Chen Y S, Steele J L
Department of Food Science, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Appl Environ Microbiol. 1998 Sep;64(9):3411-5. doi: 10.1128/AEM.64.9.3411-3415.1998.
A previously identified insert expressing an endopeptidase from a Lactobacillus helveticus CNRZ32 genomic library was characterized. Nucleotide sequence analysis revealed an open reading frame of 1,941 bp encoding a putative protein of 71.2 kDa which contained a zinc-protease motif. Protein homology searches revealed that this enzyme has 40% similarity with endopeptidase O (PepO) from Lactococcus lactis P8-2-47. Northern hybridization revealed that pepO is monocistronic and is expressed throughout the growth phase. CNRZ32 derivatives lacking PepO activity were constructed via gene replacement. Enzyme assays revealed that the PepO mutant had significantly reduced endopeptidase activity when compared to CNRZ32 with two of the three substrates examined. Growth studies indicated that PepO has no detectable effect on growth rate or acid production by Lactobacillus helveticus CNRZ32 in amino acid defined or skim milk medium.
对先前鉴定出的一个来自瑞士乳杆菌CNRZ32基因组文库、表达一种内肽酶的插入片段进行了特性分析。核苷酸序列分析揭示了一个1941 bp的开放阅读框，其编码一个推定的71.2 kDa蛋白质，该蛋白质含有一个锌蛋白酶基序。蛋白质同源性搜索显示，这种酶与乳酸乳球菌P8 - 2 - 47的内肽酶O（PepO）有40%的相似性。Northern杂交显示pepO是单顺反子的，并且在整个生长阶段都有表达。通过基因替换构建了缺乏PepO活性的CNRZ32衍生物。酶活性测定显示，与CNRZ32相比，在检测的三种底物中的两种底物存在时，PepO突变体的内肽酶活性显著降低。生长研究表明，PepO对瑞士乳杆菌CNRZ32在氨基酸限定培养基或脱脂乳培养基中的生长速率或产酸没有可检测到的影响。
