Purification and characterization of phenylalanine dehydrogenase from Bacillus lentus

Abstract

Bacillus lentus in a NAD[superscript+] dependent phenylalanine dehydrogenase (EC 1.4.1.20) producing bacteria. The optimum condition for the enzyme production was 18 hours of cultivation in 1% pepton medium, pH 7.0 supplemented with 0.4% L-phenylalanine at 37degrees Celsius. The enzyme was purified to homogeneity by DEAE-Toyopearl, Butyl-Toyopearl and Sephadex G-200 column chromatography with 18.4% yield and 166.3 purification fold. The enzyme had a molecular mass of about 360,00 Da as determined by Ferguson plots and consisted of 8 identical subunits. The enzyme showed high substrate specificity in the oxidative deamination on L-phenylalanine and the reductive amination on phenylpyruvate. The NAD[superscript +] analog 3-acetylpyridine- NAD[superscript +], gave 1.74 times higher activity than its natural coenzyme, NAD[superscript +]. The optimum pH for the the oxidative deamination and the reductive amination were 10.4 and 8.5, respectively and optimum temperature were 50degrees Celsius and 55degrees Celsius, respectively. The enzyme was stable over a broad pH range of 6.0 and 12.0. No loss of the enzyme activity was observed upon incubation at 50degrees Celsius for 4 hours. The enzyme retained 50% of the activity after incubation at the same temperature for 3 days. The enzyme activity was inactivated completely by AgNO[subscript 3] at a final concentration of 1 mM. D-Phenylalanine, D-methionine, D-leucine, D-tryptophan, hydrocinnamate and p-hydroxyphenylacetate inhibited the oxidative deamination of L-phenylalanine. The apparent K[subscript m] values for L-phenylalanine, NAD[superscript +], phenylpyruvate, NH[subscript 4]CI and NADH were 0.59, 0.55, 0.18, 300 and 0.09 mM, respectively. Initial-velocity and product inhibition studies showed that the reductive amination proceeded through a sequential ordered ternary-binary mechanism. NADH bound first to the enzyme. Followed by phenylpyruvate and then NH[subscript 4]CI, and the products were released in the order of L-phenylalanine and NAD[superscript +].