| 研究概要 |
プロリン含有ペプチドを合成するために、エキソ型アミノペプチダーゼであるプロリンアミノペプチダーゼ(PAP)に着目した。ゲノム情報を基にスクリーニングした結果、中等度好熱性放線菌であるStreptomyces thermoluteus subsp. fuscus NBRC14270からPAP遺伝子(14270PAP)を得た。14270PAPの活性中心SerをCysに置換した酵素(scPAP 14270)を作成した。プロリンベンジルエステルを基質とし、アミノリシス活性を野生型と比較したところ、scPAP 14270のみが、プロリンオリゴマーとcyclo(Pro-Pro)を合成した。アミノリシス反応におけるscPAP 14270のアシルアクセプター特異性を検討したところ、基質のlog P値と反応性は良い相関を示し、ヒドロキシプロリンベンジルエステルを除いて、log P値がゼロよりも大きい値を示すものは、基質として認識されることを明らかにした。
We focused exo-peptidase, prolyl aminopeptidase (PAP), for targeting to synthesize proline-peptides conveniently. By sequence based screening, a PAP from Streptomyces thermoluteus subsp. fuscus NBRC 14270 (PAP 14270) was obtained. PAP 14270 was substituted 144Ser with Cys (scPAP 14270) to give aminolysis activity. Comparison of the wild type of PAP 14270, 0nly scPAP 14270 produced polymer of proline benzyl ester and cyclo [Pro-Pro]. The products were confirmed of its mass by LC/MS. Several factors which affect the reaction such as pH, concentration of substrate, amount of enzyme and reaction time were measured of its effect. Furthermore, correlation between substrate specificity in the proline-peptide synthesis and the log P value of acyl acceptors on aminolysis catalyzed by scPAP 14270 was also demonstrated. The results showed that dipeptide synthesis was processed in weekly acidic environment while cyclization and polymerization were in alkaline condition. Further, it was suggested that all most of amino acid esters whose log P value is more than 0 could be recognized as acyl acceptor except Hyp-OBzl. These findings will support the use of PAPs as a leading tool for produce physiologically active proline-peptides.
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