Biochemical analysis of Centaurea depressa phenylalanine ammonia lyase (PAL) for biotechnological applications in phenylketonuria (PKU)

Context: Phenylketonuria (PKU) is the most common hereditary defect of phenylalanine hydroxylase (PAH) enzyme achieving the hydroxylation of phenylalanine (Phe). Phenylalanine ammonia lyase (PAL) converts Phe to a harmless metabolite, trans-cinnamic acid (TCA) in plants and PAL enzyme activity is fairly high in plants rich in flavonoids. Objective: The study aimed the biochemical analysis of PAL form Centaurea depressa BIEB. (Asteraceae) a flavonoid rich plant. This study may form the main frame of future research efforts for the development of a plant preparation aimed for oral intake in PKU patients in an attempt to enrich their diet by allowing them to ingest some food stuff containing Phe without being exposed to complications. Materials and methods: PAL was partially purified from the leaves of C. depressa. Enzyme activity was determined in comparison with that of other herbs that reportedly have a high PAL activity. Enzyme optimization was achieved and the PAL protein was detected by western blotting. Results: C. depressa PAL demonstrated high activity (34.9 +/- 0.6 U/mg protein). The enzyme was purified by 1.92-fold, which resulted in an activity of 53.30 +/- 0.2 U/mg protein. The high-performance liquid chromatography analyzes of the PAL activity both before and after purification were in agreement. Western blot of PAL exhibited a 70 kDa protein band. The optimum pH and temperature are pH 8.8 and 37 degrees C. The optimum activities under gastric and intestinal digestion conditions were observed at pH 4.0 and pH 8.0, respectively. Discussion and conclusion: PAL activity of C. depressa is high, and does not disappear under different environmental conditions. This enzyme could be used for the development of dietary foods and biotechnological products for patients with PKU

Biochemical Evaluation of Phenylalanine Ammonia Lyase from Endemic Plant Cyathobasis fruticulosa (Bunge) Aellen. for the Dietary Treatment of Phenylketonuria

Enzyme substitution therapy with the phenylalanine ammonia lyase (PAL) is a new approach to the treatment of patients with phenylketonuria (PKU). This enzyme is responsible for the conversion of phenylalanine to trans-cinnamic acid. We assessed the PAL enzyme of the endemic plant Cyathobasis fruticulosa (Bunge) Aellen. for its possible role in the dietary treatment of PKU. The enzyme was found to have a high activity of (64.9 +/- 0.1) U/mg, with the optimum pH, temperature and buffer (Tris HCl and L-phenylalanine) concentration levels of pH=8.8, 37 degrees C and 100 mM, respectively. Optimum enzyme activity was achieved at pH=4.0 and 7.5, corresponding to pH levels of gastric and intestinal juice, and NaCl concentration of 200 mM. The purification of the enzyme by 1.87-fold yielded an activity of 98.6 U/mg. PAL activities determined by HPLC analyses before and after purification were similar. Two protein bands, one at 70 and the other at 23 kDa, were determined by Western blot analysis of the enzyme. This enzyme is a potential candidate for serial production of dietary food and biotechnological products