Toru Nagasawa

Microbial transformations of nitriles

Abstract Nitrile-hydrolysing enzymes such as nitrile hydratase, nitrilase and amidase have great potential as catalysts in processing organic chemicals because they can convert nitriles to the corresponding higher-value amides or acids. Very recently, the use of bacterial nitrile hydratase for industrial production of the important chemical commodity, acrylamide, was pioneered in Japan. We review here both the enzymatic production of acrylamide, and other recent progress in microbial transformation of nitriles.

Cloning, nucleotide sequence and expression in Escherichia coli of two cobalt-containing nitrile hydratase genes from Rhodococcus rhodochrous J1

Rhodococcus rhodochrous J1 produces two kinds of cobalt-containing nitrile hydratases (NHases); one is a high molecular mass-NHase (H-NHase) and the other is a low molecular mass-NHase (L-NHase). Both NHases are composed of two subunits of different sizes (alpha and beta subunits). The H- and L-NHase genes were cloned into Escherichia coli by a DNA-probing method using the NHase gene of Rhodococcus sp. N-774, a ferric ion-containing NHase producing strain, as the hybridization probe and their nucleotide sequences were determined. In each of the H- and L-NHase genes, the open reading frame for the beta subunit was located just upstream of that for the alpha subunit, which probably belongs to the same operon. The amino acid sequences of each subunit of the H- and L-NHases from R. rhodochrous J1 showed generally significant similarities to those from Rhodococcus sp. N-774, but the arrangement of the coding sequences for two subunits is reverse of the order found in the NHase gene of Rhodococcus sp. N-774. Each of the NHase genes was expressed in E. coli cells under the control of lac promoter, only when they were cultured in the medium supplemented with CoCl2.

ε-caprolactam, a new powerful inducer for the formation of Rhodococcus rhodochrous J1 nitrilase

We found that ε-caprolactam is a new powerful inducer for the formation of Rhodococcus rhodochrous J1 nitrilase. When Rhodococcus rhodochrous J1 cells were cultivated at 28°C for 120 h in a nutrient medium supplemented with 0.5% (w/v) ε-caprolactam, an enormous amount of nitrilase was formed in the cells which corresponded to approximately 30% of all soluble protein. The level of ε-caprolactam in the culture broth barely decreased in the course of cultivation. γ-Butyrolactam and δ-valerolactam also caused effective induction. The induction of nitrilase formation by ε-caprolactam was also observed in some other Rhodococcus strains.

Synthesis of various aromatic amide derivatives using nitrile hydratase of Rhodococcus rhodochrous J1

Abstract Nitrile hydratase, that is produced abundantly in cells of Rhodococcus rhodochrous J1, catalyses the conversion of various aromatic nitrile derivatives to the corresponding amides. Using Rh. rhodochrous J1 resting cells, the conditions for the production of benzamide, 2,6-difluorobenzamide, indoleacetamide, thiophenecarboxamide and furanecarboxamide here optimized. Under the determined conditions, 489 g of benzamide, 306 g of 2,6-difluorobenzamide, 1045 g of 3-indoleacetamide, 210 g of 2-thiophenecarboxamide and 522 g of 2-furanecarboxamide were produced, with 100% molar conversion, from the corresponding nitriles, per litre of reaction mixture.

Optimum culture conditions for the production of cobalt-containing nitrile hydratase by Rhodococcus rhodochrous J1

SummaryWe sought the optimum conditions for production of nitrile hydratase by Rhodococcus rhodochrous J1. The addiiion of both cobalt ions and an aliphatic nitrile or amide as an inducer was indispensable for the appearance of nitrile hydratase activity in R. rhodochrous J1 cells. Crotonamide was an efficient inducer and, moreover, urea was found to be the most powerful inducer for the production of nitrile hydratase. When R. rhodochrous J1 was cultivated under optimal conditions, the enzyme activity in the culture broth and the specific activity was approximately 32,000 and 512 times higher than the initially obtained levels, respectively. The nitrile hydratase formed corresponded to more than 45% of the total soluble protein in urea-induced cells, as judged by quantitative evaluation of the gel track.

Production of acrylic acid and methacrylic acid using Rhodococcus rhodochrous J1 nitrilase

Summaryε-Caprolactam-induced Rhodococcus rhodochrous J1 cells containing abundant nitrilase were used in the production of acrylic acid and methacrylic acid from acrylonitrile and methacrylonitrile, respectively. Under a periodic substrate feeding system, the highest accumulations, 390 g acrylic acid/l and 260 g methacrylic acid/l, were attained.

Occurrence of a novel nitrilase, arylacetonitrilase in Alcaligenes faecalis JM3

A novel nitrilase which catalyzes the direct hydrolysis of arylacetonitrile derivatives to the corresponding arylacetic acids has been found in strain JM3, tentatively identified as Alcaligenes faecalis. The addition of isovaleronitrile greatly enhanced the formation of the nitrilase. The culture conditions for the best production of the nitrilase and the substrate specificity of the enzyme are described.

Large-Scale Bioconversion of Nitriles into Useful Amides and Acids

The application of the bioconversion process has been generally restricted to the production of fine chemicals that are difficult to obtain through conventional chemical methods. Recently, however, the industrial production of acrylamide, an important commodity chemical, using bacterial nitrile hydratase, was started in Japan. We describe here the first successful example, the enzymatic production of acrylamide, and the recent progress in the microbial transformation of nitriles. The cofactors and reaction mechanism of nitrile hydratase are also discussed.