Nucleotide and deduced amino acid sequences of a high-molecular-mass subtilisin from an alkaliphilic Bacillus isolate.
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.
Ogawa
Akinori
A
Tochigi Research Laboratories of Kao Corporation, 2606 Akabane, Ichikai, Haga, Tochigi 321-3497, Japan.
Sumitomo
Nobuyuki
N
Okuda
Mitsuyoshi
M
Saeki
Katsuhisa
K
Kawai
Shuji
S
Kobayashi
Tohru
T
Ito
Susumu
S
D000595:Amino Acid Sequence; D001407:Bacillus / Q000737:chemistry*; D001483:Base Sequence; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D020860:Subtilisin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013696:Temperature
D020860:Subtilisin