Liu Siqing, Rich Joseph O, Anderson Amber
RPT Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture, 1815 N. University St, Peoria, IL, 61604, USA,
J Ind Microbiol Biotechnol. 2015 Feb;42(2):229-35. doi: 10.1007/s10295-014-1557-6. Epub 2014 Dec 23.
The cell-free supernatant (CFS) from Lactobacillus paracasei NRRL B-50314 culture has been previously reported as containing antibacterial activity against a wide variety of Gram-positive bacteria. The CFS protein gel slice corresponding to antibacterial activities was subjected to trypsin digestion and ion trap MASS (Gel/LC-MS/MS) analysis. BlastP search of the resulted IQAVISIAEQQIGKP sequence led to a hypothetical cell-wall associated hydrolase (designated as CWH here) from Lactobacillus paracasei ATCC 25302. Further analyses of CWH revealed that the IQAVISIAEQQIGKP belongs to a highly conserved region of the NlpC/P60 superfamily. The L. paracasei NRRL B-50314 CWH gene, cloned in pStrepHIS1525CWH477, was introduced into Bacillus megaterium MS 941. The production of CWH477 protein was induced by xylose. The CWH477 protein was purified by using NiNTA column, and elution fraction E2 showed highest antibacterial activity. This study and bioinformatics analyses suggested that the antibacterial activity of CWH could originate from its cell wall degrading enzymatic function.
先前有报道称，副干酪乳杆菌NRRL B - 50314培养物的无细胞上清液（CFS）对多种革兰氏阳性菌具有抗菌活性。对与抗菌活性相对应的CFS蛋白凝胶切片进行胰蛋白酶消化和离子阱质谱（凝胶/液相色谱 - 串联质谱）分析。对所得IQAVISIAEQQIGKP序列进行BlastP搜索，发现其与副干酪乳杆菌ATCC 25302的一种假定的细胞壁相关水解酶（此处命名为CWH）匹配。对CWH的进一步分析表明，IQAVISIAEQQIGKP属于NlpC/P60超家族的一个高度保守区域。将克隆于pStrepHIS1525CWH477的副干酪乳杆菌NRRL B - 50314 CWH基因导入巨大芽孢杆菌MS 941。木糖诱导CWH477蛋白的产生。使用镍 - 亚氨基二乙酸（NiNTA）柱纯化CWH477蛋白，洗脱级分E2显示出最高的抗菌活性。本研究及生物信息学分析表明，CWH的抗菌活性可能源于其细胞壁降解酶功能。
