Catara G, Ruggiero G, La Cara F, Digilio F A, Capasso A, Rossi M
Institute of Protein Biochemistry, CNR, Via Marconi 10, 80125 Naples, Italy.
Extremophiles. 2003 Oct;7(5):391-9. doi: 10.1007/s00792-003-0337-4. Epub 2003 Jun 7.
A novel extracellular serine protease designated Pernisine was purified to homogeneity and characterized from the archaeon Aeropyrum pernix K1. The molecular mass, estimated by SDS-PAGE analysis and by gel filtration chromatography, was about 34 kDa suggesting that the enzyme is monomeric. Pernisine was active in a broad range of pH (5.0-12.0) and temperature (60-120 degrees C) with maximal activity at 90 degrees C and between pH 8.0 and 9.0. In the presence of 1 mM CaCl(2) the activity, as a function of the temperature, reached a maximum at 90 degrees C but at 120 degrees C the enzyme retained almost 80% of its maximal activity. Activity inhibition studies suggest that the enzyme is a serine metalloprotease and biochemical data indicate that Pernisine is a subtilisin-like enzyme. The protease gene, identified from the sequenced genome of A. pernix, was amplified from total genomic DNA by PCR technique to construct the expression plasmid pGEX-Pernisine. The Pernisine, lacking the leader sequence, was expressed in Escherichia coli BL21 strain as a fusion protein with glutathione- S-transferase. The biochemical properties of the recombinant enzyme were found to be similar to those of the native enzyme.
一种名为Pernisine的新型细胞外丝氨酸蛋白酶被纯化至同质，并从古菌嗜热栖热菌K1中进行了表征。通过SDS-PAGE分析和凝胶过滤色谱法估计，其分子量约为34 kDa，表明该酶是单体。Pernisine在广泛的pH范围（5.0 - 12.0）和温度范围（60 - 120℃）内具有活性，在90℃以及pH 8.0至9.0之间具有最大活性。在存在1 mM CaCl₂的情况下，其活性随温度变化，在90℃时达到最大值，但在120℃时该酶仍保留其最大活性的近80%。活性抑制研究表明该酶是一种丝氨酸金属蛋白酶，生化数据表明Pernisine是一种枯草杆菌蛋白酶样酶。从嗜热栖热菌的测序基因组中鉴定出的蛋白酶基因，通过PCR技术从总基因组DNA中扩增出来，构建表达质粒pGEX-Pernisine。缺少前导序列的Pernisine在大肠杆菌BL21菌株中作为与谷胱甘肽-S-转移酶的融合蛋白表达。发现重组酶的生化特性与天然酶相似。
