Zhao Y, DeLancey G B
Department of Chemical, Biochemical, Materials Engineering, Stevens Institute of Technology, Hoboken, New Jersey 07030, USA.
Biotechnol Bioeng. 1999 Aug 20;64(4):442-51.
Equilibrium conversions were observed in the range of 60.2-76.0% with different initial compositions of reaction media for the bioreduction of acetophenone using resting cells of Saccharomyces cerevisiae in aqueous solutions at 30 degrees C. The reduction of acetophenone in the cells under anaerobic conditions is considered to be coupled with the oxidation of ethanol to acetate in the cytoplasm. A biphasic thermodynamic model is proposed which includes a nonuniform distribution of reagents across the cell membrane, a transmembrane pH gradient, ideal and nonideal solution models, and a basic reaction stoichiometry (ACP + (1/2) EtOH + (1/2)H2O <--> PEA + (1/2)Ac- + (1/2)H+). The intracellular activity coefficients were based on the Lewis-Randall rule for acetophenone, phenethyl alcohol, and H2O and Henry's law for ethanol, acetate anion, and H+. The overall standard Gibbs free energy was estimated to be -0.11 kcal/mol at a pH 7, 25 degrees C, and 1 atm. The intracellular thermodynamic activity coefficients of acetophenone and phenethyl alcohol were predicted to be 471.2 and 866.4, respectively, using the measured initial distribution coefficients and calculated extracellular activity coefficients. The model reflected a zero Gibbs free energy change at calculated conversions within 4% of the measured equilibrium conversions. The analysis verified the effect of the concentration ratio of the substrate acetophenone to the co-substrate ethanol on the conversion efficiency and suggested that the intracellular pH and the pH gradient across the cell transmembrane significantly affect the predicted equilibrium conversion. The intracellular pH of resting, viable cells of Bakers' yeast at the bioconversion conditions was determined experimentally to be 5.77.
在30℃的水溶液中，使用酿酒酵母的静止细胞对苯乙酮进行生物还原时，观察到不同反应介质初始组成下的平衡转化率在60.2 - 76.0%范围内。在厌氧条件下，细胞内苯乙酮的还原被认为与细胞质中乙醇氧化为乙酸盐相偶联。提出了一个双相热力学模型，该模型包括试剂在细胞膜上的非均匀分布、跨膜pH梯度、理想和非理想溶液模型以及基本反应化学计量（苯乙酮 + (1/2)乙醇 + (1/2)水 <--> 苯乙醇 + (1/2)乙酸根 + (1/2)氢离子）。细胞内活度系数基于苯乙酮、苯乙醇和水的路易斯 - 兰德尔规则以及乙醇、乙酸根阴离子和氢离子的亨利定律。在pH 7、25℃和1个大气压下，整体标准吉布斯自由能估计为 -0.11千卡/摩尔。利用测得的初始分配系数和计算出的细胞外活度系数，预测苯乙酮和苯乙醇的细胞内热力学活度系数分别为471.2和866.4。该模型反映出在计算转化率与测得的平衡转化率相差4%以内时吉布斯自由能变化为零。分析验证了底物苯乙酮与共底物乙醇的浓度比对转化效率的影响，并表明细胞内pH和跨细胞膜的pH梯度显著影响预测的平衡转化率。在生物转化条件下，通过实验测定面包酵母静止活细胞的细胞内pH为5.77。
