Continuous preparation of low-phenylalanine formulations by treatment of edible protein with immobilized phenylalanine ammonia lyase

Abstract

Dietary restriction of phenylalanine is the predominant treatment for phenylketonuria. However, the currently available medicated formulations depend on an arduous and unsustainable chemical extraction procedure, which inflates the product’s price. Here, we describe a method that uses immobilized phenylalanine ammonia lyase (PAL) and offers a sustainable solution to generate a low-phenylalanine protein supplement for phenylketonuria patients. This enzyme deaminates phenylalanine to trans-cinnamic acid. Here, we optimized the high cell density cultivation of a recombinant Escherichia coli strain harboring Anabaena variabilis PAL (AvPAL) and achieved protein titers greater than 400 mg/l. This AvPAL protein was immobilized on multiwall carbon nanotubes, and the bioconjugate retained > 50 % activity after ten successive cycles with significantly improved thermostability. Batch deamination with the bioconjugate showed an 80 % reduction in the total phenylalanine content of soya protein hydrolysate. In a continuous packed bed column, > 98 % of the phenylalanine content was depleted at a feed flow rate of 0.1 ml/min, resulting in a space-time yield of cinnamate of 6.65 g/l/h. In a 72-h continuous run, the bioconjugate lost only 2 % of its activity with an operational inactivation constant of 3.9 × 10−5 min−1. This simple covalent immobilization method offers the potential to generate low-phenylalanine diet for phenylketonuria patients.