Phenylketonuria (PKU) is an autosomal recessive disorder, which is characterized by severe mental retardation, microcephaly and seizures. The symptoms of this disease can be prevented if detected soon after birth. Therefore, blood Phenylalanine (Phe) measurement is essential for the early diagnosis, treatment and dietary monitoring of PKU patients. The goal of this research was to introduce a r...

Abstract NADH-dependent phenylalanine amine dehydrogenase (F-AmDH) engineered from (PheDH) catalyzes the synthesis of aromatic chiral amines prochiral ketone substrates. However, its low coenzyme affinity and catalytic efficiency limit industrial application. Here, we developed a chimeric dehydrogenase, cFLF-AmDH, based on relative independence structure at domain level, combined with substrate...

In this paper, methods of sample preparation for potentiometric measurement of phenylalanine are presented. Basing on the spectrophotometric measurements of phenylalanine, the concentrations of reagents of the enzymatic reaction (10 mM L-Phe, 0,4 mM NAD, 2U L-PheDH) were determined. Then, the absorption spectrum of the reaction product, NADH, was monitored (maximum peak at 340 nm). The results ...

Aqueous two-phase systems (ATPS) have  emerged as a powerful extraction method for the downstream processing of bio-molecules. The aim of this work was to investigate the possibility of utilizing ATPS for the separation of recombinant Bacillus sphaericus phenylalanine dehydrogenase (PheDH). Polyethylene glycol (PEG) and ammonium sulfate systems were selected for our experi...

Two non-canonical amino acids (ncAAs) with bio-orthogonal reactive groups, namely, p-azido-L-phenylalanine (p-AzF) and p-propargyloxy-L-phenylalanine (p-PaF), were genetically inserted into an aldo-keto reductase (AKR) alcohol dehydrogenase (ADH), respectively, at...

Cloning and expression of the L-phenylalanine dehydrogenase gene, from B. sphaericus in E. coli were done. The gene was cloned in the vector pET16b and transformed into E. coli BL21 (DE3). The functional form of the L-phenylalanine dehydrogenase enzyme was purified by affinity purification techniques, taking advantage of the ability of this enzyme to bind to the nucleotide site affinity dye, Re...

Quantitative determination of plasma phenylalanine (Phe) is essential for the diagnosis of phenylketonuria (PKU) and the control of dietary therapy of PKU patients. We have adapted a spectrophotometric method (Clin Chim Acta 1991;201:95-8) based on phenylalanine dehydrogenase (EC 1.4.1.-) for rapid and accurate determinations of Phe with the Cobas Fara II centrifugal analyzer. The method is bas...

L-Phenylalanine dehydrogenase catalyzes the NAD(+)-dependent, reversible, oxidative deamination of L-phenylalanine to form ammonia, phenyl pyruvate, and NADH. The enzyme has been purified to homogeneity from Rhodococcus sp. M4, and a partial amino acid sequence was obtained. A cosmid library of Rhodococcus sp. M4 genomic DNA was prepared and used to isolate a 2.5-kilobase PstI fragment that con...

lignin is one of the major characteristics of plant secondary cell wall that provides structural rigidity for the cells and tissues and hydrophobicity to tracheary elements. internode tissues of aeluropus littoralis as a halophyte grass were sampled at different developmental stages (from the first to the fifth internodes ) and under different nacl concentrations. the influences of nacl and int...

Enzymatic cycling assays for the determination of L-phenylalanine and phenylpyruvate in deproteinized tissue extracts are described. Assay 1 couples glutamine transaminase K with L-phenylalanine dehydrogenase. Assay 2 combines phenylalanine dehydrogenase, L-amino acid oxidase, and catalase. In both assays, tyrosine and some other amino acids (or their alpha-keto acid analogs) can replace phenyl...