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Development of bioreactor system with highly efficient regeneration of coenzymes

辅酶高效再生生物反应器系统的开发

基本信息

批准号：
61550699
负责人：
MIYAWAKI Osato
金额：
$ 0.9万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1986
资助国家：
日本
起止时间：
1986 至 1987
项目状态：
已结题

项目摘要

1. Affinity chromatographic reactor was developed as a bioreactor with highly efficient usage of expensive coenzymes by making use of dynamic affinity between enzyme and coenzyme. Basic aspects of affinity chromatographic reactor was investigated theoretically to elucidate the necessary condition for the expression of the dynamic affinity. The results were well compared with experimental results for a system with single immobilized enzyme of alcohol dehydrogenase which catalyzed both reduction of propionaldehyde and oxidation of ethanol with NAD cycling.2. Affinity chromatographic reactor was applied to the two enzyme system with alanine dehydrogenase and lactate dehydrogenase for the production of L-alanine from lactate. The dynamic affinity between enzymes and coenzyme was expressed essentially at the same condition as the single enzyme case described above. The dynamic affinity, however, was strongly dependent on the concentration of pyruvate, the intermediate product. At a steady state operation, fairly good results were obtained for the productivity of the reactor and NAD cycling number.3. Electrochemical method with consumption of nonspecific electrical energy was investigated as an alternative way of coenzyme regeneration. From the measurement of hydrogen exchange reaction rate with NADH, phenazine methosulfate was selected as the best mediator for electrochemical oxidation of NADH. Reaction of glucose 6-phosphate dehydrogenase could be coupled with three methods of oxidative regeneration of NAD; (1) direct electrode oxidation, (2) mediated electrode oxidation and (3) mediated oxidation with oxygen as the final electron acceptor. As a method of coenzyme regeneration for bioreactors. electrochemical method should be improved in surface mass transfer rate.

1.利用酶与辅酶的动态亲和力，开发了高效利用昂贵辅酶的生物反应器。从理论上研究了亲和层析反应器的基本问题，阐明了表达动态亲和力的必要条件。实验结果与单一固定化乙醇脱氢酶体系的实验结果相吻合，该体系同时催化丙醛的还原和乙醇的氧化。将亲和层析反应器应用于丙氨酸脱氢酶和乳酸脱氢酶两种酶体系中，以乳酸为原料合成L丙氨酸。酶和辅酶之间的动态亲和力基本上是在与上述单一酶情况相同的条件下表达的。然而，动态亲和力强烈依赖于中间产物丙酮酸的浓度。在稳态运行时，反应器的生产能力和NAD循环次数都取得了较好的结果。研究了利用非特定电能的电化学法作为辅酶再生的一种替代方法。通过与NADH的氢交换反应速率的测定，确定吩嗪甲硫酸盐为NADH电化学氧化的最佳介体。葡萄糖6-磷酸脱氢酶的反应可与NAD氧化再生的三种方式耦合：(1)直接电极氧化，(2)介导电极氧化，(3)以氧为最终电子受体的介导氧化。作为生物反应器辅酶再生的一种方法。电化学法在表面传质速率方面有待改进。