Cloning and expression of phenylalanine dehydrogenase gene from Acinetobacter lwoffii and the possibility for amino acid production

Abstract

Phenylalanine dehydrogenase (EC 1.4.1.20), one of amino acid dehydrogenases, catalyzes the reversible pyridine nucleotide -dependent oxidative deamination of L-phenylalanine to form ammonia, phenylpyruvate, and NADH. Our research group has studied phenylalanine dehydrogenase from Acinetobacter lwoffii and found that L-methionine, L-tryptophan and L-norleucine could act as substrate in the oxidative deamination of the enzyme. No loss of the enzyme activity was observed upon incubation at 55 ํC, pH 7.4 for 10 minutes. From these properties, the enzyme seems to have more potential in the synthesis of various amino acids. Moreover, nucleotide sequence of phenylalanine dehydrogenase gene from A. lwoffii was already determined. Thus, in this research the phenylalanine dehydrogenase gene was cloned and expressed in E. coli BL21(DE3) and E. coli BL21(DE3)pLysS host cells using expression vector, pET-17b. The specific activity from crude extract of recombinant clones were found in the range of 0.81 4.46 units/mg protein. The highest specific activity was 55.75 fold higher than that of the enzyme from A. lwoffii. The optimum condition for phenylalanine dehydrogenase gene expression was induction with 0.4 mM IPTG for 8 hours. Stability of phenylalanine dehydrogenase gene expression in E. coli BL21(DE3) was studied. After daily subculture the recombinant clone showed the highest phenylalanine dehydrogenase activity for 20 days, it was found that phenylalanine dehydrogenase activity decreased with the increasing number of subculture. The enzyme was purified to homogeneity by 50-70 % saturated ammonium sulfate precipitation and DEAE-Toyopearl column chromatography with 29.45 % yield and 5.19 purification fold. The enzyme showed high substrate specificity in the oxidative deamination on L-phenylalanine while it acted on alpha-ketocaproate, alpha-keto-gamma-methiol-n-butyrate, alpha-ketovalerate and alpha-ketoisocaproate with 5.96, 4.12, 3.84 and 3.15 fold of its natural substrate, phenylpyruvate, respectively in reductive amination. No loss of enzyme activity was observed upon incubation at 30 ํC, pH 9.5 for 4 hours and 50 % of the activity was retained after incubation at this temperature for 12 hours. When phenylalanine dehydrogenase was used for production of amino acids using their corresponding keto acids as substrates, the product yield was in the range between 36.0-72.2 %