CAJ | 학술논문

为了降低细胞壁和细胞膜的传质阻力对全细胞催化过程的不利影响，采用有机溶剂对表达重组谷氨酸脱羧酶(GAD)的工程菌BL21(DE3)-pET28a-gadB进行了透性化处理。结果表明，菌体GAD表观催化活力的改善程度与有机溶剂的介电系数和疏水性具有较强相关性，低介电常数(<6)，高疏水性(logP>0.68)的有机溶剂可以有效提高工程菌的GAD催化活性，其中最有效的透性化试剂为二甲苯(介电常数为2.4，log P为3.1)。当二甲苯的用量为5μL?mg?1(干重细胞)、处理时间5 min时,菌体表观催化活力达到最优值，为7.94 U?mg?1(干重细胞),是处理前表观催化活力的12.4倍。为了充分利用菌体的催化活力，采用海藻酸钙包埋的方法对二甲苯处理过的菌体进行固定化，结果发现固定化的透性化菌体可以较好地保持催化活力，反复催化10次后发现，第10次的GABA产率为第1次GABA产率的72.04%。研究为系统的选择有机溶剂来有效提高GAD工程菌表观催化活性提供了参考，同时制备的固定化的透性化菌体显示了良好工业应用前景。
위료강저세포벽화세포막적전질조력대전세포최화과정적불리영향，채용유궤용제대표체중조곡안산탈최매(GAD)적공정균BL21(DE3)-pET28a-gadB진행료투성화처리。결과표명，균체GAD표관최화활력적개선정도여유궤용제적개전계수화소수성구유교강상관성，저개전상수(<6)，고소수성(logP>0.68)적유궤용제가이유효제고공정균적GAD최화활성，기중최유효적투성화시제위이갑분(개전상수위2.4，log P위3.1)。당이갑분적용량위5μL?mg?1(간중세포)、처리시간5 min시,균체표관최화활력체도최우치，위7.94 U?mg?1(간중세포),시처리전표관최화활력적12.4배。위료충분이용균체적최화활력，채용해조산개포매적방법대이갑분처리과적균체진행고정화，결과발현고정화적투성화균체가이교호지보지최화활력，반복최화10차후발현，제10차적GABA산솔위제1차GABA산솔적72.04%。연구위계통적선택유궤용제래유효제고GAD공정균표관최화활성제공료삼고，동시제비적고정화적투성화균체현시료량호공업응용전경。
RecombinantEscherichia coli cells over-expressing glutamate decarboxylase (GAD) (BL21(DE3)-pET28a-gadB)were permeabilized with organic solvents to overcome the permeability barrier of cell envelope against substrate and product diffusion. The results show that the change in apparent GAD activity is correlated with the physicochemical properties of the organic solvents used for permeabilization. Solvents with lower dielectricconstant (<6) and high hydrophobicity (logP> 0.68) are relatively effective in increasing apparent GAD activity of cells. Xylene (dielectricconstant value is 2.4, logP value is 3.1)is the most effective permeabilizer for improving the cell GAD activity, and an optimal GAD activity (7.94 U·mg-1 dry biomass) is obtained by treating the cells using xylene at 5 μL?mg?1 dry biomass for 5 min. The GAD activity of the xylene-permeabilized cells is 12.4 times higher than that of the controls. The xylene-permeabilized cells are immobilized with Ca-alginate gel and the conversion rate is 72.04% after 10 repeated batches. These results allow systematic selection of suitable solvents for permeabilization of the recombinantE. coli cells to improve apparent GAD activity and the immobilized Xylene-premeabilized recombinantE. colicells shows strong potential for industrial production of GABA.