Study on physiological role of proline containing peptide

研究结果总结如下：以从酱油曲中分离的米曲霉RIB915为试验菌株，利用二肽氨基肽酶IV（DPIV）合成m - orphiceptin（tyrr - pro - ph - pro - nh_2）制备糠曲，经盐析和凝胶过滤部分纯化得到DPIV酶粗溶液。以Tyr-Pro-OEt和ph - pro - nh_2为底物，考察了该粗酶的合成条件，得到了40%的morphiceptin。本研究首次合成了含脯氨酸的吗啡西普汀。此外，虽然一般认为氨基的保护在肽的形成中是有效的，但研究表明DPIV的肽形成不需要这种保护基团。用DPIV合成Leu- pro - tyr - pro - nh_2用合成morphiceptin的DPIV进行了(1)Tyr-Pro-OEt+Arg-NH_2→Tyr-Pro-Arg-NH_2(2)Leu- pro - oet +Tyr-Pro-Arg-NH_2→Leu…More - pro -Tyr-Pro-Arg-NH_2的DPIV合成Leu- pro -Tyr-Pro-Arg-NH_2的反应体系：水体系和添加有机溶剂体系。在水体系中，Tyr-Pro-Arg-NH_2的合成收率为31.2%，Leu-Pro-Tyr-Pro-Arg-NH_2的合成收率为3.3%。另一方面，在有机溶剂乙二醇的作用下，tyr1 - pro - arg - nh_2的收率为30.5%，leu - pro - tyr1 - pro - arg - nh_2的收率为26.7%，均有提高。这是由于在加入乙二醇作为有机溶剂的体系中，酪氨酸-原arg - nh_2和亮氨酸-原酪氨酸-原arg - nh_2的合成率都很高。虽然已知食物来源的二肽和三肽通过转运体在小肠中被吸收，但尚不清楚五肽Leu-Pro-Tyr-Pro-Arg是否也在小肠中被吸收。在大鼠体外实验体系中，灌胃后26 min门静脉血中发现该肽；LC/MS分析证实其为Leu-Pro-Tyr-Pro-Arg。少

Summary of research results is the following.1.Synthesis of m orphiceptin(Tyr-Pro-Phe-Pro-NH_2) by dipeptidylaminopeptidase IV(DPIV)Aspergillus oryzae RIB915, isolated from soy sauce koji, was used as the test strain to prepare bran koji, and crude DPIV enzyme solution was obtained through partial purification by salting-out and gel filtration. Conditions for synthesis with this crude enzyme were examined using Tyr-Pro-OEt and Phe-Pro-NH_2 as substrates, and resulted in 40% morphiceptin. This study achieved synthesis of the proline-containing morphiceptin for the first time. In addition, although it has been considered that the protection of the amino group is effective in peptide formation in general, it was shown that peptide formation by DPIV required no such protecting groups.2.Synthesis of Leu-Pro-Tyr-Pro-NH_2 by DPIVWith DPIV that was used in the synthesis of morphiceptin, a two-step reaction including(1)Tyr-Pro-OEt+Arg-NH_2 → Tyr-Pro-Arg-NH_2(2)Leu-Pro-OEt+Tyr-Pro-Arg-NH_2 → Leu … More -Pro-Tyr-Pro-Arg-NH_2to synthesize Leu-Pro-Tyr-Pro-Arg-NH_2 by DPIV was examined in commonly used reaction systems: the aqueous system and the system with addition of organic solvent. Synthesis yield in aqueous system were 31.2% for Tyr-Pro-Arg-NH_2, and 3.3% for Leu-Pro-Tyr-Pro-Arg-NH_2. On the other hand, yields in the system with organic solvent ethylene glycol were 30.5% for Tyr-Pro-Arg-NH_2, and 26.7% for Leu-Pro-Tyr-Pro-Arg-NH_2, showing improvement. This resulted from high synthesis yields for both Tyr-Pro-Arg-NH_2 and Leu-Pro-Tyr-Pro-Arg-NH_2 in the system with addition of ethylene glycol as organic solvent.3.Absorption of opioid peptidesAlthough food-derived dipeptides and tripeptides are known to be absorbed in the small intestine via a transporter, it remains unclear if the pentapeptide Leu-Pro-Tyr-Pro-Arg was also absorbed there. In the in vitro experimental system in rats, the peptide was found in portal blood 26 min after intragastric administration ; LC/MS analysis confirmed that it was Leu-Pro-Tyr-Pro-Arg. Less