Characterization and synthetic applications of recombinant AtNIT1 from Arabidopsis thaliana.
The nitrilase AtNIT1 from Arabidopsis thaliana was overexpressed in Escherichia coli with an N-terminal His6 tag and purified by zinc chelate affinity chromatography in a single step almost to homogeneity in a 68% yield with a specific activity of 34.1 U.mg-1. The native enzyme (approximately 450 kDa) consists of 11-13 subunits (38 kDa). The temperature optimum was determined to be 35 degrees C and a pH optimum of 9 was found. Thus, recombinant AtNIT1 resembles in its properties the native enzyme and the nitrilase from Brassica napus. The stability of AtNIT1 could be significantly improved by the addition of dithiothreitol and EDTA. The substrate range of AtNIT1 differs considerably from those of bacterial nitrilases. Aliphatic nitriles are the most effective substrates, showing increasing rates of hydrolysis with increasing size of the residues, as demonstrated in the series butyronitrile, octanenitrile, phenylpropionitrile. In comparison with 3-indolylacetonitrile, the rate of hydrolysis of 3-phenylpropionitrile is increased by a factor of 330, and the Km value is reduced by a factor of 23. With the exception of fluoro, substituents in the alpha position to the nitrile function completely inhibit the hydrolysis.
Wajant
Harald
H
Institut für Organische Chemie, and Institut für Zellbiologie und Immunologie, Universität Stuttgart, Germany.
Effenberger
Franz
F
D000619:Aminohydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017360:Arabidopsis / Q000235:genetics*; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D017931:DNA Primers; D011994:Recombinant Proteins; D000619:Aminohydrolases; C021867:nitrilase