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Purification and properties of an alkaline protease from alkalophilic Bacillus sp. KSM-K16

  • Biochemical Engineering
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Abstract

Alkaline protease (EC 3.4.21.14) activity, suitable for use in detergents, was detected in the alkaline culture medium of Bacillus sp. KSM-K16, which was originally isolated from soil. The enzyme, designated M protease, was purified to homogeneity from the culture broth by column chromatographies. The N-terminal amino acid sequence was Ala-Gln-Ser-Val-Pro-Trp-Gly-Ile-Ser-Arg-Val-Gln-Ala-Pro-Ala-Ala-His-Asn-Arg-Gly-Leu-Thr-Gly. The molecular mass of the protease was 28 kDa, and its isoelectric point was close to pH 10.6. Maximum activity toward casein was observed at 55°C and at pH 12.3 in 50 mM phosphate/NaOH buffer. The activity was inhibited by phenylmethylsulfonyl flouride and chymostatin. The enzyme was very stable in long-term incubation with liquid detergents at 40°C. The enzyme cleaved the oxidized insulin B chain initially at Leu15-Tyr16 and efficiently at ten more sites. Among various oligopeptidyl p-nitro-anilides (pNA) tested, N-succinyl-Ala-Ala-Pro-Phe-pNA was efficiently hydrolyzed by M protease. M protease was precipitated in (NH4)2SO4-saturated acetate buffer (pH 5.0) as plank-like cyrstals.

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References

  • Anisworth SJ (1994) Soap and detergents. Chem Eng News 72:34–59

    Google Scholar 

  • Claus D, Berkeley RCW (1986) Genus Bacillus. In: Sneath PHA, Mair NS, Sharpe ME, Holt JG (eds) Bergey's manual of systematic bacteriology, vol 2. William & Wilkins, Baltimore, pp 1105–1139

    Google Scholar 

  • Edelhoch H (1967) Spectoscopic determination of tryptophan and tyrosine in proteins. Biochemistry 6:1948–1954

    Google Scholar 

  • Gordon RE, Haynes WC, Pang CH (1973) The genus Bacillus. United States Department of Agriculture, Washington DC, USA

    Google Scholar 

  • Gray WR (1972) Endo group analysis using dansyl chloride. Methods Enzymol 25:121–138

    CAS  PubMed  Google Scholar 

  • Gray CJ, Barker SA, Dhariwal MS, Sullivan JM (1985) Assay of the high-alkaline proteinase from alkalophilic Bacillus PB92 using a chromogenic tripeptide substrate. Biotechnol Bioeng 27:1717–1720

    Google Scholar 

  • Hakamada Y, Kobayashi T, Hitomi J, Kawai S, Ito S (1994) Molecular colning and nucleotide sequence of the gene for an alkaline protease from the alkalophilic Bacillus sp. KSM-K16. J Ferment Bioeng 78:105–108

    Google Scholar 

  • Horikoshi K (1971) Production of alkaline enzymes by alkalophilic microorganisms. Part I. Alkaline protease produced by Bacillus no. 221. Agric Biol Chem 35:1407–1414

    Google Scholar 

  • Horikoshi K, Akiba T (1982) Enzymes of alkalophilic bacteria. In: Alkalophilic microorganisms. Japan Scientific Societies Press, Tokyo, pp 93–101

    Google Scholar 

  • Ito S, Ohta Y, Shimooka M, Takaiwa M, Ozaki K, Adachi S, Okamoto K (1991) Enhanced production of extracellular enzymes by mutants of Bacillus that have acquired resistance to vancomycin and ristocetin. Agric Biol Chem 55:2387–2391

    Google Scholar 

  • 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

    Google Scholar 

  • Kaneko R, Koyama N, Tsai YC, Yuang RY, Yoda K, Yamasaki M (1989) Molecular cloning of the strucutral gene for alkaline elastase YaB, a new subtilisin produced by an alkaliphilic Bacillus strain. J Bacteriol 171:5232–5236

    Google Scholar 

  • 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

    Google Scholar 

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

    PubMed  Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall JR (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 242:265–275

    Google Scholar 

  • Lyublinskaya LA, Belyaev SV, Strongin AYA, Matyash LF, Levin ED, Stepanov VM (1974) A new chromogenic substrate for subtilisin. Anal Biochem 62:371–376

    Google Scholar 

  • Maase FWJL, Tilburg R van (1983) The benefit of detergent enzymes under changing washing conditions. J Am Oil Chem Soc 60:1672–1675

