Enzymatic Synthesis of Physiological Active Peptide with simultaneous Operetions of Reaction and Product Separetion by Liquid Extraction and Crystallization

本研究以合成生理活性肽为例，提出了酶法合成阿斯巴甜前体的同时进行酶促反应和液相萃取结晶分离的新方法。新的操作方法是将含有底物的有机溶剂送入生物反应器，其中游离酶保留在水溶液中，含有产物的有机溶剂流出。同时操作由四个基本速率过程组成，即(1)将底物从有机溶剂中萃取到水溶液中，(2)在水溶液中酶促合成肽，(3)将产物从水溶液中萃取到有机溶剂中，(4)产物在有机溶剂中结晶。以z - l -天冬氨酸和l -苯丙氨酸甲酯为原料，采用热溶素酶法合成了阿斯巴甜前体。从理论上和实验上考察了操作条件对底物、水相和有机相产物浓度以及产物收率的影响。可以说，羧基底物从有机溶剂中萃取到水溶液中是控制速率的步骤，因为酶促反应速率高，同时胺基底物从有机溶剂中萃取到水溶液中，产物从水溶液中萃取到有机溶剂中的速率也高。结果表明，产品得率随停留时间和搅拌速率的增加而增加，在停留时间为10 h，转速为450 rpm时，产品得率可接近100%。可以预期，新开发的操作方法适用于今后多种生理活性肽的酶促合成。

In this studies, simultaneous operations of enzymatic reaction and product separation by liquid extraction and crystallization was newly developed for the enzymatic synthesis of aspartame precursor as an example of the synthesis of physiologic active peptide. The new operational method is that organic solvent containing substrates is fed into a bioreactor in which free enzyme is retained in aqueous solution, and the organic solvent containing the product is flowed out. The simultaneous operations are consisted of four elementary rate processes, that is, (1) extractions of substrates from organic solovent into the aqueous solution, (2) enzymatic synthesis of peptide in the aqueous, (3) extraction of the product from the aqueous solution into the organic solvent, (4) crystallization of the product in the organic solvent.The aspartame precursor was enzymatically synthesized with thermolysin by the new operationalmethod from Z-L-aspartic acid and L-phenylalanine methyl ester. Effects of operational conditions on substrates and product concentrations in both of aqueous and organic phases and product yield were theretically and experimentally investigated.It can be said that the extraction of the carboxyl substrate from the organic solvent into the aqueous solution is the rate controlling step, because of the high enzymatic reaction rate, as well as the high rates of the extraction of the amine substrate from the organic solvent into the aqueous solution and the extraction of the product from the aqueous solution into the organic solvent. It is a remarkable result that the product yield increases with increasing the residence time and the agitation rate, and nearly 100% product yield can be obtained at 10 hr of the residence time and 450 rpm of the rotational speed.It can be expected that the new developed operational method is aplocable to enzymatic synthesis of many kind of physiological active peptides in future.