具高臨界溶解溫度之高分子應用於生物技術的研究

Abstract

相較於具有低臨界溶解溫度(LCST)之高分子(LCST-type高分子)，具有高臨界溶解溫度(UCST)之高分子(UCST-type高分子)的應用比較少被開發，因其相轉變溫度通常對於溶液中環境的變化較敏感。然而，我們團隊認為具有高臨界溶解溫度的高分子(UCST-type高分子)在與生物技術相關的領域中具有龐大的發展潛力，尤其是於有機體以及其衍生物之固定化上的應用。LCST-type高分子常當作有機體之固定化基質，由於其透過溫度控制的沉澱在工業操作上的便利性，然而該分離過程中需有的升溫程序，很可能造成生物質的失活。而透過改用UCST-type高分子為基質，升溫的過程能夠被避免，解決了這樣的問題。 這份研究主要針對於UCST-type高分子在蛋白質純化以及固定化酵素方面的應用。我們選用之UCST-type高分子為烯丙基胺/烯丙基脲共聚物(Poly(allylamine)-copoly(allylurea)s, PAUs)，因其具有幾下優點：(1)合成步驟簡單。(2)具有能夠被改質的官能基，胺基。(3)在較高的鹽類條件下具有較高之相轉變溫度的特性使其分離步驟更加簡單。 在應用UCST-type高分子於金屬螯合法蛋白質純化的研究中，我們利用亞氨基二乙酸(IDA)改質了PAU，成功的合成出同時具有UCST以及螯合金屬離子能力的高分子PAU-IDA。其中，含有0.75及0.1的莫爾比之胺基分別被改質成脲基及IDA基的PAU-IDA，不帶有金屬離子、螯合鎳離子、以及螯合銅離子時，在PBS中的相轉變溫度分別為14.4°C、14.8°C、以及21.5°C，而這些溫度皆適用於蛋白質純化的操作過程中。在蛋白質捕捉實驗中，螯合有銅離子的PAU-IDA(PAU-0.75-IDA-0.10-Cu)對於帶有組氨酸標籤的蛋白A (his-tag protein A)，每毫克高分子能夠捕捉396 微克的蛋白質，並且只需要2.5毫克的高分子及能在含有100 微克蛋白A的溶液中捕捉高達98%的蛋白質。被捕捉的蛋白質能夠透過含有咪唑的溶液洗脫，透過含有50 mM咪唑的溶液洗脫一次即可洗出89.7%的被捕捉蛋白。最後，PAU-0.75-IDA-0.10-Cu被用於蛋白質分離程序中，並且成功的將蛋白A從蛋白質混和液中分離出來。 在利用UCST-type高分子用作固定化酵素基質的研究中，纖維素酶透過1-乙基-3-(3-二甲基氨基丙基)碳醯二亞胺/N-羥基丁二醯亞胺(EDC/NHS)偶和法固定在PAU上。合成出之固定化酶在醋酸緩衝液中(300 mM, pH 3)具有22.1°C的相轉變溫度，該溫度適用於纖維素之水解反應。在羧甲基纖維素之水解中，酸鹼值與溫度對於固定化酶的活性影響皆被測定。在水解反應後，固定化酶可以透過降溫與離心進行分離與再利用，並且經過五次的回收後，仍有81.1%的初始活性成功被保留。

Application of polymers with upper critical solution temperature (UCST-type polymers) was less investigated comparing to polymers with lower critical solution temperature (LCST-type polymers), as they are generally more sensitive to environmental changes, such as pH and salt condition. However, in our view, UCST-type polymers have great potential application in the territory of biotechnology. Using UCST-type polymers as supports for immobilization of organisms and their derivatives could solves the problem of deactivation caused by the requirement of increasing temperature during the separation process of LCST-type polymers. In this study, we focused on developing the application of UCST-type polymers in protein purification and enzyme immobilization. Ureido-derivatized UCST-type polymers, Poly(allylamine)-co-poly(allylurea)s (PAUs) were chosen based on the following advantages: (1) Synthesis procedure of PAUs is simple; (2) PAUs have easily-modified functional groups, amine group; (3) Increasing phase separation temperature of PAUs under elevated salt condition makes the separation process of these polymers easier. In the metal-chelate protein purification based on UCST-type polymers, we successfully modified PAUs with iminodiacetic acid (IDA) to create PAU-IDAs, polymers simultaneously present UCST behavior and have the ability to chelate metal ion. PAU-IDA with 0.75 and 0.10 ratio of original amine groups transformed to ureido groups and IDA groups respectively (PAU-0.75-IDA-0.10) show phase separation temperature (Tp) of 14.4°C without metal ion, 14.8°C with nickel ion loaded, and 21.5°C with copper ion loaded in PBS, which is suitable operation condition for protein purification. In protein capture investigation, Cu2+ loaded PAU-0.75-IDA-0.10 (PAU-0.75-IDA-0.10-Cu) displayed a his-tagged protein A binding capacities of 396 μg/ mg polymer and captured up to 98% among 100 μg his-tagged protein A in the solution by 2.5 mg of this polymer. Besides, up to 89.7% of captured his-tag protein A was eluted by one time of elution through buffer containing 50 mM imidazole. Lastly, PAU-IDA-Cu was used to treat protein mixture and successfully separated his-tagged protein A form the mixture. In the enzyme immobilization on UCST-type polymers, cellulase was immobilized on PAU through EDC/NHS coupling. The immobilized cellulase show a Tp of 22.1°C in acetate buffer (300 mM, pH 4.8) which is desirable for cellulase reaction. The influence of pH and temperature to the hydrolysis of CMC by immobilized CMC was investigated. After hydrolysis reaction, immobilized cellulase can be recovered through cooling and centrifugation. In the recycling experiment, immobilized cellulase remained undenatured after 5 cycles of hydrolysis reaction.