Thermostable d-hydantoinase from thermophilic Bacillus stearothermophilus SD-1: characteristics of purified enzyme

Abstract

One thousand thermophiles isolated from soils were screened for hydantoinase and its thermostability. One thermophilic bacterium that showed the highest thermostability and activity of hydantoinase was identified to be Bacillus stearothermophilus SD-1 according to morphological and physiological characteristics. The hydantoinase of B. stearothermophilus SD-1 was purified to homogeneity via ammonium sulfate fractionation, anion-exchange chromatography, heat treatment, hydrophobic-interaction chromatography, and preparative gel electrophoresis. The relative molecular mass of the hydantoinase was determined to be 126 kDa by gel-filtration chromatography, and a value of 54 kDa was obtained as a molecular mass of the subunit on analytical sodiumdodecylsulfate/polyacrylamide gel electrophoresis. The hydantoinase was strictly d-specific and metal-dependent. The optimal pH and temperature were about 8.0 and 65°C respectively, and the half-life of the d-hydantoinase was estimated to be 30 min at 80°C, indicating the most thermostable enzyme so far.

This is a preview of subscription content, access via your institution.

References

  1. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  2. Kathleen PB, Eugene GS (1980) Preparative polyacrylamide gel electrophoresis: removal of polyacrylate from proteins. Anal Biochem 107:182–186

    Google Scholar 

  3. Kim D-M and Kim H-S (1993) Enzymatic synthesis of d-p-hydroxyphenylglycine from dl-p-hydroxyphenylhydantoin in the presence of organic solvent. Enzyme Microb Technol 15:530–534

    Google Scholar 

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

    PubMed  Google Scholar 

  5. Morin A, Hummel W, Schutte H, Kula MR (1986) Characterization of hydantoinase from Pseudomonas fluorescens DSM 84. Biotechnol Appl Biochem 8:564–574

    Google Scholar 

  6. Morin A, Leblanc D, Paleczek A, Hummel W, Kula M-R (1990) Comparison of seven microbial d-hydantoinases. J Biotechnol 16:37–48

    Google Scholar 

  7. Morin A, Touzel J-P, Lafond A, Leblanc D (1991) Hydantoinase from anaerobic microorganisms. Appl Microbiol Biotechnol 35:536–540

    Google Scholar 

  8. Olivieri R, Fascetti E, Angelini L, Degen L (1981) Microbial transformation of racemic hydantoins to d-amino acids. Biotechnol Bioeng 23:2173–2183

    Google Scholar 

  9. Runser SM, Meyer PC (1993) Purification and biochemical characterization of the hydantoin hydrolyzing enzyme from Agrobacterium species a hydantoinase with no 5,6-dihydropyrimidine amidohydrolase activity. Eur J Biochem 213:1315–1324

    Google Scholar 

  10. Runser SM, Ohleyer E (1990) Properties of the hydantoinase from Agrobacterium sp. IP I-671. Biotechnol Lett 12:259–264

    Google Scholar 

  11. Runser S, Chinski N, Ohleyer E (1990) d-p-Hydroxyphenylglycine production from dl-5-p-hydroxyphenylhydantoin by Agrobacterium sp. Appl Microbiol Biotechnol 33:382–388

    Google Scholar 

  12. Sneath PHA (1986) Endospore-forming gram-positive rods and cocci. In: Sneath et al (eds) Bergey's manual of systematic bacteriology, vol 2. Williams & Wilkins, pp 1105–1139

  13. Suzuki T, Igarashi K, Hase K, Tuzimura K (1973) Optical rotatory dispersion and circular dichroism of amino acid hydantoin. Agric Biol Chem 37:411–416

    Google Scholar 

  14. Syldatk C, Cotoras D, Dombach G, Grob C, Kallwab H, Wagner F (1987) Substrate- and stereospecificity, induction and metallo-dependence of a microbial hydantoinase. Biotechnol Lett 9:25–30

    Google Scholar 

  15. Syldatk C, Laufer A, Muller R, Hoke H (1990) Production of optically pure d- and l-α-amino acids by bioconversion of D,l-5-monosubstituted hydantoin derivatives. In: Fiechter A (ed) Advances in biochemical engineering/biotechnology, vol 41. Springer, Berlin Heidelberg New York, p 29

    Google Scholar 

  16. Takahashi S, Kii Y, Kumagai H, Yamada H (1978) Purification, crystallization and properties of hydantoinase from Pseudomonas striata. J Ferment Technol 56:492–498

    Google Scholar 

  17. Takahashi S, Ohashi T, Kii Y, Kumagai H, Yamada H (1979) Microbial transformation of hydantoins to N-carbamyl-d-amino acids. J Ferment Technol 57:328–332

    Google Scholar 

  18. Tamaoka J, Komagata K (1984) Determination of DNA base composition by reversed phase high performance liquid chromatography. FEMS Microbiol Lett 25:125–128

    Article  CAS  Google Scholar 

  19. Yamada H, Takahashi S, Kii Y, Kumagai H (1978) Distribution of hydantoin hydrolyzing activity in microorganisms. J Ferment Technol 56:484–491

    Google Scholar 

  20. Yamada H, Shimizu S, Shimada H, Tani Y, Takahashi S, Ohashi T (1980) Production of d-phenylglycine-related amino acids by immobilized microbial cells. Biochimie 62:395–399

    Google Scholar 

  21. Yokozeki K, Nakamori S, Eguchi C, Yamada K, Mitsugi K (1987a) Screening of microorganisms producing d-p-hydroxyphenylglycine from dl-5-(p-hydroxyphenyl) hydantoin. Agric Biol Chem 51:355–362

    Google Scholar 

  22. Yokozeki K, Nakamori S, Yamanaka S, Eguchi C, Mitsugi K, Yoshinaga F (1987b) Optimal conditions for the enzymatic production of d-amino acids from the corresponding 5-substituted hydantoins. Agric Biol Chem 51:715–719

    Google Scholar 

Download references

Author information

Affiliations

Authors

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Lee, S.G., Lee, D.C., Hong, S.P. et al. Thermostable d-hydantoinase from thermophilic Bacillus stearothermophilus SD-1: characteristics of purified enzyme. Appl Microbiol Biotechnol 43, 270–276 (1995). https://doi.org/10.1007/BF00172823

Download citation

Keywords

  • Enzyme
  • Heat Treatment
  • Fractionation
  • Molecular Mass
  • Ammonium Sulfate