Purification and characterization of piperideine-6-carboxylate dehydrogenase from Pseudomonas putida

Abstract

NAD[superscript +]-dependent piperideine-6-carboxylate dehydrogenase (EC 1.2.1.31) producing bacteria were screened from soil and the isolate which produced the highest enzyme activity was identified as Pseudomonas putida. The optimum condition for the enzyme production was 15 hours of cultivation in 0.8% peptone medium, pH 8.0 supplemented with 0.6% L-lysine at 37 ºC. The enzyme was purified to homogeneity by 50-60% saturated ammonium sulfate, DEAE-Toyopearl, Butyl-Toyopearl and Hitrap Q column chromatographies with 18.7% yield and 152 purification fold. The enzyme had a molecular mass of about 301 kDa and consisted of 6 identical subunits. The enzyme showed high substrate specificity with piperideine-6-carboxylate. L-Pipecolic acid could act as a substrate. The NAD[superscript +] analog nicotinamide hypoxanthine dinucleotide gave 1.2 times higher activity than its natural coenzyme, NAD[superscript +]. The optimum pH was 8.7 and optimum temperature was 45 ºC. The enzyme was stable to a broad pH range of 6.0 to 12.0. No loss of the enzyme activity was observed upon incubation at 45 ºC for 3 hours. The enzyme retained 50% of the activity after incubation at the same temperature for 3 days. The enzyme activity was inactivated completely by CuSO4 and FeSO4 at a final concentration of 1 mM. The apparent K[subscript m] values for L-pipecolic acid and NAD[superscript +] were 1.25 and 0.18 mM, respectively.