title: Biochemical analysis of Centaurea depressa phenylalanine ammonia lyase (PAL) for biotechnological applications in phenylketonuria (PKU).
abstract: BACKGROUND
  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.
  
  
  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 °C. The optimum activities under gastric and intestinal digestion conditions were observed at pH 4.0 and pH 8.0, respectively.
  
  
  CONCLUSIONS
  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.
authors.affiliation: a Vocational High School of Health Services, Gazi University , Gölbaşı , Ankara , Turkey.
authors.forename: Selcen
authors.initials: S
authors.lastname: Babaoğlu Aydaş
authors.affiliation: b Faculty of Science, Department of Biology , Gazi University , Teknikokullar , Ankara , Turkey.
authors.forename: Seda
authors.initials: S
authors.lastname: Şirin
authors.affiliation: b Faculty of Science, Department of Biology , Gazi University , Teknikokullar , Ankara , Turkey.
authors.forename: Belma
authors.initials: B
authors.lastname: Aslim
mesh_terms: D000284:Administration, Oral; D031179:Centaurea / Q000737:chemistry* / Q000201:enzymology*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D010650:Phenylalanine Ammonia-Lyase / Q000008:administration & dosage / Q000378:metabolism*; D010661:Phenylketonurias / Q000188:drug therapy / Q000201:enzymology*; D010936:Plant Extracts / Q000302:isolation & purification / Q000378:metabolism* / Q000627:therapeutic use
chemical_list: D010936:Plant Extracts; D010650:Phenylalanine Ammonia-Lyase
keywords: Enzyme optimization; HPLC; PAL-specific activity; partial enzyme purification; western blotting