Molecular cloning of the aspartate 4-decarboxylase gene from Pseudomonas sp. ATCC 19121 and characterization of the bifunctional recombinant enzyme.
L-Aspartate 4-decarboxylase (Asd) is a major enzyme used in the industrial production of L-alanine. Its gene was cloned from Pseudomonas sp. ATCC 19121 and characterized in the present study. The 1,593-bp asd encodes a protein with a molecular mass of 59,243 Da. The Asd from this Pseudomonas strain was considerably homologous to other Asds and aminotransferases, and has evolved independently of these enzymes from gram-positive microbes. Productivity rate of the C-terminal His-tagged fusion Asd was at 33 mg/l of Escherichia coli transformant culture. The kinetic parameters K (m) and V (max) of the fusion protein were 11.50 mM and 0.11 mM/min, respectively. Gel filtration analysis demonstrated that Asd is a dodecamer at pH 5.0 while 4.4 % of the recombinant protein dissociated into dimer when the pH was increased to 7.0. Asd exhibited its maximum activity at pH 5.0 and specific activity of 280 U/mg, and remained stable over a broad range of pH. The optimum temperature for Asd reaction was 45 degrees C, and 92 % of the activity remained when the enzyme was incubated at 40 degrees C for 40 min. This enzyme did not have any preferred divalent cation for catalysis. The recombinant Asd also exhibited aminotransferase activity when D,L-Asp, L-Glu, L-Gln, and L-Ala were utilized as substrates. However, the decarboxylation activity of L-aspartate was 2,477 times higher than its aminotransferase activity. The present study is the first investigation on the important biochemical properties of the purified recombinant Asd.
Wang
Nai-Chen
NC
Graduate Institute of Agricultural Chemistry, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei 10617, Taiwan, Republic of China.
Lee
Chia-Yin
CY
D000595:Amino Acid Sequence; D002262:Carboxy-Lyases / Q000096:biosynthesis* / Q000235:genetics*; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D010957:Plasmids / Q000378:metabolism; D011549:Pseudomonas / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors; D000637:Transaminases / Q000378:metabolism
D011994:Recombinant Proteins; D000637:Transaminases; D002262:Carboxy-Lyases; C019747:aspartate 4-decarboxylase