Ogawa Akinori, Sumitomo Nobuyuki, Okuda Mitsuyoshi, Saeki Katsuhisa, Kawai Shuji, Kobayashi Tohru, Ito Susumu
Tochigi Research Laboratories of Kao Corporation, 2606 Akabane, Ichikai, Haga, Tochigi 321-3497, Japan.
Biochim Biophys Acta. 2003 Dec 5;1624(1-3):109-14. doi: 10.1016/j.bbagen.2003.10.002.
A high-molecular-mass subtilisin was found in culture broth of the alkaliphilic Bacillus sp. strain KSM-KP43. The gene encoding the enzyme (FT protease) was determined using a mixed primer designed from the N-terminal amino acid (aa) sequence of the purified enzyme. The determined nucleotide sequence of the gene consisted of a 2427-bp open reading frame (ORF) that encoded a putative prepro-peptide (152 aa) and a mature enzyme (656 aa; 68,506 Da). The deduced aa of the mature enzyme revealed a moderate homology to a subtilisin-type proteinase from Bacillus halodurans and a minor extracellular protease, Vpr, from Bacillus subtilis with 64% and 57% identity, respectively. The molecular mass of the purified recombinant FT protease was approximately 72 kDa as judged by both SDS-polyacrylamide gel electrophoresis (PAGE) and gel filtration. FT protease showed maximal activity toward glutaryl-Ala-Ala-Pro-Leu-p-nitroanilide at pH 10.5 and at 45 degrees C. The enzyme was rapidly inactivated by incubation over 45 degrees C for 15 min at both pH 7 and 10. Calcium ions were slightly protective for thermoinactivation of the enzyme.
在嗜碱芽孢杆菌属菌株KSM-KP43的培养液中发现了一种高分子量枯草杆菌蛋白酶。使用根据纯化酶的N端氨基酸序列设计的混合引物确定了编码该酶（FT蛋白酶）的基因。该基因确定的核苷酸序列由一个2427 bp的开放阅读框（ORF）组成，其编码一个推定的前原肽（152个氨基酸）和一个成熟酶（656个氨基酸；68506 Da）。成熟酶推导的氨基酸序列与嗜盐芽孢杆菌的枯草杆菌蛋白酶型蛋白酶以及枯草芽孢杆菌的一种次要细胞外蛋白酶Vpr分别具有64%和57%的同一性，显示出适度的同源性。通过SDS-聚丙烯酰胺凝胶电泳（PAGE）和凝胶过滤判断，纯化的重组FT蛋白酶的分子量约为72 kDa。FT蛋白酶在pH 10.5和45℃时对戊二酰-Ala-Ala-Pro-Leu-对硝基苯胺表现出最大活性。在pH 7和10条件下，该酶在45℃以上孵育15分钟会迅速失活。钙离子对该酶的热失活有轻微的保护作用。
