【摘要】 点状产气单胞菌点状亚种（ Ａｅｒｏｍｏｎａｓ ｐｕｎｃｔａｔａ ｓｕｂｓｐ ． ｐｕｎｃｔａｔａ） 具有脯氨酰内肽酶（ｐｒｏｌｙｌ ｅｎｄｏｐｅｐｔｉｄａｓｅ Ｐ Ｅ Ｐ） 活性。将其染色体 Ｄ Ｎ Ａ 用 Ｅｃｏ Ｒ Ｉ部分酶切后回收８ ～１６ ｋｂ 的 Ｄ Ｎ Ａ 片断， 与 Ｅｃｏ Ｒ Ｉ消化载体ｐ Ｕ Ｃ１９ 连接后转化 Ｅ． ｃｏｉｌ Ｄ Ｈ５α， 用该酶的专一性底物 Ｂｅｎｚｙｌｏｘｙｃａｒｂｏｎｙｌ Ｇｌｙ Ｐｒｏβｎａｐｈｔｈｙｌａｍｉｄｅ 从质粒库中筛选到一株阳性克隆， 该重组质粒上的１２ ｋｂ Ｅｃｏ Ｒ Ｉ片断经亚克隆后获得３ ．５ ｋｂ Ｅｃｏ Ｒ Ｉ／ Ｈｉｎｃ Ｉ Ｉ的活性片断。构建该片断的限制性内切酶谱并进行核苷酸序列的测定， 获得长度为２ ０７３ ｂｐ 的脯氨酰内肽酶基因完整的 Ｏ Ｒ Ｆ， 编码由６９０ 个氨基酸组成的酶， 计算得分子量为７６ ４６７ 。其氨基酸一级结构与已克隆的嗜水气单胞菌、脑膜炎脓毒黄杆菌、猪脑、人淋巴细胞和狂怒火球菌的 Ｐ Ｅ Ｐ分别具有９２ ．３ ％ 、５３ ．２ ％ 、３３ ．５ ％ 、３３ ．２ ％ 和２０ ．５ ％ 的同源性： 同源性比较显示该酶含有由 Ｓｅｒ５３８ 、 Ａｓｐ６２２ 和 Ｈｉｓ６５７ 组成的催化三联体。更多还原

【Abstract】 Prolyl endopeptidase activity was found in Aeromonas punctata subsp. punctata. The genomic DNA was partially digested with Eco RI and the recovered 8～16 kb DNA fragments were inserted into the Eco RI site of plasmid pUC19, and were transformated into Escherichia coli DH5α. The resulted clones were screened by using Benzyloxycarbonyl Gly Pro β naphthylamide, the specific substrate of prolyl endopeptidase and a positive clone was obtained. The 12 kb insertion fragment of recombinant plasmid was digested with Hin cII and subcloned. The PEP gene was found in the 3.5 kb Hin cII/ Eco RI fragment. Nucleotide sequence of the gene was completely sequenced by Auto Sequencer. The complete gene consisted of 2 073 bp corresponding to 690 amino acid residues with a calculated molecular weight of 76 467 Da. The amino acid sequence was 92.3% 、53.2%、33.5%、33.2% and 20.5% homologous to those of Aeromonas hydrophila, Flavobacterium meningosepticum, porcine brain, human lymphocytes and Pyrococcus furiosus respectively. From a survey of sequence homology with other members of the prolyl endopeptidase family, the amino acid residues involved in the catalytic triad were deduced to be Ser 538 、Asp 622 and His 657 .更多还原