This thesis addresses the study and development of a novel biotechnological method for the production of alternative ingredients or formulas for PKU patients. This methodology consists in the use of a biocatalyst, particularly, the enzyme L-Phenylalanine ammonia lyase (PAL, EC 4.3.1.25) from Rhodosporidium toruloides NBRC 0559. PAL catalyzes the non oxidative deamination of L-Phenylalanine into trans-cinnamic acid. This enzyme is applied in the current thesis for the reduction or deprivation of L-phenylalanine in commercially available protein hydrolysates.
The methodology proposed consists of a first stage based on biocatalyst obtention. First, in order to enhance PAL activity, the components of the culture medium were optimized by using several experimental designs. After cultivation, the biocatalyst was obtained by two different strategies: a) catalytic cells containing PAL were recovered from culture medium and then permeabilized with the aim of using them as a microreactor; b) PAL extract obtained from cell disruption was purified using a standard protocol.
In a second stage, the catalytic cells were immobilized by inclusion into polymers, using calcium alginate. This biocatalyst was compared with purified PAL immobilized by retention into cellulose membranes. In both cases the alternatives were characterized to determine its feasibility of use given its potential industrial application.
Finally, in a third step, the selected biocatalyst was employed in the treatment of commercially available protein hydrolysates, as model of its application. As an alternative approach, partially purified PAL was evaluated for the intended propose. Under each treatment, we evaluated the need for pretreatment of substrates as well as the optimum operating conditions to achieve the highest reduction in the shortest time. From the obtained results, discussed in this thesis, it was demonstrated the feasibility of the proposed process for the reduction of L-Phe content in protein hydrolysates. These studies are the preliminary knowledge for the generation of substitutes or formulas for patients with PKU, using a biotechnological method. Future studies require a technological approach in a pilot scale to transfer this knowledge to local industry.