Identification and characterization of a novel D-amidase gene from Variovorax paradoxus and its expression in Escherichia coli.
The gene for the newly described D-amidase from Variovorax paradoxus (Krieg et al. 2002) was cloned and functionally expressed in Escherichia coli. Since native enzyme was available in minute amounts only, we determined the N-terminal sequence of the enzyme and utilized the Universal GenomeWalker Approach to make use of the common internal sequence of the amidase signature family. The high GC content of the gene made it necessary to employ an appropriate DNA polymerase in the amplification reactions. Thus, the sequence of the complete gene and the flanking regions was established. In independent experiments, the gene was then amplified from genomic DNA of V. paradoxus, expressed in E. coli, and characterized. The recombinant enzyme has a specific activity of 1.7 units/mg with racemic tert-leucine amide as substrate and is a homodimer of 49.6-kDa monomers.
Institute of Enzyme Technology, Heinrich-Heine University Düsseldorf, 52426 Jülich, Germany.
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Krieg
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Slusarczyk
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Verseck
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Kula
D000581:Amidohydrolases / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D020562:Betaproteobacteria / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D000581:Amidohydrolases; C019665:amidase