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.
Buy single article
Instant access to the full article PDF.
Tax calculation will be finalised during checkout.
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
Tax calculation will be finalised during checkout.
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
Kathleen PB, Eugene GS (1980) Preparative polyacrylamide gel electrophoresis: removal of polyacrylate from proteins. Anal Biochem 107:182–186
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
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T. Nature 227:680–685
Morin A, Hummel W, Schutte H, Kula MR (1986) Characterization of hydantoinase from Pseudomonas fluorescens DSM 84. Biotechnol Appl Biochem 8:564–574
Morin A, Leblanc D, Paleczek A, Hummel W, Kula M-R (1990) Comparison of seven microbial d-hydantoinases. J Biotechnol 16:37–48
Morin A, Touzel J-P, Lafond A, Leblanc D (1991) Hydantoinase from anaerobic microorganisms. Appl Microbiol Biotechnol 35:536–540
Olivieri R, Fascetti E, Angelini L, Degen L (1981) Microbial transformation of racemic hydantoins to d-amino acids. Biotechnol Bioeng 23:2173–2183
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
Runser SM, Ohleyer E (1990) Properties of the hydantoinase from Agrobacterium sp. IP I-671. Biotechnol Lett 12:259–264
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
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
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
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
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
Takahashi S, Kii Y, Kumagai H, Yamada H (1978) Purification, crystallization and properties of hydantoinase from Pseudomonas striata. J Ferment Technol 56:492–498
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
Tamaoka J, Komagata K (1984) Determination of DNA base composition by reversed phase high performance liquid chromatography. FEMS Microbiol Lett 25:125–128
Yamada H, Takahashi S, Kii Y, Kumagai H (1978) Distribution of hydantoin hydrolyzing activity in microorganisms. J Ferment Technol 56:484–491
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
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
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
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
- Heat Treatment
- Molecular Mass
- Ammonium Sulfate