热带假丝酵母细胞内pH的测定及其与生长代谢活性的关系

Measurement of Intracellular pH in Long-chain Dicarboxylic Acid-producing Yeast Candida tropicalis and Its Growth Activity

应用荧光探针 5(6)_双醋酸羧基荧光素 (Carboxyfluoresceindiacetate)测定了产长链二元酸热带假丝酵母(Candidatropicalis)细胞内pH (pHi)值 ,确定了该探针载入C .tropicalis细胞的适宜条件。用摇瓶培养C .tropicalis细胞 ,考察了细胞外pH和生长碳源对pHi的影响 ,实验结果表明 :细胞外pH对pHi略有影响 ,而生长碳源对pHi的影响略为明显。利用 5L发酵罐进一步研究了细胞生长代谢活性与pHi的关系 ,结果表明 :细胞比生长速率、CO2 比生产速率和葡萄糖比消耗速率与pHi变化密切相关 ,pHi的增加伴随着细胞生长活力的增加 ,反之亦然。在pH 6 0条件下用葡萄糖和醋酸钠共作碳源培养C .tropicalis细胞时 ,测得的pHi值维持在 5 72～ 6

Intracellular pH (pH i ) has an important inf luence on the metabolic activity of cells or cellular processes. The intracellular pH (pH i ) of long_chain α, ω_dicarboxylic acid_producin g Candida tropicalis was determined by fluorescence technique u sing a pH_sensitive fluorescent probe 5(6)_carboxyfluorescein diacetate. Optimal loading conditions of the fluorescent probe into the cells were experimentally determined. Effects of extracellular pH and carbon sources for growth on p H i in the cell grown in a flask were studied; the results indicat ed that extracellular pH has a slight influence on pH i , wherea s carbon sources such as sucrose, glucose, acetic acid, and n_tridecane show ed moderate influences. Further work on the relationship betwee n the cell growth activity and pH i was carried out in a 5 L bioreactor. The time course of specific rates of the cell growth, glucose consumption, CO 2 production, and pH gradients across cell plasma membrane were plotted, where the cell growth was improve d by the higher pH i at 8～12 h. The measured pH i values were varied from 5 72 to 6 1 5 at medium pH 6 0 in which glucose and sodium acetate were used together as ca rbon source. The investigation of pH i can be helpful fo r understanding its effects on the kinetics of the metabolic steps involved in t he synthesis rate of α, ω_dicarboxylic acid and α, ω_dicarboxylic acid trans port across plasma membranes.