| Co-Investigator(Kenkyū-buntansha) |
NAKANISHI Kazuhiro Okayama Univ., Dept. of Biotechnol., Professor, 工学部, 教授 (90026584)
KIMURA Yukitaka Kyoto Univ., Dept. of Food Sci. & Technol., Instructor, 農学部, 助手 (70211878)
ADACHI Shuji Kyoto Univ., Dept. of Food Sci. & Technol., Associate Professor, 農学部, 助教授 (90115783)
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| Research Abstract |
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
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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
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