Enzymatic synthesis of physiologically functional oligopeptides consisting of essential amino acids

与它们的组成氨基酸相比，寡肽具有一些生理上优越的功能，如在肠刷状膜上具有更高的同化率。本研究成功地实现了酶促合成包括必需氨基酸在内的寡肽。热溶酶（TL）、α -胰凝乳酶（CT）和木瓜蛋白酶（PP）在有机溶剂或有机/水双相体系中通过缩合或转移作用合成二肽和三肽。合成三肽的一个例子是Z-GFLNH_2。用固定TL和CT串联的两列色谱柱连续合成该三肽，产率在80%以上，反应220 h。合成的寡肽包括必需氨基酸和组氨酸：Z- k (Z)IOMe （TL；缩合，约100%）、Z- twoet （PP；转移，73%）、Z- twvnh_2 （CT；缩合，约100%）、Z- lfome （TL；缩合，约100%）、Z- mlfome （TL；缩合，约100%）、Z- mlfome （TL;co . more缩合，约100%）、Boc-H(Tos)VOEt (PP；凝结,91%)。在某些情况下，反应体系中的含水量显著影响底物向固定化酶的传质速率。随着含量的增加，传质阻力增大。我们提出了一种新的酶法，通过CT、TL和PP，以氨基酸衍生物为原料合成阿片肽Leu-enkephalin， Z-YGGFLOEt前体。实验证明了该方法的有效性。胰蛋白酶在乙腈和缓冲液的混合物中合成了一些多肽，包括碱性必需氨基酸K。考察了仅由k组成的二肽或三肽的合成，发现了实现高收率和长时间保持客观的条件。本研究表明，仅用4种蛋白酶就可以高产地合成多种寡肽，并构建了以相对较少的量生产多种产物的体系，这是生物工艺开发的方向。少

Oligopeptides possess some physiologically superior functionalities such as higher assimilation rate at the brush boader membrane of the intestine, compared with their constituent amino acids. In this study,enzymatic syntheses of oligopeptides including of essential amino acids have been successfully realized. Thermolysin (TL), alpha-chymotrypsin (CT), and papain (PP) were used in organic solvent or in organic/aqueous biphasic system to synthesize di- and tripeptides by their condensation or transfer action. An example of tripeptide synthesized is Z-GFLNH_2. The tripeptide was continuously synthesized by using two columns packed with immobilized TL and CT, which were connected in series, at an yield of more than 80% for 220 h. The oligopeptides synthesized, which included essential amino acids and histidine, were as follows: Z-K(Z)IOMe (TL;condensation, ca. 100%), Z-TWOEt (PP; transfer, 73%), Z-TWVNH_2 (CT; condensation, ca. 100%), Z-LFOMe (TL; condensation, ca. 100%), Z-MLFOMe (TL; co … More ndensation, ca. 100%), Boc-H(Tos)VOEt (PP; condensation, 91%). The water content in the reaction system, in some cases, significantly affected the mass transfer rates of substrates into immobilized enzymes. An increase of the content resulted in a large mass transfer resistance. We proposed a novel enzymatic method to synthesize the precursor of an opioid peptide, Leu-enkephalin, Z-YGGFLOEt from its constituent amino acid derivatives by using CT, TL, and PP. The validity of the method was demonstrated experimentally. Some peptides including a basic essential amino acid, K were synthesized by trypsin in a mixture of acetonitrile and buffer solution. Examinations were focused on the synthesis of di- or tripeptides consisting of only K. We found the conditions where a high yield of objective could be realized and maintained for a long period. This study showed that a variety of oligopeptides could be synthesized in high yields using only four proteinases, and constructed the system to produce many kinds of products in a relatively small amount, which is directed in bioprocess development. Less