Screening Endophytic Fungi for Production of Phenylalanine Ammonia Lyase, for use as a Therapeutic Intervention to Treat Phenylketonurics

Abstract

Phenylketonuria is a rare inborn autosomal recessive disorder in which an accumulation of an amino acid, phenylalanine and receded levels of a hepato-cellular enzyme phenylalanine hydroxylase (PAH) which is accountable for regulation of phenylalanine levels in the plasma. Generally seen in neonates, the outcome of enhanced level of phenylalanine can lead to the symptoms such as brain dysfunction, eczema, hyperactivity, abnormal electroencephalogram, microcephaly and autism. The sustainable interventions such as enzyme phenylalanine ammonia lyase (PAL) that can cure phenylketonuria are in high demand. PAL catalyzes the deamination of L-phenylalanine into trans-cinnamic acid and trace amount of ammonia. PAL is widely apportioned in plants, cyanobacteria, algae and fungi. Various biological entities have been studied for the production of PAL. However, the research is limited for the production of PAL using endophytic fungi in the context of therapeutic intervention. Endophytic fungi inhabit within the living tissues of host plant in a symbiotic association without causing any overt disease symptoms to the host plant. The association between endophytic fungi and host plant enables the endophytic fungi to produce of bioactive compounds and secondary metabolites having therapeutic applications. In this study, we screened 43 endophytic fungi out of which isolate #2CCBD and #61MKLTh02 exhibited highest PAL activity using agar well diffusion (AWD) assay. Further on direct nesslerization assay isolate #2CCBD exhibited highest ammonia production closely followed by #61MKLTh02. On protein estimation isolate #2CCBD exhibited 1.93±0.02 and 1.87±0.01 mg/mL protein content using Bradford and Folin-Lowry assay whereas isolate #61MKLTh02 exhibited 1.18±0.06 and 1.17±0.05 mg/mL protein content respectively. The UV-Vis spectroscopy-based enzyme activity of crude protein of isolate #2CCBD was 0.85 U/min/mL whereas #61MKLTh02 exhibited activity of 0.42 U/min/mL of crude protein. The isolate #2CCBD also exhibited better results in DPPH activity with IC50485.213±1.204 μg/mL, total phenolic content of 594.73 ± 1.91 μg/mL and total flavonoid content 877.07 ± 20.94 μg/mL. Thus, the isolate #2CCBD was tentatively identified as a Penicillium sp. using morpho-taxonomic tools