Abstract
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 °C, and 92 % of the activity remained when the enzyme was incubated at 40 °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.
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Acknowledgements
This work was supported by grants 90AS-2.1.1-FD-Z4(1) and 91AS-3.1.1-FD-Z5(3) from the Council of Agriculture, and was partially supported by grant NSC92-2313-B-002-092 and NSC94-2313-B-002-049 from the National Science Council, Taipei, Taiwan.
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Wang, NC., Lee, CY. Molecular cloning of the aspartate 4-decarboxylase gene from Pseudomonas sp. ATCC 19121 and characterization of the bifunctional recombinant enzyme. Appl Microbiol Biotechnol 73, 339–348 (2006). https://doi.org/10.1007/s00253-006-0475-6
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DOI: https://doi.org/10.1007/s00253-006-0475-6