Yamagata Y, Ichishima E
Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, Sendai, Japan.
Curr Microbiol. 1995 Jun;30(6):357-66. doi: 10.1007/BF00369863.
To obtain a new serine protease from alkalophilic Bacillus sp. NKS-21, shotgun cloning was carried out. As a result, a new protease gene was obtained. It encoded an intracellular serine protease (ISP-1) in which there was no signal sequence. The molecular weight was 34,624. The protease showed about 50% homology with those of intracellular serine proteases (ISP-1) from Bacillus subtilis, B. polymyxa, and alkalophilic Bacillus sp. No. 221. The amino acid residues that form the catalytic triad, Ser, His and Asp, were completely conserved in comparison with subtilisins (the extracellular proteases from Bacillus). The cloned intracellular protease was expressed in Escherichia coli, and its purification and characterization were carried out. The enzyme showed stability under alkaline condition at pH 10 and tolerance to surfactants. The cloned ISP-1 digested well nucleoproteins, clupein and salmin, for the substrates.
为了从嗜碱芽孢杆菌NKS-21中获得一种新的丝氨酸蛋白酶，进行了鸟枪法克隆。结果，获得了一个新的蛋白酶基因。它编码一种没有信号序列的细胞内丝氨酸蛋白酶（ISP-1）。分子量为34,624。该蛋白酶与枯草芽孢杆菌、多粘芽孢杆菌和嗜碱芽孢杆菌221号的细胞内丝氨酸蛋白酶（ISP-1）具有约50%的同源性。与枯草杆菌蛋白酶（芽孢杆菌的细胞外蛋白酶）相比，形成催化三联体的氨基酸残基Ser、His和Asp完全保守。克隆的细胞内蛋白酶在大肠杆菌中表达，并对其进行了纯化和表征。该酶在pH 10的碱性条件下表现出稳定性，并且对表面活性剂具有耐受性。克隆的ISP-1对核蛋白、鲱精蛋白和鲑精蛋白等底物具有良好的消化作用。
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