Development of enzymatic process to synthesize biologically active peptides with a extractive separation of produc

本研究开发了一种利用可溶性酶连续合成生物活性肽的新方法，并同时进行酶促反应和产物分离。A)连续合成z - apm肽是在最佳pH条件下用酶合成的，尽管在低pH条件下酶合成肽具有许多优点，如抑制氨基酯底物的非酶分解和较少需要NaOH来调节pH。本研究在低pH水/有机双相体系中合成了Z-APM，收率达95%以上。建立了萃取反应连续合成Z-APM的数学模型，与实验结果吻合较好。B)连续合成的f - apmn -苯氧羰基- l-天冬氨酸（Z-L-Asp）和l-苯丙氨酸甲酯（L-PheOMe）一般被用作酶促合成阿斯巴甜前体的底物。n -甲酰基（N-Formyl group, F-group）比z -基团更便宜，也更容易去除，本研究研究了用n -甲酰基代替z -基团来保护天冬氨酸，以降低工业连续生产阿斯巴甜的总成本。在单物体系中，F-APM的收率约为3%。而在水/TBP双相体系中，热溶菌催化合成F-APM的转化率高达95%以上。

The novel method to synthesize biologically active peptides with a continuous use of soluble enzyme and a simultaneous operation of an enzymatic reaction with a product separation was developed in this study, and applied to the protease-catalyzed synthesis of the precursor of aspartame (Z-APM and F-APM)A) Continuous synthesis of Z-APMpeptides are synthesized with enzyme at its optimal pH,though the enzymatic synthesis of peptides at low pH has a lot of advantages for an industrial-scaled production, such as, suppression of nonenzymatic decomposition of amino ester substrate and less requirement of NaOH for pH adjustment. We synthesize Z-APM at low pH in aqueous/organic biphasic system and obtained more than 95% of yield in this study. The mathe mathematical model to predict the continuous synthesis of Z-APM with extractive reaction was proposed and agreed well with the experimental results.B) Continuous synthesis of F-APMN-Benzyloxycarbonyl-L-aspartic acid (Z-L-Asp) and L-phenylalanine methyl ester (L-PheOMe) were, in general, used as the substrates for the enzymatic synthesis of aspartame precursor. N-Formyl group (F-group), which was cheaper and could be removed more easily than Z-group, was examined to be used for the protection of aspartic acid instead of Z-group to lower the total cost of the industrially continuous production of aspartame in this study. The F-APM yield was about 3% in aqueous monophsic system. The extremely high conversion, more than 95%, was, however, obtained for the thermolysin-catalyzed synthesis of F-APM in aqueous/TBP biphasic system.