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首页> 外文期刊>Biotechnology Progress >Affinity Precipitation and Macroaffinity Ligand Facilitated Three-Phase Partitioning for Refolding and Simultaneous Purification of Urea-Denatured Pectinase
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Affinity Precipitation and Macroaffinity Ligand Facilitated Three-Phase Partitioning for Refolding and Simultaneous Purification of Urea-Denatured Pectinase

机译：亲和沉淀和Macroaffinity配体促进三相分区，以重新折叠和同时纯化尿素变性的果胶酶

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摘要

Protein refolding is an integral step in the recovery of protein activity from inclusion bodies.It is shown that affinity precipitation and macroaffinity ligand facilitated three-phase partitioning (MLFTPP)led to refolding of urea-denatured pectinase present in a commercial preparation,with simultaneous purification.Affinity precipitation consists of precipitation of the desired enzyme by complexing it with a suitable stimulus-sensitive macroaffinity ligand.This ligand in this case was alginate/esterified alginate.The complex of the polymer-pectinase could be precipitated by adding calcium ions.In MLFTPP (carried out by adding tertiary butanol and ammonium sulfate to the aqueous solution of crude enzyme and the polymer),the polymer or its complex with the enzyme form an interfacial precipitate between tert-butyl alcohol phase and aqueous phase.It is believed that in both processes,while molecular recognition of alginate/esterified alginate to pectinase facilitates their selective binding to the enzyme,the correct refolding is facilitated by preventing molecular aggregation of unfolded enzyme molecules.Three-phase partitioning with esterified alginate as the macroaffinity ligand gave 100% recovery with 4-fold purification.Affinity precipitation with 1% alginate gave 52% yield with 18-fold purification.On the other hand,use of 0.5% esterified alginate gave only 7-fold purification but with 75% recovery of activity.
机译：蛋白质重折叠是从包涵体中恢复蛋白质活性的必不可少的步骤。研究表明，亲和沉淀和巨亲和配体促进了三相分配（MLFTPP），从而导致商业制剂中尿素变性果胶酶的重折叠，同时进行纯化亲和沉淀是通过将所需的酶与合适的刺激敏感的宏观亲和配体络合来沉淀所需的酶，在这种情况下，该配体为藻酸盐/酯化藻酸盐，可以通过添加钙离子来沉淀聚合物-果胶酶的络合物。 （通过在粗酶和聚合物的水溶液中加入叔丁醇和硫酸铵进行），聚合物或其与酶的络合物在叔丁醇相和水相之间形成界面沉淀。过程中，藻酸盐/酯化藻酸盐对果胶酶的分子识别有助于它们的选择性结合在酶上，通过防止未折叠的酶分子的分子聚集可以促进正确的重折叠。以酯化藻酸盐为大亲和力配体进行的三相分配可通过4倍纯化获得100％的回收率。用1％海藻酸盐进行亲和沉淀可得到52％的产率另一方面，使用0.5％的酯化藻酸盐只能得到7倍的纯化，但活性却可以得到75％的回收。

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