    Google Scholar 

  • Markland FS, Smith EL (1967) Subtilisin BPN'. VII. Isolation of cyanogenbromide peptides and the complete amino acid sequence. J Biol Chem 242:5198–5211

    Google Scholar 

  • Markland FS, Smith EL (1971) Subtilisins: primary structure, chemical and physical properties. In: Boyer PD (ed) The enzymes, 3rd edn, vol 3, Academic Press, New York, pp 586–592

    Google Scholar 

  • Morihara K, Tsuzuki H (1969) Comparison of the specificities of various serine proteinases from microorganisms. Arch Biochem Biophys 129:620–634

    Google Scholar 

  • Nijenhuis B (1977) Alkaline protease produced by a Bacillus. US Patent no 4, 002, 572

  • Ottesen M, Svendsen I (1971) The subtilisin. Methods Enzymol 19:199–215

    Google Scholar 

  • Priest FG (1977) Extracellular enzyme synthesis in the genus Bacillus. Bacteriol Rev 41:711–753

    Google Scholar 

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

    Google Scholar 

  • Shimogaki H, Takeuchi K, Nishino T, Ohdera M, Kudo T, Ohba K, Iwama M, Irie M (1991) Purification and properties of a novel surface-active agent- and alkaline-resistant protease from Bacillus sp. Y. Agric Biol Chem 55:2251–2258

    Google Scholar 

  • Smith EL, Delange RJ, Evans WH, Landon EM, Markland FS (1968) Subtilisin Carlsberg V, The complete sequence; comparison with subtilisin BPN'; evolutionary relationships. J Biol Chem 243:2184–2191

    Google Scholar 

  • Stepanov VM, Strongin AYA, Izotova LS, Abramov ZT, Lyublinskaya LA, Ermakova LM, Baratova LA, Belyanova LP (1977) Intracellular serine protease from Bacillus subtilis. Structural comparison with extracellular serine proteases-subtilisins. Biochem Biophys Res Commun 77:298–305

    Google Scholar 

  • Taber HW, Sherman F (1964) Spectophotometric analyzers for disk electrophoresis; studies of yeast cytochrome c. Ann NY Acad Sci 121:600–615

    Google Scholar 

  • Takami H, Akiba T, Horikoshi K (1990) Characterization of an alkaline protease form Bacillus sp. no. AH-101. Appl Microbiol Biotechnol 33:519–523

    Google Scholar 

  • 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

    Google Scholar 

  • 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 alkalophilic Bacillus sp. 221. Biosci Biotechnol Biochem 56:1455–1460

    Google Scholar 

  • Tsai YC, Lin YT, Yang YB, Li YF, Yamasaki M, Tamura G (1988) Specificity of alkaline elastase Bacillus on the oxidized insulin A-and B-chains. J Biochem (Tokyo) 104:416–420

    Google Scholar 

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

    Google Scholar 

  • Yamagata Y, Ichishima E (1989) A new alkaline proteinase with pI 2.8 from alkalophilic Bacillus sp. Curr Microbiol 19:259–264

    Google Scholar 

  • Yang JT, Wu CSC, Martinez HM (1986) Calculation of protein conformation from circular dichroism. Methods Enzymol 130:208–269

    Google Scholar 

  • Yoshida K, Hidaka H, Miyado S, Shibata U, Saito K, Yamada Y (1977) Purification and some properties of Bacillus sphaericus protease. Agric Biol Chem 41:745–754

    Google Scholar 

  • Yoshimatsu T, Ozaki K, Shikata S, Ohta Y, Koike K, Kawai S, Ito S (1990) Purification and characterization of alkaline endo-1,4-β-glucanases from alkalophilic Bacillus sp. KSM-635. J Gen Microbiol 136:1973–1979

    Google Scholar 

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Authors and Affiliations

  1. Tochigi Research Laboratories of Kao Corporation, 2606 Akabane, Ichikai, Haga, 321-34, Tochigi, Japan

    T. Kobayashi, Y. Hakamada, S. Adachi, J. Hitomi, T. Yoshimatsu, K. Koike, S. Kawai & S. Ito

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  1. T. Kobayashi
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  2. Y. Hakamada
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  3. S. Adachi
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  4. J. Hitomi
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  5. T. Yoshimatsu
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  6. K. Koike
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  7. S. Kawai
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  8. S. Ito
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Kobayashi, T., Hakamada, Y., Adachi, S. et al. Purification and properties of an alkaline protease from alkalophilic Bacillus sp. KSM-K16. Appl Microbiol Biotechnol 43, 473–481 (1995). https://doi.org/10.1007/BF00218452

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  • DOI: https://doi.org/10.1007/BF00218452

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