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

Extremely high alkaline protease from a deep-subsurface bacterium, Alkaliphilus transvaalensis


A new high-alkaline protease (ALTP) was purified to homogeneity from a culture of the strictly anaerobic and extremely alkaliphilic Alkaliphilus transvaalensis. The molecular mass was 30 kDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The enzyme showed the maximal caseinolytic activity higher than pH 12.6 in KCl–NaOH buffer at 40°C. Hydrolysis of the oxidized insulin B-chain followed by mass spectrometric analysis of the cleaved products revealed that as many as 24 of the total 29 peptide bonds are hydrolyzed in a block-cutting manner, suggesting that ALTP has a widespread proteolytic functions. Calcium ion had no effect on the activity and stability of ALTP, unlike known subtilisins. The deduced amino acid sequence of the enzyme comprised 279 amino acids plus 97 prepropeptide amino acids. The amino acid sequence of mature ALTP was confirmed by capillary liquid chromatography coupled to tandem mass spectrometry, which was the 93% coverage of the deduced amino acid sequence. The mature enzyme showed moderate homology to subtilisin LD1 from the alkaliphilic Bacillus sp. strain KSM-LD1 with 64% identity, and both enzymes formed a new subcluster at an intermediate position among true subtilisins and high-alkaline proteases in a phylogenetic tree of subtilase family A. ALTP is the first high-alkaline protease reported from a strict anaerobe in this family.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6


  1. Abraham LD, Chow DT, Breuil C (1995) Characterization of the cleavage specificity of a subtilisin-like serine protease from Ophiostoma piceae by liquid chromatography/mass spectrometry and tandem MS. FEBS Lett 374:208–210

  2. Aggeler R, Coons J, Taylor SW, Ghosh SS, Garcia JJ, Capaldi RA, Marusich MF (2002) A functionally active human F1F0 ATPase can be purified by immunocapture from heart tissue and fibroblast cell lines. Subunit structure and activity studies. J Biol Chem 277:33906–33912

  3. Boone DR, Liu Y, Zhao ZJ, Balkwill DL, Drake GR, Stevens TO, Aldrich HC (1995) Bacillus infernus sp. nov., an Fe(III)- and Mn(IV)-reducing anaerobe from the deep terrestrial subsurface. Int J Syst Bacteriol 45:441–448

  4. Cleveland D (1983) Peptide mapping in one dimension by limited proteolysis of sodium dodecyl sulfate-solubilized proteins. Methods Enzymol 96:222–229

  5. 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

  6. 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 alkaliphilic Bacillus sp. KSM-K16. J Ferment Bioeng 78:105–108

  7. Hoppe HP (1991) Microbial extracellular enzyme activity: a new key parameter in aquatic ecology. In: RG Chrost (ed) Microbial enzymes in aquatic environment. Springer, Berlin Heidelberg New York, pp 61–80

  8. 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

  9. Horikoshi K (1999) Alkaliphiles: some applications of their products for biotechnology. Microbiol Mol Biol Rev 63:735–750

  10. Jang HJ, Kim BC, Pyun YR, Kim YS (2002a) A novel subtilisin-like serine protease from Thermoanaerobacter yonseiensis KB-1: its cloning, expression, and biochemical properties. Extremophiles 6:233–243

  11. Jang HJ, Lee CH, Lee W, Kim YS (2002b) Two flexible loops in subtilisin-like thermophilic protease, thermicin, from Thermoanaerobacter yonseiensis. J Biochem Mol Biol 35:498–507

  12. Johansen JT, Ottesen M, Svendsen I, Wybrandt G (1968) The degradation of the B-chain of oxidized insulin by two subtilisins and their succinylated and N-carbamylated derivatives. C R Trav Lab Carlsberg 36:365–384

  13. Kieft TL, Fredrickson JK, Onstott TC, Gorby YA, Kostandarithes HM, Bailey TJ, Kennedy DW, Li SW, Plymale AE, Spadoni CM, Gray MS (1999) Dissimilatory reduction of Fe(III) and other electron acceptors by a Thermus isolate. Appl Environ Microbiol 65:1214–1221

