Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Alkaliphilic Bacillus sp. strain KSM-LD1 contains a record number of subtilisin-like serine proteases genes


The presence of 11 genes encoding subtilisin-like serine proteases was demonstrated by cloning from the genome of alkaliphilic Bacillus sp. strain KSM-LD1. This strain exoproduces the oxidatively stable alkaline protease LD-1 (Saeki et al. Curr Microbiol, 47:337–340, 2003). Among the 11 genes, six genes encoding alkaline proteases (SA, SB, SC, SD, SE, and LD-1) were expressed in Bacillus hosts. However, the other five genes for subtilisin-like proteases (SF, SG, SH, SI, and SJ) were expressed in neither Bacillus hosts nor Escherichia coli. The deduced amino acid sequences of SA, SB, SC, SF, SG, SH, SI, and SJ showed similarity to those of other subtilisin-like proteases from Bacillus strains with only 38 to 86% identity. The deduced amino acid sequence of SD was completely identical to that of an oxidatively stable alkaline protease from Bacillus sp. strain SD521, and that of SE was almost identical to that of a high-molecular mass subtilisin from Bacillus sp. strain D-6 with 99.7% identity. There are four to nine subtilisin-like serine protease genes in the reported genomes of Bacillus strains. At least 11 genes for the enzymes present in the genome of Bacillus sp. strain KSM-LD1, and this is the greatest number identified to date.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3


  1. Betzel C, Klupsch S, Papendorf G, Hastrup S, Branner S, Wilson KS (1992) Crystal structure of the alkaline protease Savinase™ from Bacillus lentus at 1.4 Å resolution. J Mol Biol 223:427–445

  2. Chang S, Cohen SN (1979) High frequency transformation of Bacillus subtilis. Mol Gen Genet 168:111–115

  3. Egmond MR (1997) Application of proteases in detergents. In: van Ee JH, Misset O, Baas EJ (eds) Enzymes in detergency. Marcel Dekker, New York, pp 61–74

  4. Ferrari E, Henner DJ, Perego M, Hoch JA (1988) Transcription of Bacillus subtilis subtilisin and expression of subtilisin in sporulation mutants. J Bacteriol 170:289–295

  5. Fujiwara N, Masui A, Imanaka T (1993) Purification and properties of the highly thermostable alkaline protease from an alkaliphilic and thermophilic Bacillus sp. J Biotechnol 30:245–256

  6. Gupta R, Beg QK, Lorenz P (2002) Bacterial alkaline proteases: molecular approaches and industrial applications. Appl Microbiol Biotechnol 59:15–32

  7. Horikoshi K (1971) Production of alkaline enzymes by alkaliphilic microorganisms. Part I. Alkaline protease produced by Bacillus No. 221. Biosci Biotechnol Biochem 35:1407–1414

  8. Kobayashi T, Hakamada Y, Adachi S, Hitomi J, Yoshimatsu T, Koike K, Kawai S, Ito S (1995) Purification and properties of an alkaline protease from alkalophilic Bacillus sp. KSM-K16. Appl Microbiol Biotechnol 43:473–481

  9. Kobayashi T, Hakamada Y, Hitomi J, Koike K, Ito S (1996) Purification of alkaline proteases from a Bacillus strain and their possible relationship. Appl Microbiol Biotechnol 45:63–71

  10. Kobayashi T, Lu J, Li Z, Hung VS, Kurata A, Hatada Y, Takai K, Ito S, Horikoshi K (2007) Extremely high alkaline protease from a deep-subsurface bacterium, Alkaliphilus transvaalensis. Appl Microbiol Biotechnol 75(1):71–80

  11. Kunst F, Ogasawara N, Moszer I et al (1997) The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature 390:249–256

  12. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

  13. Lane DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. Wiley, New York, pp.115–175

  14. 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 alkaliphilic Bacillus isolate. Biochim Biophys Acta 1624:109–114

  15. Okuda M, Sumitomo N, Takimura Y, Ogawa A, Saeki K, Kawai S, Kobayashi T, Ito S (2004) A new subtilisin family: nucleotide and deduced amino acid sequences of new high-molecular-mass alkaline proteases from Bacillus spp. Extremophiles 8:229–235

  16. Park SS, Wong SL, Wang LF, Doi RH (1989) Bacillus subtilis subtilisin gene (aprE) is expressed from a sigma A promoter in vitro and in vivo. J Bacteriol 171:2657–2665

  17. Rey MW, Ramaiya P, Nelson BA, et al. (2004) Complete genome sequence of the industrial bacterium Bacillus licheniformis and comparisons with closely related Bacillus species. Genome Biol 5:R77

  18. 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

  19. 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

  20. 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

  21. Saito H, Miura K (1963) Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim Biophys Acta 72:619–629

  22. Saitou N, Nei M (1987) A neighbor-joining method: anew method for reconstructing phylogenetic trees. Mol Biol Evol 44:406–425

  23. Schmidt BF, Woodhouse L, Adams RM, Ward T, Mainzer SE, Lad PJ (1995) Alkalophilic Bacillus sp. strain LG12 has a series of serine protease genes. Appl Environ Microbiol 61:4490–4493

  24. Siezen RJ, Leunissen JAM (1997) Subtilases: the superfamily of subtilisin-like serine proteases. Protein Sci 6:501–523

  25. Spanka R, Fritze D (1993) Bacillus cohnii sp. nov., a new, obligately alkaliphilic, oval-spore-forming Bacillus species with ornithine and aspartic acid instead of diaminopimelic acid in the cell wall. Int J Syst Bacteriol 43:150–156

  26. Sumitomo N, Ozaki K, Hitomi J, Kawaminami S, Kobayashi T, Kawai S, Ito S (1995) Application of the upstream region of a Bacillus endoglucanase gene to high-level expression of foreign genes in Bacillus subtilis. Biosci Biotechnol Biochem 59:2172–2175

  27. 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

  28. 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

  29. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The Clustal_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882

  30. Tsai YC, Yamasaki M, Yamamoto-Suzuki Y, Tamura G (1983) A new elastase of an alkalophilic Bacillus. Biochem Int 7:577–583

  31. Tsuchida O, Yamagata Y, Ishizuka T, Ara T, Yamada J, Takeuchi M, Ichishima E (1986) An alkaline proteinase of an alkalophilic Bacillus sp. Curr Microbiol 14:7–12

  32. van der Laan JC, Gerritse G, Mulleners LJSM, Hoek RAC, Quax WJ (1991) Cloning, characterization, and multiple chromosomal integration of a Bacillus alkaline protease gene. Appl Environ Microbiol 57:901–909

  33. Wong SL, Price CW, Goldfarb DS, Doi RH (1984) The subtilisin E gene of Bacillus subtilis is transcribed from a sigma 37 promoter in vivo. Proc Natl Acad Sci U S A 81:1184–1188

Download references

Author information

Correspondence to Tohru Kobayashi.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Takimura, Y., Saito, K., Okuda, M. et al. Alkaliphilic Bacillus sp. strain KSM-LD1 contains a record number of subtilisin-like serine proteases genes. Appl Microbiol Biotechnol 76, 395–405 (2007). https://doi.org/10.1007/s00253-007-1022-9

Download citation


  • Serine protease
  • Subtilisin
  • Genome
  • Alkaliphile
  • Bacillus