Worsztynowicz Paulina, Schmidt Agnieszka Olejnik, Białas Wojciech, Grajek Włodzimierz
Department of Biotechnology and Food Microbiology, Poznań University of Life Sciences, Poznań, Poland.
Acta Biochim Pol. 2019 Feb 3;66(1):61-69. doi: 10.18388/abp.2018_2714.
Bacteria of the genus Enterococcus are lactic acid bacteria (LAB), which occur ubiquitous in many traditional fermented foods, especially artisanal cheeses, playing positive role in the development of cheese flavor. Moreover, several enterococci are successfully used as a pharmaceutical probiotic and some of them are able to produce bacteriocin and bioactive peptides, thanks to which the possibilities of application of enterococci in dairy technology and biotechnology are increased. The aims of the study were to investigate the proteolytic potential and identify the key enzymes of proteolytic system of Enterococcus faecalis isolated from artisan Polish cheeses. An extracellular - secreted (E) and a cell envelope proteinase (CEP) were isolated and enzyme activity depending on bacterial growth phase was evaluated. CEP showed a higher protease activity than E and this fraction has been purified 70-fold by a method including precipitation, diafiltration and gel filtration chromatography. The molecular mass of the enzyme has been estimated to be ~25 kDa by SDS-PAGE. Maximum enzyme activity of the proteinase has been observed at pH 6,9 and 37 ºC. The enzyme was able to hydrolyze: casein, bovine serum albumin, α-lactalbumin, β-lactoglobulin, but not Leu-pNa. The results of zymography, SDS- PAGE and LC-MS-MS/MS data allowed us to identify the key enzymes of proteolytic system of E. faecalis as coccolysin and glutamylendopeptidase. To asses microbiological safety of the tested strain, the evaluation of the presence of virulence factors and antibiotic susceptibility was also conducted.
肠球菌属细菌是乳酸菌，广泛存在于许多传统发酵食品中，尤其是手工制作的奶酪中，在奶酪风味的形成中发挥着积极作用。此外，几种肠球菌已成功用作药用益生菌，其中一些能够产生细菌素和生物活性肽，这增加了肠球菌在乳制品技术和生物技术中的应用可能性。本研究的目的是调查从波兰手工奶酪中分离出的粪肠球菌的蛋白水解潜力，并确定其蛋白水解系统的关键酶。分离出一种细胞外分泌（E）蛋白酶和一种细胞包膜蛋白酶（CEP），并评估了酶活性随细菌生长阶段的变化。CEP显示出比E更高的蛋白酶活性，并且通过包括沉淀、渗滤和凝胶过滤色谱在内的方法将该组分纯化了70倍。通过SDS-PAGE估计该酶的分子量约为25 kDa。在pH 6.9和37℃下观察到蛋白酶的最大酶活性。该酶能够水解：酪蛋白、牛血清白蛋白、α-乳白蛋白、β-乳球蛋白，但不能水解亮氨酸对硝基苯胺。酶谱、SDS-PAGE和LC-MS-MS/MS数据的结果使我们能够确定粪肠球菌蛋白水解系统的关键酶为球菌溶菌素和谷氨酰内肽酶。为了评估受试菌株的微生物安全性，还对毒力因子的存在和抗生素敏感性进行了评估。