  14. 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

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

  16. Lu J, Nakajima-Kambe T, Shigeno T, Ohbo A, Nomura N, Nakahara T (1999) Biodegradation of dibenzothiophene and 4,6-dimethyldibenzothiophene by Sphingomonas paucimobilis strain TZS-7. J Biosci Bioeng 88:293–299

  17. Morihara K, Oka T, Tsuzuki H (1969) Comparison of alpha-chymotrypsin and subtilisin BPN′: size and specificity of the active site. Biochem Biophys Res Commun 35:210–214

  18. Nakanishi T, Yamamoto T (1974) Action and specificity of a streptomyces alkaliphilic proteinase. Biosci Biotechnol Biochem 38:2391–2397

  19. Narinx E, Davail S, Feller G, Gerday C (1992) Nucleotide and derived amino acid sequence of the subtilisin from the Antarctic psychrotroph Bacillus TA39. Biochim Biophys Acta 1131:111–113

  20. Nielsen H, Engelbrecht J, Brunak S, von Heijne G (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10:1–6

  21. 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

  22. 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

  23. 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

  24. Schechter I, Berger A (1967) On the sites of the active site in protease. I. Papain. Biochem Biophys Res Commun 27:157–162

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

  26. 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

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

  28. Takai K, Horikoshi K (2000) Thermosipho japonicus sp. nov., an extremely thermophilic bacterium isolated from a deep-sea hydrothermal vent in Japan. Extremophiles 4:9–17

  29. Takai K, Moser DP, Onstott TC, Spoelstra N, Pfiffner SM, Dohnalkova A, Fredrickson JK (2001) Alkaliphilus transvaalensis gen. nov., sp. nov., an extremely alkaliphilic bacterium isolated from a deep South African gold mine. Int J Syst Evol Microbiol 51:1245–1256

  30. Takami H, Akiba T, Horikoshi K (1989) Production of extremely thermostable alkaline protease from Bacillus sp. no. AH-101. Appl Microbiol Biotechnol 30:120–124

  31. Takami H, Akiba T, Horikoshi K (1992a) Substrate specificity of thermostable alkaline protease from Bacillus sp. no. AH-101. Biosci Biotechnol Biochem 56:333–334

  32. Takami H, Kobayashi T, Kobayashi M, Yamamoto M, Nakamura S, Aono R, Horikoshi K (1992b) Molecular cloning, nucleotide sequence, and expression of the structural gene for alkaline serine protease from alkaliphilic Bacillus sp. 221. Biosci Biotechnol Biochem 56:1455–1460

  33. Washburn MP, Wolters D, Yates JR (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol 19:242–247

  34. Wati MR, Thanabalu T, Porter AG (1997) Gene from tropical Bacillus sphaericus encoding a protease closely related to subtilisins from Antarctic bacilli. Biochim Biophys Acta 1352:56–62

  35. Whitman WB, Coleman DC, Wiebe WJ (1998) Prokaryotes: the unseen majority. Proc Natl Acad Sci USA 95:6578–6583

  36. Yamagata Y, Isshiki K, Ichishima E (1995) Subtilisin Sendai from alkalophilic Bacillus sp.: molecular and enzymatic properties of the enzyme and molecular cloning and characterization of the gene, aprS. Enzyme Microb Technol 17:653–663

Download references

Author information

Correspondence to Tohru Kobayashi.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kobayashi, T., Lu, J., Li, Z. et al. Extremely high alkaline protease from a deep-subsurface bacterium, Alkaliphilus transvaalensis . Appl Microbiol Biotechnol 75, 71–80 (2007). https://doi.org/10.1007/s00253-006-0800-0

Download citation


  • Serine protease
  • Subtilisin
  • Subtilase family A
  • Deep subsurface
  • Alkaliphilus transvaalensis