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

本研究以阿斯巴甜前体的酶促合成为例，研究了酶促反应与产物分离的同时操作。新的操作方法是将含有底物的有机溶剂送入生物反应器，在生物反应器中将游离酶保留在水溶液中，然后将含有产物的有机溶剂流出。同时操作包括四个基本步骤：(1)从有机溶剂中提取底物到水溶液中；(2)在水中用酶促合成肽；(3)从水溶液中提取产物到有机溶剂中；(4)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，采用新的操作方法，用热分解酶催化合成阿斯巴甜前体。从理论和实验两方面考察了操作条件对底物、水相和有机相中产物浓度及产物收率的影响。可以说，羧基底物从有机溶剂中提取到水溶液中是速度控制步骤，因为酶反应速度快，胺底物从有机溶剂中提取到水溶液中和产物从水溶液中提取到有机溶剂中的速率很高。结果表明，随着停留时间和搅拌速度的增加，产物的产率增加，当停留时间为10小时，转速为450转/分时，产物的产率接近100%，可望用于多种生理活性多肽的酶促合成。

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.