pH调控双水相萃取色素蛋白复合体及其分配行为研究  

pH Regulation of Extraction of Pigment Protein Complexes by Aqueous Two-phase System and Investigation of Distribution Behavior

作　　者：唐雯熙 张晓蓉[1,2] 段静[1] 吴湘霞 

机构地区：[1]吉首大学生物资源与环境科学学院 [2]吉首大学植物资源保护与利用湖南省高校重点实验室,吉首416000

出　　处：《天然产物研究与开发》2016年第5期661-667,684,共8页Natural Product Research and Development

基　　金：湖南省科技计划项目(2014SK3023);湖南省高校科技成果产业化培育项目(13CY016);湖南省教育厅重点项目(12A114);湖南省高校科技创新团队(2012-318)

摘　　要：为发展色素蛋白复合体分离纯化新方法,探究pH调控PEG1000/柠檬酸钾双水相系统萃取分离纯化色素蛋白复合体。优化萃取条件,光谱法研究其分配行为,检测产物纯度和生物活性。结果表明,最佳萃取条件为调节pH9.0,相组成CPEG100019.0%/C柠檬酸钾20.0%,蛋白质加量3.42 mg/g,K和萃取率达到最大,分别为8.8及86.0%。响应面分析法揭示,PEG1000和柠檬酸钾质量浓度及pH对分配系数和萃取率影响显著。调节pH7.0,反萃取分配系数和反萃取率最小为0.15及86.6%。蛋白质总回收率为74.2%。pH对色素蛋白复合体分配行为具有调控作用,pH大于8.5体系,色素蛋白趋于分配上相,反之分配于下相,PEG1000/柠檬酸钾以及蛋白质加入量不影响色素蛋白复合体分配于上相。电泳表征发现,萃取(pH9.0)上相存在2个蛋白质组分,相对分子量(MW)为7.0 kD及14.0 kD。反萃取(pH7.0)使相对分子量7.0 kD蛋白质组分分配于下相,该组分为LH2β亚基,经萃取和反萃取产物生物活性稳定。pH可调控PEG1000/柠檬酸钾双水相系统萃取分离色素蛋白复合体,产物纯度高,生物活性稳定。To develop a novel purification method for the pigment protein complexes （PPCs）, PPCs were separated and purified using aqueous two-phase system（ATPS） of polyethylene glycol（PEG） 1000/potassium citrate controlled by pH. The extraction condition of PPCs was optimized. The distribution behavior of PPCs in ATPS was detected by UV-Vis spectroscopy, and the purity and biological activity of product were investigated. The results indicated that the optimal ex- traction condition were 19.0% （W/W） PEG1000,20.0% （W/W） potassium citrate, the proteins adding amount was 3.42 rag/g, and with pH 9.0. Under these conditions, the distribution coefficient （K） and the extraction rate of PPCs reached to the maximum at 8.8 and 86.0% ,respectively. Response surface methodology analysis revealed that the phase compositions of PEG1000 and potassium citrate,pH condition had the significant effects on K and extraction rate. In re- verse extraction process,the K of the reverse extraction came to the maximum of 0.15 ,the extraction rate of 86.6% at the pH 7.0. And the total protein yield was to 74.2%. The results of UV-Vis analysis discovered that the distribution be- havior of PPCs can be regulated by the pH of system. PPCs tended to concentrate in the top phase solution under the condition of pH 〉 8.5 ,otherwise in bottom phase solution. The factors of PEG1000, potassium citrate and proteins added had not affected on the distribution behavior in ATP$. The electrophoresis analysis result found that there were two pro- tein components in top phase（pH 9.0） with molecular weight（MW） about 7.0 kD and 14.0 kD. After reverse extrac- tion（pH 7.0） ,only a component with MW 7.0 kD was back to potassium citrate solution,whlch was the β subunit of LH2. The biological activity of the purified PPCs was stable after the routines of the extraction and re-extraction. PPCs can be separated and purified by the ATPS of PEG1000 / potassium citrate through controlling the pH of the system.

关 键 词：色素蛋白复合体 双水相萃取系统 分离因素 萃取率 生物活性 

分 类 号：R284.2[医药卫生—中药学]