出版社,2010:21.
[19]ZASLAVSKY B Y.Aqueous two-phase partitioning:physical chemistry and bioanalytical applications[M].CRC Press,1994.
[20]CAO Q,LI S,HE C,et al.Extraction and determination of papaverin in pericarpium papaveris using aqueous two-phase system of poly(ethylene glycol)-(NH4)2SO4 coupled with high-performance liquid chromatography[J].Analytica Chimica Acta,2007,590(2):187-194.
[21]谢云飞,常雅宁,谢志镭,等.用双水相体系分离血红蛋白(HB)[J].华东理工大学学报(自然科学版),2010,36(5):685-689.
[22]王倩,贾长虹,常丽新,等.双水相萃取法分离纯化狗枣猕猴桃叶总黄酮[J].食品科学,2011,32(16):167-171.