乙醇对屎肠球菌全细胞谷氨酸脱羧酶表观活力的促进作用

Ethanol improves the apparent activity of whole-cell glutamate decarboxylase in Enterococcus faecium

摘要:

【背景】细胞内外物质的交换速率对全细胞转化活力具有重要影响。【目的】探讨乙醇对全细胞谷氨酸脱羧酶(glutamate decarboxylase, GAD)表观活力的影响，提高屎肠球菌全细胞转化法合成γ-氨基丁酸的产量。【方法】通过比较乙醇对GAD纯酶和全细胞GAD酶学特性、细胞结构、细胞膜通透性等的影响，探究乙醇对全细胞GAD活力的促进机制。【结果】低浓度乙醇对全细胞GAD活力有显著促进作用，最适浓度为7.5%；在该浓度下，全细胞GAD表观活力提升41.63%，乙醇对全细胞GAD最适反应pH无影响，但降低了全细胞GAD活力对胞外pH变化的敏感性，减小了米氏常数(K_m)；7.5%乙醇对GAD纯酶影响不显著(P>0.05)，未造成GAD外泄和细胞破损，仅在与细胞共存的反应体系才具有促进作用。结果表明，7.5%乙醇可改变细胞的透性，提升细胞传质速率，从而促进全细胞GAD的表观活力。【结论】乙醇价格低廉、安全性高，对全细胞GAD的促进作用在GABA工业上具有很好的应用前景。

Abstract:

[Background] The exchange rate of substances inside and outside cells has a significant effect on the transformation activity of whole-cells. [Objective] To increase the yield of γ-aminobutyric acid (GABA) synthesized by whole-cell transformation, we investigated the improving effect of ethanol on the apparent activity of whole-cell glutamate decarboxylase (GAD) in Enterococcus faecium. [Methods] We explored the mechanism of ethanol in improving the apparent activity of whole-cell GAD by comparing the effects of ethanol on the enzymatic properties of pure GAD and whole-cell GAD, cell structure, and membrane permeability. [Results] Low-concentration ethanol significantly promoted the activity of whole-cell GAD with the optimal concentration of 7.5%. At this concentration, the apparent activity of whole-cell GAD was increased by 41.63%, and the optimal reaction pH of whole-cell GAD did not change. However, the sensitivity of whole-cell GAD to the changes of extracellular pH and the Michaelis constant (K_m) of whole-cell GAD were both reduced. This concentration of ethanol had no significant effect on pure GAD (P>0.05), and did not cause GAD leakage or cell damage. In addition, 7.5% ethanol increased the activity of GAD only when it coexisted with the cells in reaction system. The results indicated that 7.5% ethanol altered the cell permeability and enhanced the mass transfer rate of cells, thereby improving the apparent activity of whole-cell GAD. [Conclusion] Due to the low price and high safety, ethanol demonstrates great potential of application in the improvement of whole-cell GAD activity for the industrial production of GABA.