Characterization of a nitrilase from Arthrobacter aurescens CYC705 for synthesis of iminodiacetic acid.
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.
State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology.
Wenwen
W
Cai
Erzheng
E
Su
Shujing
S
Zhu
Yuhong
Y
Ren
Dongzhi
D
Wei
D000619:Aminohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001173:Arthrobacter / Q000201:enzymology* / Q000235:genetics; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D016000:Cluster Analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007098:Imino Acids / Q000378:metabolism*; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D055503:Protein Multimerization; D021122:Protein Subunits / Q000032:analysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D013696:Temperature
D004269:DNA, Bacterial; D007098:Imino Acids; D021122:Protein Subunits; D011994:Recombinant Proteins; D000619:Aminohydrolases; C021867:nitrilase; C008109:iminodiacetic acid