Thermostable alanine racemase from Bacillus stearothermophilus: molecular cloning of the gene, enzyme purification, and characterization.
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 degrees C), the enzyme has a specific activity of 1800 units/mg (Vmax, D- to L-alanine). Resolution and reconstitution experiments together with the absorption spectrum of the enzyme clearly indicate that alanine racemase of B. stearothermophilus is a pyridoxal 5'-phosphate enzyme.
D000411:Alanine Racemase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000591:Amino Acid Isomerases / Q000235:genetics*; D000596:Amino Acids / Q000032:analysis; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004355:Drug Stability; D005796:Genes; D005798:Genes, Bacterial; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D006358:Hot Temperature; D007106:Immune Sera; D005779:Immunodiffusion; D010957:Plasmids; D013053:Spectrophotometry
D000596:Amino Acids; D007106:Immune Sera; D004262:DNA Restriction Enzymes; D000591:Amino Acid Isomerases; D000411:Alanine Racemase