Extraction of phenylalanine ammonia lyase from <i>Anabaena variabilis</i> isolates from Iran

Phenylketonuria is an autosomal genetic disorder characterized by a deficiency of phenylalanine hydroxylase, leading to the accumulation of phenylalanine in the blood instead of its metabolism and excretion. This accumulation can result in intellectual disabilities in infants. Phenylalanine ammonia lyase (PAL; EC 4.3.1.24) was originally identified from plants, and today its cyanobacterial type is considered as a candidate to help in the treatment of phenylketonuria. In this study, three isolates of the cyanobacterium Anabaena variabilis from the Karaj Azad University collection were cultured in BG-11 culture medium under appropriate light conditions, including 24-h artificial illumination. Following growth for 3–4 weeks, the cells were lysed via sonication, and protein was extracted from the lysate. The extract yielded the PAL enzyme, which catalyses the conversion of phenylalanine into cinnamic acid. The concentration of cinnamic acid was quantified at 285 nm with a spectrophotometer. Enzyme activity was assessed at three temperatures relevant to physiological and environmental conditions, 25°C, 37°C and 40°C, for 30 and 60 min. Additionally, since tyrosine serves as another substrate for PAL, yielding fumaric acid, its activity was measured at a wavelength of 270 nm from pH 2 to pH 12. Variable enzyme activities at different pH levels and temperatures over both time intervals showed the sensitivity of the enzyme to temperature and pH. To further characterize the enzyme, the molecular weight of PAL extracted from Iranian cyanobacteria was compared against published data for the SDS-PAGE method, confirming a molecular weight consistent with the literature of 60–70 kDa. HPLC showed that levels of cinnamic acid in the cyanobacterial extract were very low before adding phenylalanine and they were much higher after addition of phenylalanine.