Reduction of l-phenylalanine in protein hydrolysates using l-phenylalanine ammonia-lyase from Rhodosporidium toruloides

Abstract

L-Phenylalanine ammonia-lyase (PAL, EC 4.3.1.25) from Rhodosporidium toruloides was utilized to remove L-phenylalanine (L-Phe) from different commercial protein hydrolysates. A casein acid hydrolysate (CAH, L-Phe ~2.28 %) was employed as a model substrate. t-Cinnamic acid resulting from deamination of L-Phe was extracted, analyzed at λ = 290 nm, and used for PAL activity determination. Optimum reaction conditions, optimized using successive Doehlert design, were 35 mg mL(-1) of CAH and 800 mU mL(-1) of PAL, while temperature and pH were 42 °C and 8.7, respectively. Reaction kinetics of PAL with CAH was determined under optimized conditions. Then, removal of L-Phe from CAH was tested. Results showed that more than 92 % of initial L-Phe was eliminated. Similar results were obtained with other protein hydrolysates. These findings demonstrate that PAL is a useful biocatalyst for L-Phe removal from protein hydrolysates, which can be evaluated as potential ingredients in foodstuffs for PKU patients.

Synthetic and Therapeutic Applications of Ammonia-lyases and Aminomutases

“…Enzymes with PAL activity have been widely studied for the treatment of PKU, with various methods employed to improve the biotherapeutic properties of a handful of proteins. Although some work exists on the treatment of foodstuffs by PAL to reduce dietary phenylalanine 304 and thus hyperphenylalaninemia, most research aims to use the enzymes within the body. This is because current treatments for PKU already encompass strict dietary restrictions which are difficult to follow and/or implement in patient groups.…”

Directed evolution of Anabaena variabilis phenylalanine ammonia-lyase (PAL) identifies mutants with enhanced activities

“…Daily subcutaneous injection of a purified and PEGylated recombinant PAL from Anabaena variabilis (PEG-rAvPAL; Palynziq ® , BioMarin Pharmaceutical Inc.) was approved by the US FDA in 2018 as an enzyme substitution therapy for PKU 13 . Concurrently, an orally administered engineered PAL-expressing probiotic Escherichia coli Nissle 1917 is currently under investigation by Synlogic Inc 14 .…”

Food Regime for Phenylketonuria: Presenting Complications and Possible Solutions

“…The estimates of prevalence rates in Asia varies from about one per fifteen thousand to one per one hundred and fifteen thousand and in USA the prevalence rate is one in 15,000. 19 , 38 , 39 , 41–43 Some countries like Iran and Turkey are not only known to have high prevalence ratio but along with that are known to display extreme proportions of consanguinity who are suffering from PKU. 44 , 45 Studies conducted between 1964–2017 report the highest PKU prevalence in Turkey (38.13), while the lowest was observed in Thailand (0.3) ( Figure 1B ).…”

“…Moreover, LNAAs compete with blood phenylalanine levels only in the blood-brain barrier, having no impact on the blood phenylalanine levels in other parts of the body. 10 , 19 Generally 20–30% of the protein from LNAAs is co-supplemented along with traditional formulations of diet.…”

“…10 The other sources of low phenylalanine diet are glycomacropeptides and large neutral amino acids (LNAA). 10,15,18,19 Despite this, its extensive production and usage is limited owing to (1) its tedious chemical processes and sophisticated purification techniques; (2) palatability of phenylalanine free diet; (3) nutritional deficiencies in commercial mixtures; and (4) expensive formulated end product. [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35] To this end, this review article discusses efforts laid by different research groups in developing palatable and affordable diet for PKU patients.…”