渗透剂与人工伴侣耦合辅助苹果酸脱氢酶包含体的复性  

Cooperation of osmolytes and artificial chaperone on malate dehydrogenase refolding

作　　者：张拓宇[1] 董海龙[1] 董晓燕[1] 

机构地区：[1]天津大学化工学院,天津300072

出　　处：《化工学报》2008年第4期988-994,共7页CIESC Journal

基　　金：国家自然科学基金项目(20476081;20676098);河北省教育厅科研计划项目(2004107);唐山市科学技术研究与发展计划项目(04204501C-20)~~

摘　　要：苹果酸脱氢酶不仅是一种重要的糖代谢酶,而且具有较高的经济价值和理论研究价值。为了获得苹果酸脱氢酶包含体的有效体外复性方法,本文将渗透剂(蔗糖,甜菜碱,海藻糖)与人工伴侣体系(CTAB/β-CD)耦合,进行了大肠杆菌苹果酸脱氢酶(eMDH)包含体的体外复性研究。结果发现,人工伴侣与蔗糖或甜菜碱耦合,可明显提高eMDH包含体的复性效果,复性后产物的活性较稀释复性提高了2倍以上;但未发现海藻糖与人工伴侣存在这种耦合作用。同时发现,耦合作用还可降低人工伴侣体系中CTAB与β-CD的最佳配比(从1∶8下降到1∶6),从而可提升人工伴侣体系的总用量,实现较高酶浓度条件下的有效复性。另外,利用渗透剂对蛋白质的热保护作用,实现了在较高温度(15℃)下eMDH包含体的复性。对复性产物结构的初步分析发现,耦合作用可使eMDH分子的α-螺旋及二聚体含量明显增多,因此复性产物的活性也显著提高。Malate dehydrogenase （MDH） is an essential metabolic enzyme, which has high economic value in protein research. To find an efficient method for MDH IBs refolding, the cooperation of osmolytes （sucrose, betaine and trehalose） and artificial chaperone （CTAB/ β-CD） in eMDH IBs refolding was investigated. The results showed that when sucrose and betaine were added with artificial chaperone, the refolding of eMDH was facilitated and the activity was twice higher than the refolded eMDH by dilution. But trehalose did not have any influence. Then, the optimal ratio of CTAB to β-CD was decreased from 1 ： 8 to 1 ： 6 by the cooperation. As a result, more artificial chaperone could be used to facilitate the refolding with a higher eMDH concentration. Because of the thermo-protection of osmolytes, the refolding of eMDH at a higher temperature （15℃） was achieved. According to structure analysis, the contents of α- helix and dimer structure in refolded eMDH were increased by the cooperation, and eMDH activity was enhanced.

关 键 词：苹果酸脱氢酶(MDH) 复性 渗透剂 人工伴侣 耦合作用 

分 类 号：O629.73[理学—有机化学] O643.1[理学—化学]