Reconstitution of fragmentary form of thermostable alanine racemase

Hirohide Toyama, Young Hee Lim, Young Hee Lim, Tohru Yoshimura, Kenji Soda

Research output: Contribution to journalArticle

Abstract

The fragmentary form of alanine racemase from Bacillus stearothermophilus is composed of two sets of two different polypeptides corresponding to the two domains of the subunit of wild-type enzyme. It was denatured with 6 M guanidium hydrochloride to be separated into pieces, and renatured by dilution with about 50% recovery of the activity. The two kinds of polypeptides (i.e., large and a small fragments) were isolated by gel filtration in the presence of 4 M guanidium hydrochloride. The CD spectra obtained by summation of the spectra of the refolded fragments were closely similar to that of the native fragmentary enzyme. The lysine residue to which PLP is bound in the wild-type enzyme occurs in the large peptide of the fragmentary enzyme containing the amino terminus of the wild-type enzyme. The visible spectrum of the large peptide refolded, however, indicated that PLP was not bound to it. The large peptide alone had no significant activity, but it was activated by incubation with the small peptide. Accordingly, co-existene of both peptide fragments is necessary for folding of a complex of the two kinds of peptide to form an active structure.

Original languageEnglish
Pages (from-to)1118-1120
Number of pages3
JournalBioscience, Biotechnology and Biochemistry
Volume59
Issue number6
DOIs
Publication statusPublished - 1995
Externally publishedYes

Fingerprint

Alanine Racemase
alanine
peptides
Peptides
enzymes
Enzymes
polypeptides
Geobacillus stearothermophilus
Peptide Fragments
Bacilli
lysine
Dilution
Lysine
Gel Chromatography
gels
Gels
Recovery

ASJC Scopus subject areas

  • Biotechnology
  • Analytical Chemistry
  • Organic Chemistry
  • Applied Microbiology and Biotechnology
  • Biochemistry
  • Molecular Biology
  • Food Science
  • Biochemistry, Genetics and Molecular Biology(all)
  • Chemistry (miscellaneous)

Cite this

Reconstitution of fragmentary form of thermostable alanine racemase. / Toyama, Hirohide; Lim, Young Hee; Lim, Young Hee; Yoshimura, Tohru; Soda, Kenji.

In: Bioscience, Biotechnology and Biochemistry, Vol. 59, No. 6, 1995, p. 1118-1120.

Research output: Contribution to journalArticle

Toyama, Hirohide ; Lim, Young Hee ; Lim, Young Hee ; Yoshimura, Tohru ; Soda, Kenji. / Reconstitution of fragmentary form of thermostable alanine racemase. In: Bioscience, Biotechnology and Biochemistry. 1995 ; Vol. 59, No. 6. pp. 1118-1120.
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