No abstract

Background The conversion of nitriles into amides or carboxylic acids by nitrilase has taken its application into consideration, as the scope of its applications has recently been extended. Objectives In this study, P. aeruginosa RZ44 was isolated from sewage in the Kerman which has Nitrile-degradation activity. In order to improve the nitrilase production, several optimization were done on environmental condition. Nitrilase activity was characterized against different pHs, temperatures, ions, and substrates. Materials and Methods Enzyme activity was evaluated by determining the production of ammonia following to the modification of the phenol/hypochlorite method. Different factors that affect production of the enzyme by P. aeruginosa RZ44 were optimized and evaluated in the culture mediums. Results The results showed that degradation of the acetonitrile by P. aeruginosa RZ44 increased the pH of the growth medium from the initial pH 7.0 to 9.37. Optimizing the medium for P. aeruginosa RZ44, it was found that glucose and starch (5 g.L-1) have strongly supported nitrilase production, compared to the control. As well, urea (5 g.L-1) and yeast extract (15 g.L-1) have favored an increased biomass and nitrilase production, as the nitrogen sources. These results show that nitrilase production increases in the pH range 5.0 to 7.0 and then start decreasing. Addition of the Mg2+, Fe2+ and Na+ has supported the biomass and nitrilase production. Co2+, Mn2+ and Cu2+ were confirmed to inhibit cell growth and enzyme production. Enzyme characterization results show that, P. aeruginosa RZ44 nitrilase exhibits comparatively high activity and stability at pH 7.0 and 40°C. Nitrilase was completely inhibited by CoCl2 and CaCl2, whereas, the inhibition in the presence of MnSO4 and CuSO4 was about 60%. Time course analysis of the nitrile conversion by the resting P. aeruginosa RZ44 cells showed that nitrile substrates (i.e. acetonitrile) was hydrolyzed within 8 h. Conclusions these results indicate that P. aeruginosa RZ44 has the potential to be applied in the biotransformation of nitrile compounds.

Nitrile-hydrolyzing bacteria have the potential to perform useful biotransformations such as the production of industrially useful acids and amides. In this study, we report a nitrile-degrading bacterium with signifi cant nitrile metabolism. Molecular characterization of 16S rDNA gene characterized this strain as Bacillus cereus . Medium optimization of B. cereus FA12 showed that biomass and nitrilase production was strongly supported by glucose (10 gL Ϫ 1 ) and yeast extract (10 gL Ϫ 1 ). Enzymatic production improved slightly in the pH range from 6.0 to 7.0. The addition of Mg ϩ 2 , Fe ϩ 2 , and Na ϩ supported biomass and nitrilase production; however, other metal ions, Co ϩ 2 and Cu ϩ 2 , inhibited production. The apparent molecular mass of the puri fi ed FA12 nitrilase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was about 45 kDa. Nitrilase FA12 shows relatively high activity and stability at pH 7.0 and 40 ° C. Nitrilase FA12 was marginally inhibited with Ca ϩ 2 and Co ϩ 2 , whereas inhibition in the presence of dithiothreitol or DTT was 80%. The pseudo K m (mM) values of resting cells (i.e., treating whole cells as if they were an enzyme) for acetonitrile and acetamide were determined to be 2.36 and 1.81, respectively. Under optimum situations, B. cereus FA12 resting cells produced 83 and 58 (U/mg) acetonitrile/acetamide degrading activity, respectively. Ammonia production from acetamide and acetonitrile by the B. cereus FA12 was maximum after 5 and 7 h of incubation, respectively. These results indicate that B. cereus FA12 resting cells may be used in nitrile biotransformations to produce commercially useful compounds.