Immobilized Phosphoenolpyruvate Carboxylase Beads for Catalytic Carboxylation in Carbon Dioxide-Containing Gas－Liquid Flow
Immobilized Phosphoenolpyruvate Carboxylase Beads for Catalytic Carboxylation in Carbon Dioxide-Containing Gas－Liquid Flow
复制标题
固定化磷酸烯醇丙酮酸羧化酶珠在含二氧化碳气液流中催化羧化
DOI：
10.1021/acs.iecr.3c00715
复制
发表时间：
2023
影响因子：
4.2
通讯作者：
Fumiya Tanaka and Makoto Yoshimoto
中科院分区：
文献类型：
--
作者：
Kojima Yusuke;Okuzaki Yuya;Nishijima Ken-ichi;Moriwaki Shuichiro;Asai Seiya;Kaneoka Hidenori;Iijima Shinji;庄司衛太，齋藤大河，琵琶哲志，久保正樹，塚田隆夫，笘居高明，阿尻雅文;Fumiya Tanaka and Makoto Yoshimoto
磷酸烯醇式丙酮酸羧化酶（PEPC）在水相中以碳源催化Mg 2+依赖的羧化反应。在具有气-液接触的生物反应器中进行酶促反应是具有挑战性的，因为蛋白质在气-液界面处可能经历构象变化。制备了固定化磷酸烯醇式丙酮酸羧化酶聚苯乙烯小球（PEPC-PSB），并将其应用于外循环气升式鼓泡塔（ELBC）中催化磷酸烯醇式丙酮酸（PEP）羧化生成草酰乙酸（OAA）。对于典型的反应，向ELBC中加入10 mL最初含有3.1 mM PEP和9.7 mM MgSO 4的0.2 M磷酸钾缓冲溶液（pH = 7.5）。在25 °C、20个PEPC-PSB（[PEPC] = 2.0 μg/mL）、以1.0 cm/s的表观气速连续通入CO2气泡的ELBC反应条件下，反应300 min，PEP的转化率可达77%，反应结束后，PEPC-PSB可从反应混合物中回收，洗涤后可重复使用。PEPC-PSB可在4 °C下储存，并且物理上足够坚固以进行处理。在气液两相流中，反应时间为900-1080 min时，PEPC-PSB的催化活性较好。另一方面，在与PEPC-PSB催化的反应相当的条件下操作的ELBC中，用游离PEPC没有产生可检测量的OAA。CO2气泡对PEPC的吸附导致ELBC中具有生物活性的PEPC浓度迅速下降。粗糙表面的聚苯乙烯微球可以为固定化酶提供一个稳定的环境，使其在连续提供含CO2的气泡的条件下稳定地催化PEP的长时间羧化反应。
Phosphoenolpyruvate carboxylase (PEPC) catalyzes the Mg2+-dependent carboxylation reaction in aqueous phase with a carbon source. Performing enzymatic reactions in a bioreactor with gas–liquid contacting is challenging because proteins potentially undergo conformational change at the gas–liquid interface. In the present work, immobilized phosphoenolpyruvate carboxylase polystyrene beads (PEPC-PSBs) were prepared and applied to catalyze the carboxylation of phosphoenolpyruvate (PEP) producing oxaloacetate (OAA) in an external loop airlift bubble column (ELBC). For a typical reaction, ELBC was charged with 10 mL of a 0.2 M potassium phosphate buffer solution (pH = 7.5) initially containing 3.1 mM PEP and 9.7 mM MgSO4. When ELBC was operated at 25 °C with 20 PEPC-PSBs ([PEPC] = 2.0 μg/mL) by continuously introducing CO2gas bubbles at the superficial gas velocity of 1.0 cm/s, the conversion of PEP of 77% was obtained at the reaction time of 300 min. After the reaction, PEPC-PSBs could be recovered from the reaction mixture followed by washing for reuse. PEPC-PSBs were storable at 4 °C and physically robust enough for handling. The catalytic activity of PEPC-PSBs was observed for the total reaction time of 900–1080 min in the gas–liquid flow. On the other hand, no detectable amount of OAA was produced with free PEPC in ELBC operated under the comparable condition to the PEPC-PSB-catalyzed reaction. Adsorption of free PEPC to CO2gas bubbles was indicated to cause a rapid decrease in the concentration of biologically active PEPC in ELBC. The polystyrene beads with rough surface were suggested to offer an environment for the immobilized enzyme to stably catalyze the prolonged carboxylation of PEP with continuously supplied CO2-containing gas bubbles.