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

ABSTRACT

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: