Thermostable Alanine Racemase from Bacillus stearothermophilus: Molecular Cloning of the Gene, Enzyme Purification, and Characterization

Kenji Inagaki, Katsuyuki Tanizawa, Bernard Badet, Christopher T. Walsh, Hidehiko Tanaka, Kenji Soda

Research output: Contribution to journalArticle

116 Citations (Scopus)

Abstract

The alanine racemase (EC 5.1.1.1) gene of a thermophilic bacterium, Bacillus stearothermophilus, was cloned and expressed in Escherichia coli C600 with vector plasmid pICR301, which was constructed from pBR322 and the L-alanine dehydrogenase gene derived from B. stearothermophilus. A coupled assay method with L-alanine dehydrogenase and tetrazolium salts was used to detect visually the alanine racemase activity in the clones. Alanine racemase overproduced in a clone carrying the plasmid pICR4, 12 kilobases of DNA, was purified from cell extracts about 340-fold to homogeneity by five steps including heat treatment. The overproduced enzyme was confirmed to originate from B. stearothermophilus by an immunochemical cross-reaction with the enzyme of B. stearothermophilus. The purified enzyme has a molecular weight of about 78 000 and consists of two identical subunits of Mr of 39 000. At the optimum temperature (50 °C), the enzyme has a specific activity of 1800 units/mg (Vmax, D-to L-alanine). Resolution and reconsititution experiments together with the absorption spectrum of the enzyme clearly indicate that alanine racemase of B. stearothermophilus is a pyridoxal 5'-phosphate enzyme.

Original languageEnglish
Pages (from-to)3268-3274
Number of pages7
JournalBiochemistry
Volume25
Issue number11
DOIs
Publication statusPublished - Jun 1986
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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