Development of bioreactor system with highly efficient regeneration of coenzymes

1. 亲和层析反应器是利用酶与辅酶之间的动态亲和关系而开发的一种高效利用昂贵辅酶的生物反应器。对亲和色谱反应器的基本方面进行了理论研究，阐明了动态亲和反应的必要条件。用NAD循环催化丙醛还原和乙醇氧化的实验结果与单固定化乙醇脱氢酶体系的实验结果进行了比较。采用亲和层析反应器对丙氨酸脱氢酶和乳酸脱氢酶两种酶体系进行研究，以乳酸为原料生产l -丙氨酸。酶和辅酶之间的动态亲和力在基本与上述单酶情况相同的条件下表达。然而，动态亲和力强烈依赖于中间产物丙酮酸的浓度。在稳态运行下，反应器的生产率和NAD循环次数均取得了较好的结果。研究了消耗非特定电能的电化学方法作为辅酶再生的替代方法。通过对NADH交换氢反应速率的测定，选择了苯那嗪甲硫代硫酸钠作为NADH电化学氧化的最佳介质。葡萄糖6-磷酸脱氢酶反应可与三种氧化再生NAD的方法相结合；(1)直接电极氧化，(2)介导电极氧化和(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提高。

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.