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Thermostable alanine racemase. Its structural stability.

K Soda1, K Tanizawa

  • 1Institute for Chemical Research, Kyoto University, Japan.

Annals of the New York Academy of Sciences
|January 1, 1990
PubMed
Summary
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The gene for thermostable alanine racemase from Bacillus stearothermophilus was cloned and expressed. This enzyme shows structural stability and retains activity even after significant proteolysis, indicating robust domain structure.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Thermostable enzymes are valuable for industrial applications.
  • Alanine racemase plays a crucial role in amino acid metabolism.

Purpose of the Study:

  • To clone and express the gene for thermostable alanine racemase from Bacillus stearothermophilus.
  • To characterize the biochemical and structural properties of the purified enzyme.

Main Methods:

  • Gene cloning and expression in E. coli.
  • Enzyme purification and homogeneity assessment.
  • Circular dichroism (CD) spectroscopy for structural analysis.
  • Limited proteolysis to investigate domain structure.

Main Results:

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  • The gene was successfully cloned and expressed, yielding a purified enzyme subunit of 43,341 Da.
  • CD data indicated approximately 34% alpha-helix and 26% beta-structure.
  • Denaturation studies revealed a stable intermediate.
  • Limited proteolysis suggested a two-domain structure, with retained activity despite fragmentation.

Conclusions:

  • The thermostable alanine racemase subunit consists of two structurally distinct domains.
  • The enzyme exhibits remarkable conformational stability and functional resilience to proteolysis.