Adenosine Triphosphate and Pyrophosphate Dependent Phenylalanine Racemase of Bacillus brevis NAGANO*

Abstract

A phenylalanine racemase [EC 5. 1. 1 group] was found to be present in the cell-free extract of Bacillus brevis NAGANO, which produces gramicidin S. This enzyme was partially purified by ultracentrifugation, ammonium sulfate precipitation, and calcium phosphate gel adsorption and elution. The enzyme showed an absolute requirement of adenosine triphosphate, inorganic pyrophosphate, and magnesium or manganese ion for its activity. Thiol compounds, such as dithiothreitol and 2-mercaptoethanol stimulated the enzyme activity. The pH optimum of the reaction was between 8.2 and 8.5. The rate of the formation of L-phenylalanine from D-phenylalanine catalyzed by this enzyme slower than that of D-phenylalanine from L-phenylalanine. When the reaction reached an apparent equilibrium, L-phenylalanine and D-phenylalanine were found to be in the ratio of 3 to 7. This racemase activity appeared abruptly at the middle of the logarithmic phase of growth, and increased towards the end of the logarithmic phase of growth. When the cell entered the stationary phase, the enzyme activity disappeared rapidly. The similar change of the rate of gramicidin S production and of the activities of gramicidin S synthesizing system and L- and D-phenylalanine-activating enzyme was also observed.