Department of Biotechnology, Human Nutrition and Science of Food Commodities, University of Life Sciences, Lublin, Poland.
Prep Biochem Biotechnol. 2012;42(5):476-88. doi: 10.1080/10826068.2012.656869.
A proteinase produced by the human gastrointestinal isolate Lactobacillus rhamnosus strain OXY was identified and characterized. The prtR2 gene coding for proteinase activity was detected in the examined strain. The PCR primers used were constructed on the basis of the sequence of the prtR2 proteinase gene from Lactobacillus rhamnosus GG. The enzyme was purified by fast protein liquid chromatography (FPLC) using CM-Sepharose Fast Flow and Sephacryl S-300 columns. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the enzyme had a relatively low molecular mass of 60 kD. Protease activity was observed at a pH range from 6.5 to 7.5 with optimum k(cat)/K(m) values at pH 7.0 and 40°C. Maximum proteolytic activity (59 U mL(-1)) was achieved after 48 hr of cultivation. The activity of the enzyme was inhibited only by irreversible inhibitors specific for serine proteinases (PMSF and 3,4-dichloro-isocumarine), suggesting that the enzyme was a serine proteinase. Proteinase activity was increased by Ca(2+) and Mg(2+), and inhibited by Cu(2+), Zn(2+), Cd(2+), and Fe(2+).
一种由人类胃肠道分离的罗伊氏乳杆菌菌株 OXY 产生的蛋白酶被鉴定和表征。在所研究的菌株中检测到编码蛋白酶活性的 prtR2 基因。用于 PCR 的引物是基于罗伊氏乳杆菌 GG 的 prtR2 蛋白酶基因序列构建的。该酶通过快速蛋白液相色谱 (FPLC) 用 CM-Sepharose Fast Flow 和 Sephacryl S-300 柱进行纯化。十二烷基硫酸钠聚丙烯酰胺凝胶电泳 (SDS-PAGE) 显示该酶的相对分子质量较低，为 60 kD。在 pH 值为 6.5 至 7.5 的范围内观察到蛋白酶活性，在 pH 值为 7.0 和 40°C 时具有最佳 k(cat)/K(m) 值。培养 48 小时后，达到最大的蛋白水解活性（59 U mL(-1)）。该酶的活性仅被针对丝氨酸蛋白酶的不可逆抑制剂（PMSF 和 3,4-二氯异吲哚啉）抑制，表明该酶是一种丝氨酸蛋白酶。蛋白酶活性被 Ca(2+)和 Mg(2+) 增加，被 Cu(2+)、Zn(2+)、Cd(2+) 和 Fe(2+) 抑制。
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