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. 1989 Sep;171(9):5232–5236. doi: 10.1128/jb.171.9.5232-5236.1989

Molecular cloning of the structural gene for alkaline elastase YaB, a new subtilisin produced by an alkalophilic Bacillus strain.

R Kaneko 1, N Koyama 1, Y C Tsai 1, R Y Juang 1, K Yoda 1, M Yamasaki 1
PMCID: PMC210351  PMID: 2670913

Abstract

Alkaline elastase YaB is an extracellular serine protease of the alkalophilic Bacillus strain YaB. We cloned the structural gene, ale, and determined the nucleotide sequence. The mature enzyme (268 amino acids) was preceded by a putative signal sequence and a prosequence (27 and 83 amino acids, respectively). The mature enzyme was 55% homologous to subtilisin BPN'. Almost all the positively charged residues are predicted to be on the surface of the molecule, which would facilitate binding to elastin. The P1 substrate site-related sequences differed between alkaline elastase YaB and subtilisin BPN'.

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Selected References

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  1. Carter P., Wells J. A. Dissecting the catalytic triad of a serine protease. Nature. 1988 Apr 7;332(6164):564–568. doi: 10.1038/332564a0. [DOI] [PubMed] [Google Scholar]
  2. Estell D. A., Graycar T. P., Miller J. V., Powers D. B., Wells J. A., Burnier J. P., Ng P. G. Probing steric and hydrophobic effects on enzyme-substrate interactions by protein engineering. Science. 1986 Aug 8;233(4764):659–663. doi: 10.1126/science.233.4764.659. [DOI] [PubMed] [Google Scholar]
  3. Gold L., Pribnow D., Schneider T., Shinedling S., Singer B. S., Stormo G. Translational initiation in prokaryotes. Annu Rev Microbiol. 1981;35:365–403. doi: 10.1146/annurev.mi.35.100181.002053. [DOI] [PubMed] [Google Scholar]
  4. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  5. Imanaka T., Oshihara W., Himeno T., Aiba S. Comparative studies on extracellular penicillinases of the same structural gene, penP, expressed in Bacillus licheniformis and Bacillus subtilis. J Gen Microbiol. 1983 Aug;129(8):2621–2628. doi: 10.1099/00221287-129-8-2621. [DOI] [PubMed] [Google Scholar]
  6. Inouye M., Halegoua S. Secretion and membrane localization of proteins in Escherichia coli. CRC Crit Rev Biochem. 1980;7(4):339–371. doi: 10.3109/10409238009105465. [DOI] [PubMed] [Google Scholar]
  7. Jacobs M., Eliasson M., Uhlén M., Flock J. I. Cloning, sequencing and expression of subtilisin Carlsberg from Bacillus licheniformis. Nucleic Acids Res. 1985 Dec 20;13(24):8913–8926. doi: 10.1093/nar/13.24.8913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kawamura F., Doi R. H. Construction of a Bacillus subtilis double mutant deficient in extracellular alkaline and neutral proteases. J Bacteriol. 1984 Oct;160(1):442–444. doi: 10.1128/jb.160.1.442-444.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kwon S. T., Terada I., Matsuzawa H., Ohta T. Nucleotide sequence of the gene for aqualysin I (a thermophilic alkaline serine protease) of Thermus aquaticus YT-1 and characteristics of the deduced primary structure of the enzyme. Eur J Biochem. 1988 May 2;173(3):491–497. doi: 10.1111/j.1432-1033.1988.tb14025.x. [DOI] [PubMed] [Google Scholar]
  10. Matsuzawa H., Tokugawa K., Hamaoki M., Mizoguchi M., Taguchi H., Terada I., Kwon S. T., Ohta T. Purification and characterization of aqualysin I (a thermophilic alkaline serine protease) produced by Thermus aquaticus YT-1. Eur J Biochem. 1988 Feb 1;171(3):441–447. doi: 10.1111/j.1432-1033.1988.tb13809.x. [DOI] [PubMed] [Google Scholar]
