Glutaryl-7-ACA

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... 

Similar results were obtained for the immobilization of glutaryl-7-ACA-acy-lase (own laboratory experiments). Figure 6 demonstrates the decrease of activity in the supernatant of the coupling reaction mixture and the concomitant increase in carrier-bound activity. Maximum activity was measured after only 6 h, leaving about 20 % of the initial activity in solution. During the next 14 h the remaining soluble enzyme was immobilized. However, an increase in activity could not be measured under standard conditions due to diffusional limitation and internal pH-shifts in the biocatalyst particles. According to these data. 

Fig. 6. Kinetics of immobilization of glutaryl-7-ACA-acylase on epoxy-activated polymethacrylate. The Gl-7-ACA-acylase was incubated with the epoxy-activated carrier. At definite times aliquots were taken from the reaction suspension. Supernatant and carrier-fixed enzyme were separated by centrifugation. The carrier-fixed enzyme was washed with water to remove non-covalently linked enzyme. The activities of the immobilized enzyme and supernatant were determined (5 mM potassium phosphate buffer pH 8,37°C, 2% glutaryl-7-amino cepha-losporanic acid, pH-stat 8.0). Simultaneously, an aliquot of carrier-fixed enzyme was boiled in sodium dodecylsulfate (SDS)/glycine buffer and the supernatant was subjected to SDS-polyacrylamide electrophoresis (see insert from left to right lane 1 Carrier-fixed enzyme, 2 h lane 2 Carrier-fixed enzyme, 4 h lane 3 Carrier-fixed enzyme, 6 h lane 4 Carrier-fixed enzyme, 21 h lane 5 Carrier-fixed enzyme, 69 h lane 6 Dialyzed enzyme lane 7 Supernatant, 2 h lane 8 Supernatant, 21 h lane 9 Supernatant, 69 h lane 10 Molecular weight calibration markers)...

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 keto intermediate decarboxylates spontaneously to 7-/ -(4-carboxybutanami-do)-cephalosporanic acid (glutaryl-7-ACA) and carbon dioxide by chemical reaction with the hydrogen peroxide formed during the enzymatic reaction. The occurrence of the byproduct 7-ACA-sulfoxide is possible. In general a reaction mixture is produced, caused by the catalase also produced by the yeast. However, the a-ke-toacid intermediate reacts poorly with the deacylation enzyme in the second step. 

In a second step the glutaryl-7-ACA is hydrolyzed to 7-ACA and glutaric acid (Fig. 6) by means of a glutaryl-7-ACA acylase (GA). The enzyme can be isolated... 

Unfortunately the enzyme titer in the fermentation broth, even after classical mutation and several years of process development, was too low for the biocatalyt-ic process. With respect to the economics, the gene encoding for glutaryl-7-ACA acylase has been cloned, sequenced and expressed in E. coli to produce sufficient amounts. The enzyme titer could be increased by a factor of more than 100. Even the purification of the enzyme became much easier and resulted in higher yields with less side-activities, e.g., esterases. Two chromatographic purification steps were substituted by crystallization of the enzyme. The enzyme crystals could be stored long term without deactivation. To allow for reuse, the glutaryl-7-ACA acylase was immobilized on a polymeric carrier. 

Tab. 1 Production of glutaryl-7-ACA acylase natural source versus GMO (natural source = 100%).

The biocatalytic process takes place in stirred tank reactors with sieves in the bottom and a working volume of several cubic meters (Fig. 7). The first biocatalytic step is a three-phase reaction with a solid biocatalyst, immobilized DAO on a spherical carrier, the liquid Ceph C solution and oxygen or air as the gaseous phase. The second step is a two-phase reaction with immobilized GA on a solid carrier and the glutaryl-7-ACA in solution. 

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. 

The glutaryl-7-ACA-containing solution is separated by filtration from the immobilized biocatalyst and directly transferred to the deacylation step in the second reactor. The oxidation reactor is ready to start for the next cycle. If production stops, storage of the DAO catalyst in a buffer solution is possible. 

The 7-ACA produced is purified and isolated by crystallization at the isoelectric point. Of course, the quality of the 7-ACA produced by the biocatalytic synthesis has to be comparable to the chemical product with approximately 95% purity. Therefore the occurrence of byproducts, such as desacetyl-7-ACA, desacetoxy-7-ACA, 7-ACA-sulfoxide, glutaryl-7-ACA or a-ketoadipyl-7-ACA generated during the process or depending on the quality of the starting material, has to be watched very critically during the process development. 

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) ... 

Acamori I, Fukagawa M, Tsumura M, Iwami M, Ono H, Ishitani Y, Kojo H, Kohsaka M, Ucda Y, Imanaka H. Comparative characterization of new glutaryl 7 ACA and cephalosporin C acylases. J Fennent Bioeng 1992 73 185-192. 

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