Purification and characterization of a novel nitrilase of Rhodococcus rhodochrous K22 that acts on aliphatic nitriles.
A novel nitrilase that preferentially catalyzes the hydrolysis of aliphatic nitriles to the corresponding carboxylic acids and ammonia was found in the cells of a facultative crotononitrile-utilizing actinomycete isolated from soil. The strain was taxonomically studied and identified as Rhodococcus rhodochrous. The nitrilase was purified, with 9.08% overall recovery, through five steps from a cell extract of the stain. After the last step, the purified enzyme appeared to be homogeneous, as judged by polyacrylamide gel electrophoresis, analytical centrifugation, and double immunodiffusion in agarose. The relative molecular weight values for the native enzyme, estimated from the ultracentrifugal equilibrium and by high-performance liquid chromatography, were approximately 604,000 +/- 30,000 and 650,000, respectively, and the enzyme consisted of 15 to 16 subunits identical in molecular weight (41,000). The enzyme acted on aliphatic olefinic nitriles such as crotononitrile and acrylonitrile as the most suitable substrates. The apparent Km values for crotononitrile and acrylonitrile were 18.9 and 1.14 mM, respectively. The nitrilase also catalyzed the direct hydrolysis of saturated aliphatic nitriles, such as valeronitrile, 4-chlorobutyronitrile, and glutaronitrile, to the corresponding acids without the formation of amide intermediates. Hence, the R. rhodochrous K22 nitrilase is a new type distinct from all other nitrilases that act on aromatic and related nitriles.
Kobayashi
M
M
Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University, Japan.
Yanaka
N
N
Nagasawa
T
T
Yamada
H
H
D000596:Amino Acids / Q000032:analysis; D000619:Aminohydrolases / Q000302:isolation & purification* / Q000378:metabolism; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008970:Molecular Weight; D009570:Nitriles / Q000378:metabolism*; D012240:Rhodococcus / Q000201:enzymology*; D013379:Substrate Specificity
D000596:Amino Acids; D009570:Nitriles; D000619:Aminohydrolases; C021867:nitrilase