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 frommicroorganisms isolated from soil in Iran. Isolation and screening of L-proline degradative enzymes from soilsamples was carried out. The isolate was characterized by biochemical markers and 16S rRNA geneanalysis. The target ProDH was purified and the effects of pH and temperature on the activity and stabilitywere tested. Among the 150 isolates recovered from 30 soil samples, only one was identified asPseudomonas putida displayed the highest enzyme activity toward L-proline (2200U/l). The enzyme wasidentified as a ProDH and had Km value of 35 mM for L-proline. The molecular mass of the purified ProDHwas about 40 kDa, and determined to be a monomeric protein. The N-terminal amino acid sequences of thesubunit of P. putida enzyme were determined to be MLTSSLTRIIGKSGE. ProDH exhibited high activity attemperature range of 25 to 35ºC and the highest activity was achieved at 30ºC. It was almost stable at temperatures between 25-30ºC 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. Thespecificity of P. putida enzyme toward L-proline is advantageous for the application to the L-prolineanalysis.