Isolation, purification and characterization of proline dehydrogenase from a Pseudomonas putida POS-F84 isolate

The purpose of this study was to isolate and characterize Proline Dehydrogenase [ProDH] enzyme from Iranian soil microorganisms. Isolation and screening of L-proline degradative enzymes from soil samples was carried out. The isolate was characterized by biochemical markers and 16S rRNA gene analysis. The target ProDH was purified and the effects of pH and temperature on the activity and stability were tested. Among the 150 isolates recovered from 30 soil samples, only one was characterized as Pseudomonas putida displayed the highest enzyme activity toward L-proline [2200U/l]. The enzyme of interest was identified as a ProDH and had K[m] value of 35 mM for L-proline. The molecular mass of the purified ProDH was about 40 kDa, and determined to be a monomeric protein. The N-terminal amino acid sequences of the subunit of P. putida enzyme were determined to be MLTSSLTRIIGKSGE. ProDH exhibited high activity at temperature range of 25 to 35°C and the highest activity was achieved at 30°C. It was almost stable at temperatures between 25-30°C for 2 h. The optimum pH of ProDH activity was determined in pH=8.5 and it was stable in pH range of 8.0-9.0 up to 24 h. The enzyme was purified with a yield of 8.5% and a purification factor of 37.7. Briefly, a ProDH flavonzyme was purified and characterized from a P. putida bacterium. The specificity of P. putida enzyme toward L-proline may be advantageous for the application to the L-proline analysis

Preliminary report of NAD[+]-dependent amino acid dehydrogenase producing bacteria isolated from soil

Amino acid dehydrogenases [L-amino acid: oxidoreductase deaminating; EC 1.4.1.X] are members of the wider superfamily of oxidoreductases that catalyze the reversible oxidative deamination of an amino acid to its keto acid and ammonia with the concomitant reduction of either NAD[+], NADP[+] or FAD. These enzymes have been received much attention as biocatalysts for use in biosensors or diagnostic kits to screen amino acid metabolism disorders such as phenylketonuria [PKU], maple syrup urine disease [MSUD], homocystinuria [HCY] and hyperprolinemia. This study was aimed to isolation and screening of novel amino acid dehydrogenases from soil bacteria. The enzyme producing bacteria were selected among L-methionine and L-phenylalanine utilizers isolated from soil by thin layer chromatography, activity staining and confirmed by enzyme assay. Bacterial strains were identified by phenotypic and biochemical characteristics. The steady-state kinetic studies of enzymes were also performed. In total of 230 tested strains, four of them were recognized as amino acid dehydrogenase producers that belong to species of Pseudomonas, Citrobacter and Proteus. They exhibited the desired NAD[+]-dependent dehydrogenase activities toward L-isoleucine, L-methionine, L-cysteine, L-serine and L-glutamine in oxidative deamination reaction. The specific activity of L-isoleucine dehydrogenase, L-methionine dehydrogenase and L-glutamine dehydrogenase for oxidative deamination of L-isoleucine, L-methionine and L-glutamine were 1.59, 1.2 and 0.73 U/mg, respectively. The Kcat /Km [s-1.mM -1] values in these strains were as follows: L-isoleucine, 113.6, L-methionine, 62.05 and L-glutamine, 95.83. This is the first report of occurrence a specific isoleucine dehydrogenase, glutamine dehydrogenase and methionine dehydrogenase in bacteria