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Nucleotide and deduced amino acid sequences of a subtilisin-like serine protease from a deep-sea bacterium, Alkalimonas collagenimarina AC40T

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The acpI gene encoding an alkaline protease (AcpI) from a deep-sea bacterium, Alkalimonas collagenimarina AC40T, was shotgun-cloned and sequenced. It had a 1,617-bp open reading frame encoding a protein of 538 amino acids. Based on analysis of the deduced amino acid sequence, AcpI is a subtilisin-like serine protease belonging to subtilase family A. It consists of a prepropeptide, a catalytic domain, and a prepeptidase C-terminal domain like other serine proteases from the genera Pseudomonas, Shewanella, Alteromonas, and Xanthomonas. Heterologous expression of the acpI gene in Escherichia coli cells yielded a 28-kDa recombinant AcpI (rAcpI), suggesting that both the prepropeptide and prepeptidase C-terminal domains were cleaved off to give the mature form. Analysis of N-terminal and C-terminal amino acid sequences of purified rAcpI showed that the mature enzyme would be composed of 273 amino acids. The optimal pH and temperature for the caseinolytic activity of the purified rAcpI were 9.0–9.5 and 45°C in 100 mM glycine–NaOH buffer. Calcium ions slightly enhanced the enzyme activity and stability. The enzyme favorably hydrolyzed gelatin, collagen, and casein. AcpI from A. collagenimarina AC40T was also purified from culture broth, and its molecular mass was around 28 kDa, indicating that the cleavage manner of the enzyme is similar to that in E. coli cells.

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  1. Bendtsen JD, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340:783–795

  2. Chung S, Kim H, Shin Y, Jin G, Lee H (2001) Molecular characterization of gene encoding an extracellular alkaline protease in Vibrio metschnikovii. Biotechnol Lett 23:1175–1182

  3. Ducret A, Van Oostveen I, Eng JK, Yates JR 3rd, Aebersold R (1998) High throughput protein characterization by automated reverse-phase chromatography/electrospray tandem mass spectrometry. Protein Sci 7:706–719

  4. Fu X, Inouye M, Shinde U (2000) Folding pathway mediated by an intramolecular chaperone. The inhibitory and chaperone functions of the subtilisin propeptide are not obligatorily linked. J Biol Chem 275:16871–16878

  5. Hatada Y, Hidaka Y, Nogi Y, Uchimura K, Katayama K, Li Z, Akita M, Ohta Y, Goda S, Ito H, Matsui H, Ito S, Horikoshi K (2004) Hyper-production of an isomalto-dextranase of an Arthrobacter sp. by a proteases-deficient Bacillus subtilis: sequencing, properties, and crystallization of the recombinant enzyme. Appl Microbiol Biotechnol 65:583–592

  6. Khare SD, Krco CJ, Griffiths MM, Luthra HS, David CS (1995) Oral administration of an immunodominant human collagen peptide modulates collagen-induced arthritis. J Immunol 155:3653–3659

  7. Ko JH, Jang WH, Kim EK, Lee HB, Park KD, Chung JH, Yoo OJ (1996) Enhancement of thermostability and catalytic efficiency of AprP, an alkaline protease from Pseudomonas sp., by the introduction of a disulfide bond. Biochem Biophys Res Commun 221:631–635

  8. Kurata A, Miyazaki M, Kobayashi T, Nogi Y, Horikoshi K (2007) Alkalimonas collagenimarina sp. nov., a psychrotolerant, obligate alkaliphile isolated from deep-sea sediment. Int J Syst Evol Microbiol 57:1549–1553

  9. Kwon YT, Kim JO, Moon SY, Lee HL, Rho HM (1994) Extracellular alkaline proteases from alkalophilic Vibrio metschnikovii strain RH530. Biotechnol Lett 16:413–418

  10. Kwon YT, Kim JO, Moon SY, Yoo YD, Rho HM (1995) Cloning and characterization of the gene encoding an extracellular alkaline serine protease from Vibrio metschnikovii strain RH530. Gene 152:59–63

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

  12. Liu YN, Tang JL, Clarke BR, Dow JM, Daniels MJ (1990) A multipurpose broad host range cloning vector and its use to characterise an extracellular protease gene of Xanthomonas campestris pathovar campestris. Mol Gen Genet 220:433–440

  13. Minakuchi Y, Takeshita F, Kosaka N, Sasaki H, Yamamoto Y, Kouno M, Honma K, Nagahara S, Hanai K, Sano A, Kato T, Terada M, Ochiya T (2004) Atelocollagen-mediated synthetic small interfering RNA delivery for effective gene silencing in vitro and in vivo. Nucleic Acids Res 32:e109

  14. Miyamoto K, Nukui E, Itoh H, Sato T, Kobayashi T, Imada C, Watanabe E, Inamori Y, Tsujibo H (2002a) Molecular analysis of the gene encoding a novel chitin-binding protease from Alteromonas sp. strain O-7 and its role in the chitinolytic system. J Bacteriol 184:1865–1872

  15. Miyamoto K, Tsujibo H, Nukui E, Itoh H, Kaidzu Y, Inamori Y (2002b) Isolation and characterization of the genes encoding two metalloproteases (MprI and MprII) from a marine bacterium, Alteromonas sp. strain O-7. Biosci Biotechnol Biochem 66:416–421

  16. Myers LK, Rosloniec EF, Seyer JM, Stuart JM, Kang AH (1993) A synthetic peptide analogue of a determinant of type II collagen prevents the onset of collagen-induced arthritis. J Immunol 150:4652–4658

  17. Neidhart DJ, Petsko GA (1988) The refined crystal structure of subtilisin Carlsberg at 2.5 Å resolution. Protein Eng 2:271–276

  18. 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

  19. Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68:850–858

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

  21. Tsujibo H, Miyamoto K, Tanaka K, Kawai M, Tainaka K, Imada C, Okami Y, Inamori Y (1993) Cloning and sequence of an alkaline serine protease-encoding gene from the marine bacterium Alteromonas sp. strain O-7. Gene 136:247–251

  22. Tsujibo H, Miyamoto K, Tanaka K, Kaidzu Y, Imada C, Okami Y, Inamori Y (1996) Cloning and sequence analysis of a protease-encoding gene from the marine bacterium Alteromonas sp. strain O-7. Biosci Biotechnol Biochem 60:1284–1288

  23. Wandersman C (1989) Secretion, processing and activation of bacterial extracellular proteases. Mol Microbiol 3:1825–1831

  24. Washburn MP, Wolters D, Yates JR 3rd (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol 19:242–247

  25. Yeats C, Bentley S, Bateman A (2003) New knowledge from old: in silico discovery of novel protein domains in Streptomyces coelicolor. BMC Microbiol 3:3

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Correspondence to Atsushi Kurata.

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Kurata, A., Uchimura, K., Shimamura, S. et al. Nucleotide and deduced amino acid sequences of a subtilisin-like serine protease from a deep-sea bacterium, Alkalimonas collagenimarina AC40T . Appl Microbiol Biotechnol 77, 311–319 (2007). https://doi.org/10.1007/s00253-007-1164-9

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  • Serine protease
  • Subtilisin
  • Subtilase family A
  • Deep sea
  • Alkalimonas collagenimarina