Proline-peptide synthesis with a mutant of prolyl aminopeptidase via aminolysis reaction.

我们特别研究了一种胞外肽酶，即脯氨酸氨基肽酶(PAP)，以方便地合成脯氨酸肽。通过基于序列的筛选，从热链霉菌亚种中分离到一种PAP。获得了Fuscus NBRC14270(PAP14270)。在PAP14270的基础上，用Cys(ScPAP14270)取代了144Ser，使其具有氨解活性。与野生型PAP14270不同，scPAP14270产生了脯氨酸苄酯和环[Pro-Pro]的聚合物。用LC/MS对产物进行了确证，考察了pH、底物浓度、反应时间等因素对反应的影响。此外，还证明了底物专一性与酰基受体在scPAP14270催化氨解反应中的对数D值之间存在相关性。结果表明，二肽的合成在弱酸性环境中进行，环化和聚合反应在碱性条件下进行。此外，结果表明，除Hyp-OBzl外，几乎所有对数值大于0的氨基酸酯都可以被识别为酰基受体。这些发现支持将PAPs用作生产具有生理活性的脯氨酸肽的主要工具。

We specifically examined an exopeptidase, prolyl aminopeptidase (PAP), as a target to synthesize proline-peptides conveniently. Using sequence-based screening, a PAP from Stieptomyces thermoluteus subsp. fuscus NBRC14270 (PAP14270) was obtained. From the PAP14270, 144Ser was replaced by Cys (scPAP14270) to give aminolysis activity. In contrast to wild type PAP14270, scPAP14270 produced polymer of proline benzyl ester and cyclo[Pro-Pro]. The products of mass were confirmed using LC/MS. Several factors affecting the reaction such as pH, concentration of substrate, and reaction time were measured for their effects. Furthermore, the correlation between substrate specificity in the proline-peptide synthesis and the log D value of acyl acceptors on aminolysis catalyzed by scPAP14270 was demonstrated. The results showed that dipeptide synthesis proceeded in a weakly acidic environment and that cyclization and polymerization took place in alkaline conditions. Furthermore, results suggest that almost all amino acid esters whose log D value is greater than 0 can be recognized as acyl acceptors except Hyp-OBzl. These findings support the use of PAPs as a leading tool for production of physiologically active proline-peptides.