Okayama Prefectural Technology Center for Agriculture, Forestry and Fisheries, Research Institute for Biological Sciences (RIBS), Kaga-gun, Okayama 716-1241, Japan.
Appl Environ Microbiol. 2010 Sep;76(18):6180-5. doi: 10.1128/AEM.01242-10. Epub 2010 Jul 30.
We specifically examined an exopeptidase, prolyl aminopeptidase (PAP), as a target for synthesis of proline-containing peptides. A PAP from Streptomyces thermoluteus subsp. fuscus NBRC14270 (PAP14270) was obtained using sequence-based screening. From PAP14270, 144Ser was replaced by Cys (scPAP14270) to give aminolysis activity. In contrast to wild-type PAP14270, scPAP14270 produced a polymer of proline benzyl ester and cyclo[Pro-Pro]. The product mass was confirmed using liquid chromatography-mass spectrometry (LC/MS). Several factors affecting the reaction, such as the pH, concentration of the substrate, and reaction time, were measured to determine their effects. Furthermore, a correlation was found between substrate specificity in proline peptide synthesis and the log D value of acyl acceptors in aminolysis catalyzed by scPAP14270. Results showed that dipeptide synthesis proceeded in a weakly acidic environment and that cyclization and polymerization occurred under alkaline conditions. Furthermore, results suggest that almost all amino acid esters whose log D value is greater than 0, except hydroxyproline benzyl ester (Hyp-OBzl), can be recognized as acyl acceptors. These findings support the use of PAPs as a tool for production of physiologically active proline peptides.
我们专门研究了外肽酶脯氨酰氨基肽酶（PAP），将其作为合成含脯氨酸肽的靶标。通过基于序列的筛选，获得了来自嗜热链霉菌亚种fuscus NBRC14270（PAP14270）的 PAP。从 PAP14270 中，将 144 位丝氨酸替换为半胱氨酸（scPAP14270），从而获得氨解活性。与野生型 PAP14270 相比，scPAP14270 产生了脯氨酸苄酯和环[Pro-Pro]的聚合物。使用液相色谱-质谱（LC/MS）确认产物质量。测量了影响反应的几个因素，如 pH、底物浓度和反应时间，以确定它们的影响。此外，还发现 scPAP14270 催化的氨解中脯氨酸肽合成的底物特异性与酰基受体的 log D 值之间存在相关性。结果表明，二肽合成在弱酸性环境中进行，而环化和聚合在碱性条件下发生。此外，结果表明，除了羟脯氨酸苄酯（Hyp-OBzl）外，log D 值大于 0 的几乎所有氨基酸酯都可以被识别为酰基受体。这些发现支持将 PAP 用作生产具有生理活性的脯氨酸肽的工具。
