逐级盐析法结合双水相萃取纯化葛仙米藻蓝蛋白

doi:10.7506/spkx1002-6630-201524003

食品科学

逐级盐析法结合双水相萃取纯化葛仙米藻蓝蛋白

田盼盼1，程超1,2，汪兴平1,2,*

1.湖北民族学院生物科学与技术学院，湖北恩施 445000；
2.湖北民族学院生物资源保护与利用湖北省重点实验室，湖北恩施 445000

出版日期:2015-12-25 发布日期:2015-12-24
通讯作者: 汪兴平
基金资助:
国家自然科学基金地区科学基金项目（31260365）；湖北省教育厅团队项目（T201312）

Purification of Phycocyanin from Nostoc spharoids Kützing by Stepwise Salting-out and Aqueous Two-Phase Extraction

TIAN Panpan1, CHENG Chao1,2, WANG Xingping1,2,*

1. School of Biological Science and Technology, Hubei University for Nationalities, Enshi 445000, China; 2. Key Laboratory of
Biological Resources Protection and Utilization of Hubei Province, Hubei University for Nationalities, Enshi 445000, China

Online:2015-12-25 Published:2015-12-24
Contact: WANG Xingping

摘要/Abstract

摘要：

利用逐级盐析法结合双水相萃取对葛仙米藻蓝蛋白进行纯化，设计三因素二次回归正交旋转组合试验对提取工艺进行优化，并应用Design-Expert 7.0软件建立了各影响因素与萃取纯度的数学回归模型。结果显示最佳的提取工艺为：20% (NH4)2SO4盐析去沉淀后用40%和50% (NH4)2SO4盐析得藻蓝蛋白，再用质量分数10%的聚乙二醇6 000、质量分数18%的(NH4)2SO4进行双水相萃取。所得产物纯度可达到8.6，回收率为71.40%。对所得产物进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析，得到清晰的两条条带（条带1和条带2），其分子质量分别在35 ku和15 ku左右。通过质谱分析可知，条带2的分子质量为15 ku左右，等电点5.35，部分肽的氨基酸序列为TPLTEAVAAADSQGR和DIGYYLR。通过SWISS-MODEL网站模拟蛋白质结构得出，条带2可能是葛仙米藻蓝蛋白的α亚基。

关键词: 葛仙米, 藻蓝蛋白, 逐级盐析, 双水相萃取, 十二烷基硫酸钠-聚丙烯酰胺凝胶电泳, 质谱分析

Abstract:

In this paper, stepwise salting-out combined with aqueous two-phase extraction was used to purify phycocyanin
from Nostoc spharoids Kützing. The extraction process was optimized by using a three-factor quadratic regression
orthogonal rotation combination design. Using the Design-Expert 7.0 software, a mathematical regression model was
established indicating the effect of various extraction conditions on phycocyanin purity. The optimal extraction process was
determined as follows: after discarding of the precipitate salted out by 20% (NH4)2SO4, phycobiliproteins were salted out
from the supernatant by 40% and 50% (NH4)2SO4 before being subjected to aqueous two-phase extraction with 10% PEG
6 000 and 18% (NH4)2SO4. The purity (the absorbance ratio at 615 nm and 280 nm) and recovery of the purified
product under the optimized conditions were 8.6 and 71.40%, respectively. Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) analysis of the product revealed two clear bands having molecular weights of about 35 and
15 ku, respectively. Mass spectrometry (MS) analysis confirmed that the molecular weight of band 2 was about 15 ku . Its
isoelectric point was 5.35 and its partial amino acid sequences were TPLTEAVAAADSQGR and DIGYYLR, respectively.
Through simulation of protein structure according to the SWISS-MODEL website, we speculated that band 2 might be the
α-subunit of phycocyanin from Nostoc spharoids Kützing.

Key words: Nostoc spharoids Kützing, phycocyanin, stepwise salting-out, aqueous two-phase extraction, SDS-PAGE electrophoresis, mass spectrometry

中图分类号:

TS201.1

田盼盼，程超，汪兴平. 逐级盐析法结合双水相萃取纯化葛仙米藻蓝蛋白[J]. 食品科学, doi: 10.7506/spkx1002-6630-201524003.

TIAN Panpan, CHENG Chao, WANG Xingping. Purification of Phycocyanin from Nostoc spharoids Kützing by Stepwise Salting-out and Aqueous Two-Phase Extraction[J]. FOOD SCIENCE, doi: 10.7506/spkx1002-6630-201524003.

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逐级盐析法结合双水相萃取纯化葛仙米藻蓝蛋白

Purification of Phycocyanin from Nostoc spharoids Kützing by Stepwise Salting-out and Aqueous Two-Phase Extraction

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