Ayora S, Lindgren P E, Götz F
Universität Tübingen, Germany.
J Bacteriol. 1994 Jun;176(11):3218-23. doi: 10.1128/jb.176.11.3218-3223.1994.
Two extracellular proteases from Staphylococcus hyicus subsp. hyicus, ShpI and ShpII, have been characterized. ShpI is a neutral metalloprotease with broad substrate specificity; the gene has been cloned and sequenced. ShpII, characterized here, is mainly produced in the late logarithmic growth phase in contrast to ShpI, which is mainly produced in the late stationary growth phase. ShpII was purified from culture medium of S. hyicus by ammonium sulfate precipitation and DEAE-Sepharose chromatography. The molecular mass, estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 34 kDa. The temperature optimum of ShpII was 55 degrees C, and the pH optimum was 7.4. ShpII, a neutral metalloprotease, was strongly inhibited by zinc and calcium chelators. The amino-terminal sequence of the active enzyme was similar to the corresponding region of a Staphylococcus epidermidis metalloprotease. The substrate specificity of ShpII was similar to that of thermolysin-like proteases, with the exception that ShpII also recognized aromatic amino acids. We demonstrated in vitro that ShpII, but not ShpI, cleaved the 86-kDa S. hyicus subsp. hyicus prolipase between Thr-245 and Val-246 to generate the mature 46-kDa lipase. Results of additional in vivo experiments supported the model that ShpII is necessary for the extracellular processing and maturation of S. hyicus subsp. hyicus lipase.
来自猪葡萄球菌亚种猪葡萄球菌的两种细胞外蛋白酶ShpI和ShpII已得到鉴定。ShpI是一种具有广泛底物特异性的中性金属蛋白酶；该基因已被克隆和测序。与主要在稳定期后期产生的ShpI不同，本文所鉴定的ShpII主要在对数生长期后期产生。通过硫酸铵沉淀和DEAE-琼脂糖层析从猪葡萄球菌的培养基中纯化出ShpII。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳估计其分子量为34 kDa。ShpII的最适温度为55℃，最适pH为7.4。ShpII作为一种中性金属蛋白酶，受到锌和钙螯合剂的强烈抑制。活性酶的氨基末端序列与表皮葡萄球菌金属蛋白酶的相应区域相似。ShpII的底物特异性与嗜热菌蛋白酶样蛋白酶相似，不同之处在于ShpII还识别芳香族氨基酸。我们在体外证明，ShpII而非ShpI在苏氨酸-245和缬氨酸-246之间切割86 kDa的猪葡萄球菌亚种猪葡萄球菌前脂肪酶，以产生成熟的46 kDa脂肪酶。额外的体内实验结果支持了ShpII是猪葡萄球菌亚种猪葡萄球菌脂肪酶细胞外加工和成熟所必需的这一模型。
