Glutaryl amidase

The enzymatic process uses water as the solvent and two immobilized enzymes as catalysts at room temperature. In a first step cephalosporin C is deaminated to a-ketoadipyl-7-ACA using a carrier-fixed D-amino acid oxidase in the presence of oxygen. Under reaction conditions the a-keto intermediate is oxidatively decarboxy-lated to glutaryl-7-ACA. In a second step the glutaryl-7-ACA is then hydrolyzed to 7-ACA by a carrier-fixed glutaryl amidase. 

Later in the process this side chain is removed enzymatically, using an enzyme quite similar to the glutaryl amidase from Pseudomonas sp. as in the enzymatic production of 7-ACA. For the production of 7-ADCA and the dicarboxylic acid amidase, new plants are currently under construction at DSM in The Netherlands. Compared with the old process for the production of 7-ADCA, the major advantages of this process are higher purity of the end product, much greater energy efficiency and almost complete absence of organic solvents. 

Aminocephalosporanic Acid Formation by Amide Hydrolysis Catalyzed by Glutaryl Amidase (E.C. 3.1.1.41 ) 66 691... 

The second step of the 7-aminocephalosporanic acid (7-ACA) process is the deamidation of glutaryl-7-ACA (Fig. 19-22), the first step is described in Sect. 19.3.2.2. 7-ACA is an intermediate for semi-synthetic cephalosporins. Hoechst Marion Roussel uses the glutaryl amidase immobilized on a spherical carrier. Toyo Jozo and Asahi Chemical immobilize the glutaryl amidase on porous styrene anion exchange resin with subsequent cross-linking with 1% glutardialdehyde. The catalyst is applied in a fixed bed reactor in a repetitive batch mode (70 cycles). Here, an enzymatic process has replaced an existing chemical process for environmental reasons (Fig. 19-23) ... 

E = glutaryl amidase, enzyme from Escherichia coli... 

In the second stage of the process, the glutaric acid is split off by the enzyme glutaryl amidase, and 7-ACS is liberated (see Fig. 28). 

In the production of the enzyme glutaryl amidase, only a low concentration was produced by the wild strain Pseudomonas. An economically interesting concentration (greater than fiftyfold increase per volume unit) can be produced only by genetic engineering (i.e., cloning the enzyme glutaryl amidase in Escherichia coli). 

Previous efforts have failed to identify an enzyme with robust Ceph C amidase activity. Some glutaryl-7-ACA acylases can directly convert Ceph C to 7-ACA, but they do so with very poor efficiency and have not been considered for a single-enzyme manufacturing process.30-33 Nonetheless, glutary 1-7-AC A acylases with measurable activity on Ceph C are classified as cephalosporin C acylases. Mutagenesis approaches such as ePCR have been used in an attempt to improve the activity of these enzymes on Ceph C, but only marginal improvements in the desired activity have... 

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