Nucleotide and deduced amino acid sequences of a high-molecular-mass subtilisin from an alkaliphilic Bacillus isolate

• Published in: Biochimica et biophysica acta Vol. 1624; no. 1; pp. 109 - 114
• Main Authors: Ogawa, Akinori, Sumitomo, Nobuyuki, Okuda, Mitsuyoshi, Saeki, Katsuhisa, Kawai, Shuji, Kobayashi, Tohru, Ito, Susumu
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 05.12.2003
• Subjects: Alkaliphilic; Amino Acid Sequence; Bacillus; Bacillus - chemistry; Base Sequence; Cloning; Hydrogen-Ion Concentration; Molecular Sequence Data; Serine protease; Substrate Specificity; Subtilisin; Subtilisin - chemistry; Subtilisin - genetics; Subtilisin - metabolism; Temperature; Serine protease; Subtilisin; Cloning; Alkaliphilic; Bacillus
• ISSN: 0304-4165; 0006-3002; 1872-8006
• 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°C. The enzyme was rapidly inactivated by incubation over 45°C for 15 min at both pH 7 and 10. Calcium ions were slightly protective for thermoinactivation of the enzyme.