Study On Isolation And Identification Of A Phenylalanine Dehydrogenase-producing Bacterium, Prodution Conditions And Characteristics Of Its Enzyme

Phenylalanine dehydrogenase (EC 1.4.1.20) is employed for the determination of Phenylketonuria (PKU) in the clinic. At present, the enzymatic tool to determine PKU has been reported overseas, but there is not the enzymatic product in our country.In this study, a strain of bacteria capable of producing Phenylalanine dehydrogenase was isolated and was identified as Achromobacter sp. The condition for the enzymatic fermentation, purification and characteristics of the enzyme were carried out in this work. The results were showed as follows:1. Isolation and identification of a bacterium producing Phenylalanine dehydrogenase activity: Five cultures of producing PheDH were isolated with medium using phenylalanine as the main carbon source. The strain X5-3 was selected according to its activity of phenylalanine dehydrogenase. This new isolate was identified as Achromobacter sp. based on its morphological, physiological characteristics and G+Cmol%, as well as 16S rDNA sequence and phylogenetic analysis,2. Optimal conditions for Phenylalanine dehydrogenase production: Based on the studies for the conditions of PheDH production, the optimal fermentation condition was established. The composition of the medium was: glutamine sodium 18.0 g, glucose 18.0 g, L-Phenylalanine 5.0 g, K₂HPO₄·3H₂O 2.0 g, KH₂PO₄ 2.0 g, MgSO₄·7H₂O 0.1 g, NaCl 2.0 g, yeast extract 0.1 g, H₂O 1000ml and adjusted to pH 7.5. Each inoculated 100 ml flask contaning 25 ml medium was incubated on a shaker of 150 rpm at 30°C for 48 h. The PheDH activity peaked at 0.25 U/ml.3. The purification and characterization of phenylalanine dehydrogenase: The specific activity of the crude, cell-free extract of PheDH was increased 268-fold by a series of purification steps including ammonium sulfate precipitation, DEAE-Sepharoese column, Toyopearl HW-65C hydrophobic chromatography and G-100 column. The purified enzyme was three bands as judged by SDS-PAGE. It...