Abstract
Six genes encoding high-molecular-mass subtilisins (HMSs) of alkaliphilic Bacillus spp. were cloned and sequenced. Their open reading frames of 2,394–2,424 bp encoded prosubtilisins of 798–808 amino acids (aa) consisting of the prepropeptides of 151–158 aa and the mature enzymes of 640–656 aa. The deduced aa sequences of the mature enzymes exhibited 60–95% identity to those of FT protease of Bacillus sp. strain KSM-KP43, a subtilisin-like serine protease, and a minor serine protease, Vpr, of Bacillus strains. Three of the six recombinant enzymes were susceptible to proteolysis, but the others were autodigestion resistant. All enzymes had optimal pH values of 10.5–11.0, optimal temperatures of 40–45°C for hydrolysis of a synthetic substrate, and were heat labile. These alkaline proteases seem to form a new subtilisin family, as judged by their aa sequences and phylogenetic analysis.
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Hakamada Y, Kobayashi T, Hitomi J, Kawai S, Ito S (1994) Molecular cloning and nucleotide sequence of the gene for an alkaline protease from the alkalophilic Bacillus sp. KSM-K16. J Ferment Bioeng 78:105–108
Ito S, Kobayashi T, Ara K, Ozaki K, Kawai S, Hatada Y (1998) Alkaline detergent enzymes from alkaliphiles: enzymatic properties, genetics, and structure. Extremophiles 2:185–190
Kato T, Yamagata Y, Arai T, Ichishima E (1992) Purification of a new extracellular 90-kDa serine proteinase with isoelectric point of 3.9 from Bacillus subtilis (natto) and elucidation of its distinct mode of action. Biosci Biotechnol Biochem 56:1166–1168
Kobayashi T, Hakamada Y, Adachi S, Hitomi J, Yoshimatsu T, Koike K, Kawai S, Ito S (1995) Purification and some properties of an alkaline protease from alkalophilic Bacillus sp. KSM-K16. Appl Microbiol Biotechnol 43:473–481
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Ogawa A, Sumitomo N, Okuda M, Saeki K, Kawai S, Kobayashi T, Ito S (2003) Nucleotide and deduced amino acid sequences of a high-molecular-mass subtilisin from an alkaliphilic Bacillus strain. Biochim Biophys Acta 1624:109–114
Pearson WR, Lipman DJ (1988) Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 85:2444–2448
Saeki K, Okuda M, Hatada Y, Kobayashi T, Ito S, Takami H, Horikoshi K (2000) Novel oxidatively stable subtilisin-like serine proteases from alkaliphilic Bacillus spp.: enzymatic properties, sequences, and evolutionary relationships. Biochem Biophys Res Commun 279:313–319
Saeki K, Hitomi J, Okuda M, Hatada Y, Kageyama Y, Takaiwa M, Kubota H, Hagihara H, Kobayashi T, Kawai S, Ito S (2002) A novel species of alkaliphilic Bacillus that produces an oxidatively stable alkaline serine protease. Extremophiles 6:65–72
Saeki K, Magallones MV, Takimura Y, Hatada Y, Kobayashi T, Kawai S, Ito S (2003) Nucleotide and deduced amino acid sequences of a new subtilisin from an alkaliphilic Bacillus isolate. Curr Microbiol 47:337–340
Saito H, Miura K (1963) Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim Biophys Acta 72:619–629
Saitou N, Nei M (1987) A neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 44:406–425
Shirai T, Suzuki A, Yamane T, Ashida T, Kobayashi T, Hitomi J, Ito S (1997) High-resolution crystal structure of M-protease: phylogeny-aided analysis of the high-alkaline adaptation mechanism. Protein Eng 10:627–634
Siezen RJ, Leunissen JAM (1997) Subtilases: the superfamily of subtilisin-like serine proteases. Protein Sci 6:501–523
Sloma A, Rufo GA Jr, Rudolph CF, Sullivan BJ, Theriault KA, Pero J (1990) Bacillopeptidase F of Bacillus subtilis: purification of the protein and cloning of the gene. J Bacteriol 172:1470–1477
Sloma A, Rufo GA Jr, Theriault KA, Dwyer M, Wilson SW, Pero J (1991) Cloning and characterization of the gene for an additional extracellular serine protease of Bacillus subtilis. J Bacteriol 173:6889–6895
Takami H, Nakasone K, Takagi Y, Maeno G, Sasaki R, Masui N, Fuji F, Hirama C, Nakamura Y, Ogasawara N, Kuhara S, Horikoshi K (2000) Complete genome sequence of the alkaliphilic bacterium Bacillus halodurans and genomic sequence comparison with Bacillus subtilis. Nucleic Acids Res 28:4317–4331
Takami H, Takagi Y, Uchiyama I (2002) Genome sequence of Oceanobacillus iheyensis isolated from the Iheya Ridge and its unexpected adaptive capabilities to extreme environment. Nucleic Acids Res 30:3927–3935
Thompson JD, Higgins DG, Gibson TJ (1994) ClustalW: improving the sensitivity of progressive multiple sequence alignments through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22: 4673–4680
Tinoco I Jr, Borer PN, Dengler B, Levine MD, Uhlenbeck OC, Crothers DM, Gralla J (1973) Improved estimation of secondary structure of ribonucleic acids. Nat New Biol 246:40–41
Tjalsma H, Bolhuis A, Jongbloed JHD, Bron S, van Dijl JM (2000) Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome. Microbiol Mol Biol Rev 64:515–547
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Okuda, M., Sumitomo, N., Takimura, Y. et al. A new subtilisin family: nucleotide and deduced amino acid sequences of new high-molecular-mass alkaline proteases from Bacillus spp.. Extremophiles 8, 229–235 (2004). https://doi.org/10.1007/s00792-004-0381-8
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DOI: https://doi.org/10.1007/s00792-004-0381-8