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

MARÍA T. CASTAÑEDA; OSAO ADACHI; CARLOS MIGNONE; ROQUE HOURS

Rosario

Congreso; XIV Congreso Argentino de Ciencia y Tecnología de Alimentos; 2013

Asociación Argentina de Tecnólogos Alimentarios

In order to obtain low L-Phe ingredients for food formulation, suitable for PKU patients, a partially purified PAL (~2.800 mU/ml) from Rhodosporidium toruloides IFO 0559 was used for the treatment of a model protein hydrolysate(acid casein peptone, Phe ~ 2%). The reaction mixture contained variable amounts of peptone dissolved in 0.1 M Tris-HCl buffer, and enzyme under saturating concentrations. t-Cinnamic acid in the samples, resulting of the bioconversion of L-Phe by PAL, was extracted using ethyl acetate and analyzed spectrophotometrically at λ=290 nm. The reaction conditions were optimized in terms of: substrate, enzyme concentrations, buffer pH and reaction temperature, using successive Doehlert designs. Then, the reaction kinetic of PAL usingpeptone was determined under optimized conditions. Finally, TLC was used to detect the presence of reaction products. Doehlert designs revealed that optimum reaction conditions were achieved using 77 g/l of peptone and 140 mU/ml of PAL, while the optimum temperature was found to be 42°C and buffer pH = 10.5. Under these conditions, no further increment in OD290 was found after 6 h of treatment. The presence of t-cinnamic acid was evidenced in samples treated with PAL by TLC. These results demonstrate that PAL from R. toruloides IFO 0559 is capable of reducing the content of L-Phe in a hydrolyzed protein used as a model substrate.