|Budget Amount *help
¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1993 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 1992 : ¥1,500,000 (Direct Cost : ¥1,500,000)
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 op
erational 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.