Alanine racemase from the green alga Chlamydomonas reinhardtii.
Chlamydomonas reinhardtii, a unicellular green microalga, could grow to a stationary phase having optical density of 2.0-2.5 at 750 nm in Tris-acetate-phosphate (TAP) medium containing 0.1% D-alanine. D-alanine has no inhibitory effect on growth and induced alanine racemase activity 130-fold more than without D-alanine in the green alga. Although C. reinhardtii cultured in the TAP medium showed alanine racemase activity, the content of free D-alanine was only 0.14%. The enzyme was partially purified by ammonium sulfate fractionation followed by three kinds of liquid chromatography using DEAE Toyopearl, Phenyl Sepharose, and TSK G3000 SWXL columns. The specific activity for L-alanine of the partially purified alanine racemase was 3.8 micromol/min/mg. The molecular weight of the enzyme was determined to be approximately 72,000 by gel filtration. The enzyme showed a maximum activity at 45 degrees C and pH 8.4 and requires pyridoxal 5'-phosphate as a coenzyme.
Nishimura
K
K
Department of Applied Chemistry, Junior College, Nihon University, Chiba, Japan. nisimura@chem.cst.nihon-u.ac.jp
Tomoda
Y
Y
Nakamoto
Y
Y
Kawada
T
T
Ishii
Y
Y
Nagata
Y
Y
D000409:Alanine / Q000737:chemistry / Q000378:metabolism; D000411:Alanine Racemase / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D020418:Algal Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000201:enzymology* / Q000254:growth & development; D003067:Coenzymes / Q000737:chemistry* / Q000378:metabolism; D015800:Protozoan Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
D020418:Algal Proteins; D003067:Coenzymes; D015800:Protozoan Proteins; C009122:pyridoxal phosphonate; D011732:Pyridoxal Phosphate; D000411:Alanine Racemase; D000409:Alanine