Janer C, Arigoni F, Lee B H, Peláez C, Requena T
Departamento de Ciencia y Tecnología de Productos Lácteos, Instituto del Frío (CSIC), José Antonio Novais, 10, 28040 Madrid, Spain.
Appl Environ Microbiol. 2005 Dec;71(12):8460-5. doi: 10.1128/AEM.71.12.8460-8465.2005.
The proteolytic system of Bifidobacterium animalis subsp. lactis was analyzed, and an intracellular endopeptidase (PepO) was identified and characterized. This work reports the first complete cloning, purification, and characterization of a proteolytic enzyme in Bifidobacterium spp. Aminopeptidase activities (general aminopeptidases, proline iminopeptidase, X-prolyl dipeptidylaminopeptidase) found in cell extracts of B. animalis subsp. lactis were higher for cells that had been grown in a milk-based medium than for those grown in MRS. A high specific proline iminopeptidase activity was observed in B. animalis subsp. lactis. Whole cells and cell wall-bound protein fractions showed no caseinolytic activity; however, the combined action of intracellular proteolytic enzymes could hydrolyze casein fractions rapidly. The endopeptidase activity of B. animalis subsp. lactis was examined in more detail, and the gene encoding an endopeptidase O in B. animalis subsp. lactis was cloned and overexpressed in Escherichia coli. The deduced amino acid sequence for B. animalis subsp. lactis PepO indicated that it is a member of the M13 peptidase family of zinc metallopeptidases and displays 67.4% sequence homology with the predicted PepO protein from Bifidobacterium longum. The recombinant enzyme was shown to be a 74-kDa monomer. Activity of B. animalis subsp. lactis PepO was found with oligopeptide substrates of at least 5 amino acid residues, such as met-enkephalin, and with larger substrates, such as the 23-amino-acid peptide alpha s1-casein(f1-23). The predominant peptide bond cleaved by B. animalis subsp. lactis PepO was on the N-terminal side of phenylalanine residues. The enzyme also showed a post-proline secondary cleavage site.
对动物双歧杆菌乳酸亚种的蛋白水解系统进行了分析，鉴定并表征了一种细胞内肽酶（PepO）。这项工作报道了双歧杆菌属中一种蛋白水解酶的首次完整克隆、纯化及表征。在动物双歧杆菌乳酸亚种细胞提取物中发现的氨肽酶活性（一般氨肽酶、脯氨酸亚氨基肽酶、X-脯氨酰二肽基氨肽酶），以在基于牛奶的培养基中生长的细胞比在MRS培养基中生长的细胞更高。在动物双歧杆菌乳酸亚种中观察到高特异性脯氨酸亚氨基肽酶活性。完整细胞和细胞壁结合蛋白组分未显示酪蛋白分解活性；然而，细胞内蛋白水解酶的联合作用可迅速水解酪蛋白组分。对动物双歧杆菌乳酸亚种的内肽酶活性进行了更详细的研究，并克隆了动物双歧杆菌乳酸亚种中编码内肽酶O的基因，并在大肠杆菌中进行了过表达。动物双歧杆菌乳酸亚种PepO的推导氨基酸序列表明它是锌金属肽酶M13肽酶家族的成员，与长双歧杆菌预测的PepO蛋白具有67.4%的序列同源性。重组酶显示为74 kDa的单体。动物双歧杆菌乳酸亚种PepO对至少5个氨基酸残基的寡肽底物（如甲硫氨酸脑啡肽）以及较大的底物（如23个氨基酸的肽αs1-酪蛋白（f1-23））具有活性。动物双歧杆菌乳酸亚种PepO切割的主要肽键位于苯丙氨酸残基的N端侧。该酶还显示出脯氨酸后的二级切割位点。
