Determination of enzyme activity, optimization and characterization of the biotechnologically important phenylalanine ammonia lyase from some plants under stress

Phenylketonuria disease (PKU) is an autosomal recessive disorder. This genetic disorder is characterized by the accumulation of high amounts of phenylalanine amino acid in the blood and in the body fluids because of low levels of phenylalanine hydroxylase (PAH) enzyme. Today, the studies are focused on phenylalanine ammonia lyase (PAL) enzyme that is thought to be a surrogate for PKU. In the study, PAL enzyme and specific activity were screened in 13 endemic or disseminated plant samples that showed a distribution under salt-stress conditions, which have been obtained in the district of Beypazarı near Ankara and, based on these results, the analyses were continued to be performed with the plants with higher PAL specific activity. Highest PAL specific activity values were found in Cyathobasis fruticulosa (64.9 U/mg), Salsola nitraria (55.3 U/mg), Salsola grandis (49.0 U/mg) and Salvia halophila (47.8 U/mg). For PAL enzyme belonging to C. fruticulosa and S. nitraria that showed high PAL specific activity, optimization and characterization were done. For PAL enzyme, optimum pH was 8.8, optimum temperature was 37oC and optimum buffer (Tris-HCl) concentration was 100 mM (p<0.05). It was seen that the enzyme had the minimum activity loss at pH 4.0 in the artificial gastric fluid and at pH 7.5 in the artificial intestinal fluid (p<0.05). Optimum NaCl concentration of the PAL enzyme obtained from the plants was determined to be 200 mM (p<0.05). PAL enzyme obtained from both plants showed an activity loss by nearly 50 % at 2nd day at room temperature, at 5th day at +4oC and at 12th month at -20oC (p<0.05). For PAL enzyme obtained from C. fruticulosa, S. nitraria, S. grandis and S. halophila, the conversion to L-phenylalanine trans-cinnamic acid was determined as both amount and percentage using high pressure liquid chromatography (HPLC). Following the partial purification of PAL enzyme using Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western Blot methods, its molecular weight was found to be approximately 70 kDa. Furthermore, HPLC analysis performed after the partial purification revealed that PAL enzyme did not lose its activity. Consequently, in this thesis study, PAL enzyme obtained from C. fruticulosa, S. nitraria, S. grandis and S. halophila became prominent because its activity was higher, it did not lose its activity under different environmental conditions, it was more stable, it functioned at 37oC, which is the normal body temperature, and it maintained its activity in the artificial gastric fluid and intestinal fluid. This enzyme is thought to have the potential for becoming prominent with its product-intended use especially in food and pharmaceutical industry and to be likely to be used for the production of biotechnological products.