Enzymatic synthesis of physiologically functional oligopeptides consisting of essential amino acids

与其组成的氨基酸相比，寡肽具有一些生理上更优越的功能，如在肠刷状膜上的同化率更高。在这项研究中，成功地实现了酶促合成包括必需氨基酸的寡肽。以热裂解酶(TL)、α-胰凝乳蛋白酶(CT)和木瓜蛋白酶(PP)为原料，在有机溶剂或有机/水两相体系中，通过缩合或转移作用合成双肽和三肽。合成三肽的一个例子是Z-GFLNH_2。用固定化TL和CT串联的两个色谱柱连续合成三肽，产率超过80%，反应220h，合成的寡肽如下：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…更多缩合，约100%)，Boc-H(Tos)Voet(PP；缩合，91%)。在某些情况下，反应体系中的水含量显著影响底物向固定化酶的传质速率。随着质量分数的增加，传质阻力增大。我们提出了一种新的酶促合成阿片肽前体亮氨酸脑啡肽Z-YGGFLOEt的方法，该方法由其组成的氨基酸衍生物通过CT、TL和PP合成。实验证明了该方法的有效性。用胰酶在乙腈和缓冲溶液的混合溶液中合成了含有碱性必需氨基酸K的多肽。重点研究了只含K的双肽或三肽的合成，找到了实现并长期保持目标高产率的条件。本研究表明，仅用4种酶即可高产率地合成多种寡肽，并构建了以较少的量生产多种产品的体系，为生物工艺的开发提供了方向。较少

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