Thermolabile Alanine Racemase from a Psychrotroph, Pseudomonas fluorescens: Purification and Properties

A psychrotrophic bacterium that produces a thermolabile alanine racemase was isolated from raw milk, and identified as Pseudomonas fluorescens TM5-2. The enzyme was purified to homogeneity from the cell extract, and characterized to be compared with enzymes from mesophiles (Bacillus subtilis and Salmonella typhimurium) and a thermophile (Bacillus stearothermophilus). The enzyme has a molecular weight of about 76, 000 and consists of two subunits identical in molecular weight (38, 000). The enzyme contains two mol of pyridoxal 5′-phosphate per mol as a coenzyme. The amino acid composition was different from those of other alanine racemases in content of valine. The amino acid sequence of the amino terminal region (from ¹Met to ²⁵Gly) had 21—33% homology with those of other alanine racemases. Kinetic parameters of the enzyme were similar to those of other alanine racemases. The enzyme is extremely labile over 30°C, and shows the high catalytic activity even at 0°C; it is thermolabile and psychrotrophic.