Bioproduction of L-Aspartic Acid and Cinnamic Acid by L-Aspartate Ammonia Lyase from Pseudomonas aeruginosa PAO1

Aspartase (l-aspartate ammonia lyase, EC 4.3.1.1) catalyses the reversible amination and deamination of l-aspartic acid to fumaric acid which can be used to produce important biochemical. In this study, we have explored the characteristics of aspartase from Pseudomonas aeruginosa PAO1 (PA-AspA). To overproduce PA-AspA, the 1425-bp gene was introduced in Escherichia coli BL21 and purified. A 51.0-kDa protein was observed as a homogenous purified protein on SDS-PAGE. The enzyme was optimally active at pH 8.0 and 35 degrees C. PA-AspA has retained 56% activity after 7 days of incubation at 35 degrees C, which displays the hyperthermostablility characteristics of the enzyme. PA-AspA is activated in the presence of metal ions and Mg2+ is found to be most effective. Among the substrates tested for specificity of PA-AspA, l-phenylalanine (38.35 +/- 2.68) showed the highest specific activity followed by l-aspartic acid (31.21 +/- 3.31) and fumarate (5.42 +/- 2.94). K (m) values for l-phenylalanine, l-aspartic acid and fumarate were 1.71 mM, 0.346 mu M and 2 M, respectively. The catalytic efficiency (k (cat)/K (m)) for l-aspartic acid (14.18 s(-1) mM(-1)) was higher than that for l-phenylalanine (4.65 s(-1) mM(-1)). For bioconversion, from an initial concentration of 1000 mM of fumarate and 30 mM of l-phenylalanine, PA-AspA was found to convert 395.31 mu M l-aspartic acid and 3.47 mM cinnamic acid, respectively.