Palmieri G, Bianco C, Cennamo G, Giardina P, Marino G, Monti M, Sannia G
IABBAM Consiglio Nazionale delle Ricerche, 80147 Naples, Italy.
Appl Environ Microbiol. 2001 Jun;67(6):2754-9. doi: 10.1128/AEM.67.6.2754-2759.2001.
A new extracellular protease (PoSl; Pleurotus ostreatus subtilisin-like protease) from P. ostreatus culture broth has been purified and characterized. PoSl is a monomeric glycoprotein with a molecular mass of 75 kDa, a pI of 4.5, and an optimum pH in the alkaline range. The inhibitory profile indicates that PoSl is a serine protease. The N-terminal and three tryptic peptide sequences of PoSl have been determined. The homology of one internal peptide with conserved sequence around the Asp residue of the catalytic triad in the subtilase family suggests that PoSl is a subtilisin-like protease. This hypothesis is further supported by the finding that PoSl hydrolysis sites of the insulin B chain match those of subtilisin. PoSl activity is positively affected by calcium. A 10-fold decrease in the K(m) value in the presence of calcium ions can reflect an induced structural change in the substrate recognition site region. Furthermore, Ca(2+) binding slows PoSl autolysis, triggering the protein to form a more compact structure. These effects have already been observed for subtilisin and other serine proteases. Moreover, PoSl protease seems to play a key role in the regulation of P. ostreatus laccase activity by degrading and/or activating different isoenzymes.
已从平菇培养液中纯化并鉴定出一种新的细胞外蛋白酶（PoSl；平菇枯草杆菌蛋白酶样蛋白酶）。PoSl是一种单体糖蛋白，分子量为75 kDa，pI为4.5，最适pH在碱性范围内。抑制谱表明PoSl是一种丝氨酸蛋白酶。已确定了PoSl的N端和三个胰蛋白酶肽序列。PoSl的一个内部肽与枯草杆菌蛋白酶家族催化三联体Asp残基周围保守序列的同源性表明PoSl是一种枯草杆菌蛋白酶样蛋白酶。胰岛素B链的PoSl水解位点与枯草杆菌蛋白酶的水解位点相匹配这一发现进一步支持了这一假设。PoSl的活性受到钙的正向影响。在存在钙离子的情况下，K(m)值降低10倍可以反映底物识别位点区域的结构变化。此外，Ca(2+)结合减缓了PoSl的自溶，促使蛋白质形成更紧密的结构。枯草杆菌蛋白酶和其他丝氨酸蛋白酶也已观察到这些效应。此外，PoSl蛋白酶似乎通过降解和/或激活不同的同工酶在平菇漆酶活性的调节中起关键作用。
