Nitrilase ofRhodococcus rhodochrousJ1

Abstract

Nitrilase‐containing resting cells of Rhodococcus rhodochrous J1 converted acrylonitrile and benzonitrile to the corresponding acids, but the purified nitrilase hydrolyzed only benzonitrile, and not acrylonitrile. The activity of the purified enzyme towards acrylonitrile was recovered by preincubation with 10 mm benzonitrile, but not by preincubation with aliphatic nitriles such as acrylonitrile. It was shown by light‐scattering experiments, that preincubation with benzonitrile led to the assembly of the inactive, purified and homodimeric 80‐kDa enzyme to its active 410‐kDa aggregate, which was proposed to be a decamer. Furthermore, the association concomitant with the activation was reached after dialysis of the enzyme against various salts and organic solvents, with the highest recovery reached at 10% saturated ammonium sulfate and 50% (v/v) glycerol, and by preincubation at increased temperatures or enzyme concentrations.

Evolution of heteromeric nitrilase complexes in Poaceae with new functions in nitrile metabolism

“…Nitrilases belong to the superfamily of C-N hydrolases (21,22), members of which are known to form homomeric complexes consisting of 2-18 subunits (23)(24)(25)(26)(27)(28)(29). The copurification of SbNIT4A and B2 from sorghum seedlings raised the question whether these enzymes exist as heteromeric complexes in vivo and whether this interaction had an influence on their activity.…”

Thermodynamics of the hydrolysis reactions of nitriles

“…1 The purities of the nitriles were assessed by using a Hewlett Packard 5890 g.c. equipped with a flame ionization detector and a fused silica Phenomenex ZB-FFAP capillary column (30 m long, 0.53 mm i.d.).…”

“…The structures of the substances and the reactions studied herein. 1 Certain commercial equipment, or instruments, or materials are identified in this paper to specify the experimental procedures adequately. Such identification is not intended to imply recommendation, or endorsement by the National Institute of Standards and Technology (NIST), nor is it intended to imply that the materials or equipment identified are necessarily the best available for the purpose.…”

“…These studies used the h.p.l.c method described in Section 2.2. For reactions (1) to (6), the solutions used for the forward direction of reaction consisted of {nitrile(m = 0.0048 to 0.0094 mol AE kg À1 )} in phosphate buffer {m(K 2 HPO 4 ) = 0.092 mol AE kg À1 + m(H 3 PO 4 ) = 0.015 mol AE kg À1 + (4), NIT106 for reactions (2), (5), and (6), NIT1001 for reaction (3), and NIT1005 for reaction (1). NH 4 Cl(m = 0.10 mol AE kg À1 ) at pH = 7.5}.…”

“…The use of nitrilase enzymes has attracted substantial interest in the biotechnology community [1][2][3][4] because conventional chemical methods for nitrile hydrolysis entail the use of severe conditions such as the use of concentrated acid or base and high temperatures. Such harsh conditions are generally not useful when sensitive complex molecules, or chiral compounds are involved.…”