  11. Morihara K., Tsuzuki H. Elastolytic properties of various proteinases from microbial origin. Arch Biochem Biophys. 1967 Apr;120(1):68–78. doi: 10.1016/0003-9861(67)90599-1. [DOI] [PubMed] [Google Scholar]
  12. Power S. D., Adams R. M., Wells J. A. Secretion and autoproteolytic maturation of subtilisin. Proc Natl Acad Sci U S A. 1986 May;83(10):3096–3100. doi: 10.1073/pnas.83.10.3096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Shine J., Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. doi: 10.1073/pnas.71.4.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  16. Stahl M. L., Ferrari E. Replacement of the Bacillus subtilis subtilisin structural gene with an In vitro-derived deletion mutation. J Bacteriol. 1984 May;158(2):411–418. doi: 10.1128/jb.158.2.411-418.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Stone P. J., Franzblau C., Kagan H. M. Proteolysis of insoluble elastin. Methods Enzymol. 1982;82(Pt A):588–605. doi: 10.1016/0076-6879(82)82089-2. [DOI] [PubMed] [Google Scholar]
  18. Stormo G. D., Schneider T. D., Gold L. M. Characterization of translational initiation sites in E. coli. Nucleic Acids Res. 1982 May 11;10(9):2971–2996. doi: 10.1093/nar/10.9.2971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Tsai Y. C., Juang R. Y., Lin S. F., Chen S. W., Yamasaki M., Tamura G. Production and further characterization of an alkaline elastase produced by alkalophilic bacillus strain ya-B. Appl Environ Microbiol. 1988 Dec;54(12):3156–3161. doi: 10.1128/aem.54.12.3156-3161.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Tsai Y. C., Lin Y. T., Yang Y. B., Li Y. F., Yamasaki M., Tamura G. Specificity of alkaline elastase Bacillus on the oxidized insulin A- and B-chains. J Biochem. 1988 Sep;104(3):416–420. doi: 10.1093/oxfordjournals.jbchem.a122482. [DOI] [PubMed] [Google Scholar]
  21. Tsai Y. C., Yamasaki M., Tamura G. Substrate specificity of a new alkaline elastase from an alkalophilic bacillus. Biochem Int. 1984 Feb;8(2):283–288. [PubMed] [Google Scholar]
  22. Tsai Y. C., Yamasaki M., Yamamoto-Suzuki Y., Tamura G. A new alkaline elastase of an alkalophilic bacillus. Biochem Int. 1983 Nov;7(5):577–583. [PubMed] [Google Scholar]
  23. Vlasuk G. P., Inouye S., Ito H., Itakura K., Inouye M. Effects of the complete removal of basic amino acid residues from the signal peptide on secretion of lipoprotein in Escherichia coli. J Biol Chem. 1983 Jun 10;258(11):7141–7148. [PubMed] [Google Scholar]
  24. Watson M. E. Compilation of published signal sequences. Nucleic Acids Res. 1984 Jul 11;12(13):5145–5164. doi: 10.1093/nar/12.13.5145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wells J. A., Ferrari E., Henner D. J., Estell D. A., Chen E. Y. Cloning, sequencing, and secretion of Bacillus amyloliquefaciens subtilisin in Bacillus subtilis. Nucleic Acids Res. 1983 Nov 25;11(22):7911–7925. doi: 10.1093/nar/11.22.7911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Wong S. L., Doi R. H. Determination of the signal peptidase cleavage site in the preprosubtilisin of Bacillus subtilis. J Biol Chem. 1986 Aug 5;261(22):10176–10181. [PubMed] [Google Scholar]
  27. Wright C. S., Alden R. A., Kraut J. Structure of subtilisin BPN' at 2.5 angström resolution. Nature. 1969 Jan 18;221(5177):235–242. doi: 10.1038/221235a0. [DOI] [PubMed] [Google Scholar]
  28. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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