Glutaryl oxidase

ACA cephalosporin C D-amino acid oxidase + glutaryl acylase... 

Aminocephalosporanic acid (15, Scheme 9) is an important intermediate in the production of many semisynthetic cephalosporin antibiotics (66, 67). However, direct deacylation of cephalosporin C (13) to 15 by cephalosporin C acy-lase is unfavorable, so an enzymatic process is used involving D-amino acid oxidase (DAAO) oxidation of 13 to A-glutaryl-7-aminocephalosporanic acid (14, GL-7-ACA) followed by deacylation to 15 and glutaric acid, catalyzed by GL-7-ACA acylase from Pseudomonas sp. 130 (Scheme 9) (68, 69). GL-7-ACA acylase underwent pseudo first-order time-dependent inactivation by 7 3-bromoacetyl aminocephalos-poranic acid (16) (70). Dialysis did not regenerate enzyme activity, indicating irreversible inhibition. The rate of inactivation was lowered by the presence of either glutaric acid or 15,... 

Luo H, Yu H, Li Q, Shen Z. Cloning and co-expression of D-amino acid oxidase and glutaryl-7-aminocephalosporanic acid acylase genes in Escherichia coli. Enz. Microb. Technol. 2004 35(6-7) 514-518. 

ACA (36) is produced in a three-step process using two enzymes, starting from cephalosporin C (Scheme 20). After deamination by a D-amino acid oxidase, the a-ketoadipyl-7-ACA spontaneously decarboxylates and the remaining glutaryl side-chain is hydrolyzed by glutary 1-7-ACA-acylase. 

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. 

The reuse of the expensive biocatalysts is a prerequisite for the economy of the biocatalytic process. On a lab-scale the carrier-fixed enzymes can be used for more than 100 cycles (DAO) and 180 cycles (GA), before reaching half of the starting activity [15]. Prolonging the reaction time can compensate for the decreasing activity. As claimed by reference [15] for the lab-scale preparation of 1 kg 7-ACA about 1.2 kU D-amino acid oxidase and 1.5 kU glutaryl-7-ACA acylase are consumed, but operational stability is dependent on scale. In production vessels gradients, e.g., pH value and shear stress, are different and could influence the operational stability of the biocatalysts, therefore a higher biocatalyst consumption is usually realistic. 

Cephalosporin C is supplied directly to the enzymatic stage. The side chain of the molecule is oxidatively deaminated by a D-amino acid oxidase (obtained from yeast). Oxygen is consumed, and ketoadipinyl-7-ACS, ammonia, and hydrogen peroxide are formed. Under these reaction conditions, oxidative decarboxylation to form glutaryl-7-ACS occurs spontaneously. 

Two other oxidase activities have been described in rat liver peroxisomes, one acting on glutaryl-CoA, the other on valproyl-CoA. The glutaryl-CoA oxidase activity, however, co-purifies with the inducible pahnitoyl-CoA oxidase. The valproyl-CoA oxidase was claimed to differ from the above described acyl-CoA oxidases, but in our hands this activity was recovered mainly in the cytosol (Casteels, M. Van Veldhoven P. P., unpublished data). 

Bennett, M.J., Pollitt, R.J., Goodman, S.I., Hale, D.E. Vamecq, J. (1991)7. Inherit. Metab. Dis. 14, 165-73. Atypical riboflavin-responsive glutaric aciduria, and deflcient peroxisomal glutaryl-CoA oxidase activity a new peroxisomal disorder. 

Glutaric aciduria type III (glutaryl-CoA oxidase deficiency)... 

Apart from glutaric aciduria II (ETF or ETF dehydrogenase deficiency, see Sect. 14.10) a third disorder with peroxisomal glutaryl-CoA oxidase deficiency leading to glutaric aciduria has been described (glutaric aciduria III, see Chap. 24). 

A one-pot chemoenzymatic synthesis of 3 -functionalized cephalosporin C or cepha-zolin involving three consecutive biotransformations has been reported [16]. The enzymatic deacylation of cephalosporin C in one batch was performed by the use of D-amino acid oxidase (DAO) from Trigonopsis variabilis, glutaryl acylase (GA) from Acetobacter sp., and a continuous flow of oxygen. DAO catalyzed the oxidative deamination of the a-... 

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