有机溶剂透性化处理谷氨酸脱羧酶工程菌制备γ-氨基丁酸的研究

Biosynthesis of γ-aminobutyric Acid Using Organic Solvent-Permeabilized and Glutamate Decarboxylase Expressing Recombinant Escherichia Coli Cells

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)-p ET28a-gad B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(log P>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。

Recombinant Escherichia 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 （log 19〉 0.68） are relatively effective in increasing apparent GAD activity of cells. Xylene （dielectricconstant value is 2.4, log P 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 recombinant E. coli cells to improve apparent GAD activity and the immobilized Xylene-premeabilized recombinant E. coli cells shows strong potential for industrial production of GABA.