Improvement of the phenylalanine dehydrogenase immobilization method for the production of phenylalanine

Abstract

Phenylalanine dehydrogeanase (PheDH) produced from recombinant Escherichia coli was partially purified by ammonium sulfate precipitation and DEAE-Toyopearl column chromatography with a 20.2% yield and 2.6 purification fold. The enzyme had a molecular weight of 44.5 kDa. The enzyme was chemically modified with a series of group-specific reagents to identify essential amino acid residues. It was found that tryptophan, methionine, histidine and lysine residues may play an important role in the enzyme catalysis. Different immobilization methods were then used, i.e. immobilization via its amino groups, carboxylic groups and ionic interaction on various supports including silica, alumina, chitosan, epoxy-alumina and epoxy-chitsan. The immobilization of PheDH via its carboxylic groups by using carbodiimine gave the highest immobilized activity on silica. The optimum condition for enzyme immobilization was to activate carboxylic groups of the enzyme for 6 hours using 10 mM carbodiimide. Twenty five units of activated PheDH were then added to the silica (500 mg) which was activated with 2% (v/v) -aminopropyltriethoxysilane and incubated for 21 hours at 4°C. The activity of the immobilized enzyme was 1.41 U/g support with 5.17% of immobilization yield. When compared to the free enzyme, using coupled reaction with diaphorase, both enzymes showed the same optimum pH at 9.5 and the optimum temperature at 40ºC. The immobilized enzyme was stable over the pH range of 5.0-12.0 whereas the free enzyme was in the range 5.0-8.5. The immobilized enzyme had slightly higher temperature stability and storage stability at room temperature over the free enzyme. When the immobilized PheDH was used in batch production of L-phenylalanine, the immobilized PheDH produced L-phenylalanine 58% yield when incubated with 5 mol of phenylpyruvate at room temperature for 6 hours and retained its activity up to 84% after being used for three cycles. The immobilized PheDH was further applied for the production of amino acids using their corresponding keto acids as substrates, the product yields were ranging between 62.5-100%.