Reduction of L-Phe content in protein hydrolysates using fenilalanine ammonia-lyase

CASTAÑEDA, M.T.; ADACHI, O.; MIGNONE, C.F.; HOURS, R.A.

Rosario

Congreso; XIV Congreso Argentino de Ciencia y Tecnología de Alimentos (XIV CYTAL); 2013

Asociación Argentina de Tecnólogos Alimentarios (AATA)

L-Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) is an enzyme that catalyzes the biotransformation of L-phenylalanine (L-Phe) in t-cinnamic acid and ammonia. Similarly, PAL is capable of deaminate L-tyrosine to p-coumaric acid. A potential application of PAL includes its use in the treatment of phenylketonuria (PKU), an autosomal recessive disease characterized by the inability to metabolize Phe. If not treated, it leads to problems at the level of the central nervous system, causing mental retardation of the patient. Nowadays, the only available treatment is the reduction of Phe in the patient's diet. The present work aims to use PAL to reduce L-Phe content in a protein hydrolyzate with potential application as an ingredient in food preparation for PKU patients. For this purpose, a partially purified PAL (~ 2,800 mU/ml) from Rhodosporidium toruloides IFO 0559 was used for the treatment of a model substrate (acid casein peptone, Britania, AN/TN ~ 61%, Phe ~ 2% and Tyr ~ 1.7%). The reaction mixture contained variable amounts of peptone dissolved in 0.1 M Tris-HCl buffer, pH = 8.5, and enzyme under saturating concentrations. The reaction mixtures were incubated at 30oC, during 2 h. Then, t-cinnamic acid in the samples was extracted using ethyl acetate and analyzed spectrophotometrically at OD290. Additionally, the reaction conditions were optimized in terms of: substrate (0-110 mg/ml) and enzyme concentrations (0 to 140 mU/ml), buffer pH (8.5 to 10.5) and reaction temperature (30 to 70ºC), using successive Doehlert hexagonal designs. Then, the reaction kinetics of PAL using peptone was determined under optimized conditions. Finally, L-Phe bioconversion was evidenced by qualitative detection of t-cinnamic acid by TLC in silica gel plates with chloroform-methanol-acetic acid (90:10:1) as solvent and FeCl3 as developing solution. Results obtained from the Doehlert designs revealed that optimum reaction conditions were achieved using 77 g/l of peptone and 140 mU/ml of PAL (R2 = 99.9%), while the optimum temperature was found to be 42°C and buffer pH =10.5 (R2 = 96.4). Under these conditions, an increase up to ~ 300% of the PAL activity was achieved in relation to the initial experiment. No further increment in OD290 was found after 6 h of treatment. Finally, the presence of t-cinnamic acid was evidenced in samples treated with PAL by TLC. In addition, p-coumaric acid, resulting from the bioconversion of L-tyrosine by PAL, was also detected. These results demonstrate that PAL from R. toruloides IFO 0559 is capable to reduce the content of L-Phe in a hydrolyzed protein used as a model substrate, as evidenced by the formation of t-cinnamic acid after enzyme treatment. The resulting material can be considered as a potential ingredient for the formulation of food for PKU patients. A method to increment the substrate specificity of PAL in order to reduce the parallel reaction with L-tyrosine requires further studies.