Nitrile-metabolizing potential ofBacilluscereus strain FA12; Nitrilase production, purification, and characterization

Abstract

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.

Production and Characterization of a Nitrilase from Pseudomonas aeruginosa RZ44 and its Potential for Nitrile Biotransformation

“…The apparent molecular mass of the purified RZ44 nitrilase as obtained by SDS-PAGE is about 30 kDa ( Figure 4 ). The molecular weights of nitrilase from other bacterial species have been reported as 45 kDa for Bacillus cereus strain FA12 ( 41 ), 43 kDa for Pseudomonas putida ( 42 ), and 33 kDa for P. fluorescens Pf5 ( 23 ).…”

“…It was also revealed that the quantity of acrylic acid and acetic acid identified from the conversion of the correspond nitrile is maximum after 4 and 6 hours of incubation with acrylonitrile and acetonitrile, respectively. The time course of acetonitrile (3.5 mM) and acrylonitrile (5 mM) conversion into their corresponding acids by B. cereus FA12 resting cells showed that these nitriles were hydrolyzed within 12 h, and the amount of ammonia produced from acetamide and acetonitrile was maximum after 5 and 7 hours of the incubation, respectively ( 41 ). Remarkably, the quantity of the acetic and acrylic acids identified from the conversion of acetonitrile and acrylonitrile showed a small aberration from the theoretical quantity which was equivalent to the quantity of acetonitrile and acrylonitrile added (3.5 and 5 mM, respectively).…”

Effect of Sublethal Concentrations of Zinc Oxide Nanoparticles on Bacillus cereus

“…Another important observation arising from our experiments is that zinc oxide nanoparticles influence metabolic processes in Bacillus which are important for the biotransformation of various compounds. B. cereus is capable of decomposing anthropogenic pollutants, including azo dyes, nitriles, and pesticides such as beta-cypermethrin and pentachlorophenol [ 78 , 79 , 80 ]. It also plays an important role in the environment by breaking down natural complex chemical compounds such as resveratrol or polyphenolic compounds [ 58 , 59 ].…”

Exploring a broad spectrum nitrilase from moderately halophilic bacterium Halomonas sp. IIIMB2797 isolated from saline lake

“…M24D13 and optimum activity was determined at 85 °C. Ramezani‐Pour et al reported nitrile metabolizing isolate Bacillus cereus strain FA12 for nitrilase activity at 40 °C. Chen et al reported Clostridium thermocellum nitrilase expressed on Bacillus subtilis spores and this recombinant nitrilase catalyzed the conversion of 3‐cyanopyridine to niacin and displayed maximum catalytic activity (8.22 U mg −1 protein) at 40 °C and pH 7.4.…”