Okayama Prefectural Technology Center for Agriculture, Forestry, and Fisheries, Research Institute for Biological Sciences (RIBS), Okayama, Kibichuo-cho, Kaga-gun, Japan.
Appl Biochem Biotechnol. 2011 Jun;164(4):475-86. doi: 10.1007/s12010-010-9149-z. Epub 2011 Jan 5.
X-prolyl dipeptidyl aminopeptidases (X-PDAPs) are useful in various food industries. In this study, we performed sequence-based screening to obtain a stable X-PDAP enzyme from thermophilic Streptomyces strains. We found three genes that encoded X-PDAP from Streptomyces thermoluteus subsp. fuscus NBRC 14270 (14270 X-PDAP), Streptomyces thermocyaneoviolaceus NBRC 14271 (14271 X-PDAP), and Streptomyces thermocoerulescens NBRC 14273, which were subsequently cloned and sequenced. The deduced amino acid sequences of these genes showed high similarity, with ~80% identity with each other. The isolated X-PDAPs and an X-PDAP from Streptomyces coelicolor were expressed in Streptomyces lividans using a hyperexpression vector: pTONA5a. Among these genes, only 14270 and 14271 X-PDAPs caused overexpression and extracellular production without artificial signal peptides. We also characterized the biochemical properties of purified 14271 X-PDAP. In addition, we found that, in peptide synthesis via an aminolysis reaction, this enzyme recognized D-amino acid derivatives as acyl acceptors, similar to L-amino acid derivatives.
X-脯氨酰二肽氨基肽酶（X-PDAPs）在各种食品工业中都有广泛的应用。在本研究中，我们通过基于序列的筛选，从嗜热链霉菌中获得了一种稳定的 X-PDAP 酶。我们从嗜热链霉菌亚种 NBRC 14270（14270 X-PDAP）、NBRC 14271（14271 X-PDAP）和 NBRC 14273 中发现了三个编码 X-PDAP 的基因，随后对这些基因进行了克隆和测序。这些基因的推导氨基酸序列具有高度相似性，彼此之间的同源性约为 80%。从这些基因中分离出的 X-PDAP 以及来自链霉菌的 X-PDAP 在链霉菌中通过超表达载体 pTONA5a 进行表达。在这些基因中，只有 14270 和 14271 X-PDAP 导致过度表达和细胞外产生，而无需人工信号肽。我们还对纯化的 14271 X-PDAP 的生化特性进行了表征。此外，我们发现，在通过氨解反应进行肽合成时，该酶将 D-氨基酸衍生物识别为酰基受体，类似于 L-氨基酸衍生物。
