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

应用荧光探针5(6)-双醋酸羧基荧光素 (Carboxyfluorescein diacetate) 测定了产长链二元酸热带假丝酵母 (Candida tropicalis) 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pH_I的影响，实验结果表明：细胞外pH对pH_I略有影响，而生长碳源对pH_I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，pH_I的增加伴随着细胞生长活力的增加，反之亦然。在pH6.0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pH_I值维持在5.72～6.15范围内。

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

Intracellular pH (pHi) has an important influence on the metabolic activity of cells or cellular processes. The intracellular pH (pHi) of long-chain alpha,omega-dicarboxylic acid-producing Candida tropicalis was determined by fluorescence technique using 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 pHi in the cell grown in a flask were studied; the results indicated that extracellular pH has a slight influence on pHi, whereas carbon sources such as sucrose, glucose, acetic acid, and n-tridecane showed moderate influences. Further work on the relationship between the cell growth activity and pHi was carried out in a 5 L bioreactor. The time course of specific rates of the cell growth, glucose consumption, CO2 production, and pH gradients across cell plasma membrane were plotted, where the cell growth was improved by the higher pHi at 8 - 12 h. The measured pHi values were varied from 5.72 to 6.15 at medium pH 6.0 in which glucose and sodium acetate were used together as carbon source. The investigation of pHi can be helpful for understanding its effects on the kinetics of the metabolic steps involved in the synthesis rate of alpha,omega-dicarboxylic acid and alpha,omega-dicarboxylic acid transport across plasma membranes.