Phylogenic Study of Proline Dehydrogenase Producing Pseudomonas putida Bacterium and Bioinformatics Analysis of Isolated Enzyme

Proline dehydrogenase falvoenzyme (ProDH; EC 1. 5. 99. 8) plays an important role in the biosensors and diagnostic kits for the diagnosis of proline related metabolic disorders. The purpose of this research was to isolate and characterize proline metabolizing enzymes producing microorganisms from Iranian soil samples. Isolation and screening of L-proline degradative enzymes from soil samples was carried out in three steps employing enrichment culture technique، thin layer chromatography (TLC) and enzyme activity assay. The isolate was characterized by biochemical reactions، 16S rRNA gene analysis and 16S-23S rRNA Internal Transcribed Spacer (ITS) sequencing. Kinetic studies were performed and 3-D structure of the enzyme of interest was predicted through bioinformatics methods. Among the 150 soil isolates recovered from 30 samples، only one strain displayed the highest enzyme activity toward L-proline (10 U/ml) than others. The Km and Vmax values were calculated as 0. 12 mM and 0. 11 mM/min. The strain was characterized as a new biotype of Pseudomonas putida using phylogenetic analysis of 16s rRNA and 16S-23S rRNA ITS. The predicted 3-D structure of target enzyme also confirmed the similarity of isolated ProDH to the family of P. putida enzymes. This is the first report concerning the ProDH production by a P. putida bacterium isolated from soil sample.