GENETIC AND BIOCHEMICAL-PROPERTIES OF AN EXTRACELLULAR NEUTRAL METALLOPROTEASE FROM STAPHYLOCOCCUS-HYICUS SUBSP HYICUS
GENETIC AND BIOCHEMICAL-PROPERTIES OF AN EXTRACELLULAR NEUTRAL METALLOPROTEASE FROM STAPHYLOCOCCUS-HYICUS SUBSP HYICUS
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DOI：
10.1007/bf00281792
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发表时间：
1994-02-01
期刊：
影响因子：
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通讯作者：
GOTZ, F
中科院分区：
文献类型：
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作者：
AYORA, S;GOTZ, F
编码猪葡萄球菌（Staphylococcus hyicus subsp.）hyicus是克隆的。DNA序列分析表明，该基因的开放阅读框（ORF）为1317个核苷酸，编码438个氨基酸，相对分子质量为49698。当克隆的基因在肉葡萄球菌中表达时，在培养基中发现42 kDa的蛋白酶。该蛋白酶是从两种S.肉样链球菌（pCAshp 1）和S.猪亚种猪。两种蛋白酶的N-末端氨基酸序列显示，ShpI被组织为前原酶，具有建议的26个氨基酸的信号肽，75个氨基酸的亲水性前区，和337个氨基酸的胞外成熟形式，计算的Mr为38394。N-末端在两种宿主菌株中均表现出微异质性。ShpI在55 ℃和pH 7.4-8.5时具有最大蛋白水解活性。该蛋白酶对底物的专一性较低，能被EDTA和1，10-菲咯啉等金属和锌特异性抑制剂抑制。对phosphoramidon的不敏感性将ShpI与嗜热菌蛋白酶样家族分开。保守的Zn ~（2+）结合基序是该蛋白酶与其它蛋白酶的唯一同源性，Zn ~（2+）对脱辅基酶的再活化表明Zn ~（2+）是催化离子。Ca 2+很可能起稳定剂的作用。我们还证实了在S.猪亚种猪。
The gene encoding the extracellular neutral metalloprotease ShpI from Staphylococcus hyicus subsp. hyicus was cloned. DNA sequencing revealed an ORF of 1317 nucleotides encoding a 438 amino acid protein with Mr of 49698. When the cloned gene was expressed in Staphylococcus carnosus, a 42 kDa protease was found in the culture medium. The protease was purified from both S. carnosus (pCAshp1) and S. hyicus subsp. hyicus. The N-terminal amino acid sequences of the two proteases revealed that ShpI is organized as a pre-pro-enzyme with a proposed 26 amino acid signal peptide, a 75 amino acid hydrophilic pro-region, and a 337 amino acid extracellular mature form with a calculated Mr of 38394. The N-termini showed microheterogeneity in both host strains. ShpI had a maximum proteolytic activity at 55 degrees C and pH 7.4-8.5. The protease, which had a low substrate specificity, could be inhibited by metal- and zinc-specific inhibitors, such as EDTA and 1,10-phenanthroline. Insensitivity to phosphoramidon separates ShpI from the thermolysin-like family. The conserved Zn2+ binding motif, the only homology to other proteases, and the reactivation of the apoenzyme by Zn2+, indicated that Zn2+ is the catalytic ion. Ca2+ very probably acts as a stabilizer. We also demonstrated the presence of a second extracellular protease in S. hyicus subsp. hyicus.