Brevibacterium hydratase

Desymmetrization is not restricted to a single class of enzyme. For example, Madrell et reported the gram-scale preparation of a key intermediate of the lovastatin lactone through the desymmetrization of 3-(benzyloxy)glutaronitrile using whole cells from Brevibacterium R312. The transformation occurs via a dual nitrile hydratase/amidase-catalysed hydrolysis to afford acid in 65 % yield and 88 % ee (Scheme 1.49). 

This process has been operated successfully by the Tanabe Seiyaku Co. in Japan since 1973. Similar processes have since been commercialised by other companies, such as the Kyowa Hakko Co., often using different immobilisation methods such as polyurethane. The same iimnobilized cell approach has also been used by Tanabe since 1974 in their coimnercial process for the production of L-malic acid from fumarate using the hydratase activity of Brevibacterium ammoniagenes cells. 

A similar process is also used for the production of L-malic acid from fumarate, in this case using a hydratase enzyme derived from Brevibacterium ammoniagenes. Another variation of the Tanabe technology involves the synthesis of L-alanine from L-aspartic acid through the use of immobilized whole cells (P dacunae) containing aspartate-decarboxylase. 

Alfani, R, Cantarella, M., Spera, A., and Viparelli, P. 2001. Operational stability of Brevibacterium imperialis CBS 489-74 nitrile hydratase. Journal of Molecular Catalysis B-Enzymes, 11 687-97. 

Mayaux, J., Cerbelaud, E., Soubrier, R, et al. 1990. Purification, cloning, and primary structure of an enantiomer selective amidase from Brevibacterium sp. R312 Structural evidence for genetic coupling with nitrile hydratase. Journal of Bacteriology, 172 6764-73. 

L-Amino adds could be produced from D,L-aminonitriles with 50% conversion using Pseudomonas putida and Brevibacterium sp respectively, the remainder being the corresponding D-amino add amide. However, this does not prove the presence of a stereoselective nitrilase. It is more likely that the nitrile hydratase converts the D,L-nitrile into the D,L-amino add amide, where upon a L-spedfic amidase converts the amide further into 50% L-amino add and 50% D-amino add amide. In this respect the method has no real advantage over the process of using a stereospedfic L-aminopeptidase (vide supra). 

The degradation of nitriles by nitrilases (EC 3.5.5.1) has been the subject of intense study, especially as it relates to the preparation of the commodity chemical acrylamide. Nitrilases catalyze the hydrolysis of nitriles to the corresponding acid plus ammonia (Figure 1 reaction 5), whereas nitrile hydratases (EC 4.2.1.84) add water to form the amide. Strains such as Rhodococcus rhodo-chrous Jl, Brevibacterium sp., and Pseudomonas chlororaphis have been used to prepare acrylamide from acrylonitrile, which contain the hydratase and not nitrilase activity [12]. A comparison of these strains has been discussed elsewhere [98]. Other uses of nitrilases, however, have primarily been directed at resolution processes to stereoselectively hydrolyze one enantiomer over another or regiose-lectively hydrolyze dinitriles [99-101]. 

With both these strains, the enzymatic system is composed of nitrile hydratases and amidases. The nitrile hydratase gene of Brevibacterium R312 is cloned, sequenced (ref. 17) and over expressed in Rhodococcus rhodochrous ATCC12674 (pKRNH2) (ref. 18). The best selectivity which can be hoped for with this nitrile hydratase is 93 % (ref. 4). Moreover, the cyanovaleramide with its poor solubility must not be accumulated and requires a biocatalyst with a superactivated amidase activity. The nitrile hydratase is less stable than amidase and the biocatalyst with these two enzymes would not be sufficiently robust for an industrial application. 

Aliphatic nitrile hydratases, that catalyzed the hydrataiion of nitriles to amides, were purified and characterized in Arthrobacter sp. J1 [128], Brevibacterium R312 [129], and Rhodococcus sp. N774 [130]. In tixe first strain, the activity of an amidase, which forms acetic add and ammonia stoichiometrically from acetamide, was also detected [131]. Bioconversion of dinitrile to mononitrile catalyzed by nitrile hydratase and amidase was obtained from Corynebacterium sp. C5. The two enzymes were constitutively formed in cells [132]. 

Scheme 2.100 Coordination sphere of the ferric ion in Brevibacterium sp. nitrile hydratase...

---