Characterization of an inducible nitrilase from a thermophilic bacillus

Abstract

Nitrilase activity was induced in the thermophilic bacterium Bacillus pallidus strain Dac521 by growth on benzonitrile-supplemented minimal medium. The enzyme had a subunit relative molecular mass of 41 kDa but was purified as a complex with a putative GroEL protein (total M(r), 600 kDa). The enzyme catalyzed the hydrolysis of aliphatic, aromatic, and heterocyclic nitriles with widely varying kcat/KM values, primarily the result of differences in substrate affinity. Of the nitriles tested, 4-cyanopyridine was hydrolyzed at the fastest rate. Substitution of benzonitrile at the meta or para position either had no effect on catalytic rate or enhanced kcat, while orthosubstitution was strongly inhibitory, probably because of steric hindrance. The effect of catalytic inhibitors was consistent with the presence of active site thiol residues although activity was little affected by putative thiol reagents such as iodoacetate, iodoacetamide, and N-methylmaleimide. Enzymatic activity was constant between pH 6 and 9 with an optimum at pH 7.6. The optimal temperature for activity was 65 degrees C with rapid activity loss at higher temperatures. The purified nitrilase-GroEL complex had the following half-lives of activity: 8.4 h at 50 degrees C, 2.5 h at 60 degrees C, 13 min at 70 degrees C, and less than 3 min at 80 degrees C.

Oligomerization of Sulfolobus solfataricus signature amidase is promoted by acidic pH and high temperature

“…At all concentrations the protein maintained full activity and when injected on the Superdex column, no dissociation of the dimer occurred. Likewise, incubation of the two proteins in 50 mM Tris adjusted to pH 11.0 (Almatawah et al 1999) did not cause dissociation of the dimers.…”

“…Also shown are the catalytic triad (K96 (orange), S171 (yellow) and S195 (green)) at the active site, E337 (light blue) and D442 (red) at the entrance of the active site and Y41 in brilliant green. chaothropes that affect the hydrogen bond network of a protein), a lipophilic salt such as thiocyanate (which can interfere with ion pairs located either on the surface or in the hydrophobic core of folded proteins (Ptitsyn et al 1990)) or with treatment at pH 11.0 (Almatawah et al 1999), suggesting that the two subunits are strongly bound, likely through the electrostatic interactions depicted in the dimer model of Figure 7. Maximal activity of the recombinant wt amidase and its mutant Y41C was found at pH 5.0.…”

A Cyanide-Induced 3-Cyanoalanine Nitrilase in the Cyanide-Assimilating Bacterium Pseudomonas pseudoalcaligenes Strain CECT 5344

“…Moreover, fumarase and aconitase enzymes are also thermoresistant up to 60 degrees (not shown). Although it is expected to find a thermoresistant nitrilase in thermophilic bacteria (45), some mesophilic bacteria harboring thermoresistant nitrilases have been also described (46).…”

Biodegradation, Biotransformation, and Biocatalysis (B3)

“…He reported the purification, characterization, and applications of two hydratases and a nitrilase from two thermophilic Bacillus strains (1,2,19,36,74). Each of the enzymes was more thermostable than corresponding enzymes isolated from mesophiles, but their temperature optima were marginally lower than the growth temperatures of the bacterial strains from which they were isolated (55 to 65°C).…”