Abstract
A nitrilase gene cyc705 from Arthrobacter aurescens CYC705 for synthesis of iminodiacetic acid (IDA) was cloned. This gene contained a 930 bp ORF, which encoded a polypeptide of 310 amino acids. A recombinant Escherichia coli BL21(DE3)/pET28a-cyc705 was constructed to achieve the heterologous expression of cyc705. This recombinant nitrilase was purified to homogeneity with a molecular weight of 36.7 kDa on SDS-PAGE and mass spectrometry, and characterized to be an oligomer of 14 subunits by gel permeation chromatography. Using iminodiacetonitrile (IDAN) as the substrate, the Vmax, Km, kcat and kcat/Km were 9.05 U mg(-1), 43.17 mM(-1), 94.1 min(-1) and 2.18×10(3) min(-1) M(-1), respectively. The optimum temperature and pH were 25°C and 5.8. The suitable substrates for the purified nitrilase were short-chain aliphatic dinitriles. High concentration of IDAN could be hydrolyzed to IDA in a shorter time.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Aminohydrolases / chemistry
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Aminohydrolases / genetics
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Aminohydrolases / metabolism*
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Arthrobacter / enzymology*
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Arthrobacter / genetics
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Chromatography, Gel
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Cloning, Molecular
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Cluster Analysis
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DNA, Bacterial / chemistry
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DNA, Bacterial / genetics
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Electrophoresis, Polyacrylamide Gel
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Enzyme Stability
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Escherichia coli / genetics
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Escherichia coli / metabolism
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Gene Expression
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Hydrogen-Ion Concentration
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Imino Acids / metabolism*
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Kinetics
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Mass Spectrometry
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Molecular Sequence Data
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Molecular Weight
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Phylogeny
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Protein Multimerization
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Protein Subunits / analysis
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Sequence Analysis, DNA
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Sequence Homology
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Temperature
Substances
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DNA, Bacterial
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Imino Acids
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Protein Subunits
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Recombinant Proteins
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Aminohydrolases
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nitrilase
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iminodiacetic acid