ACA acylase

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... 

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,... 

Scheme 9 D-Amino acid oxidase (DAAO) oxidation of 13 to N-glutaiyl-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.

Yang YL, Yun DE, Guan YQ, Peng HL, Chen JM, He YS, Jiao RC. Cloning of GL-7-ACA acylase gene from Pseudomonas sp. 130 and its expression in Escherichia coli. Chin. J. Biotechnol. 1991 7(2) 93-104. 

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. 

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)...

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 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. 

L-Phenylalanine has been produced continuously from ACA with the help of ACA acylase in an enzyme membrane reactor (EMR) with a space-time-yield of 277 g L 1 d1 83 . With ACL acylase, L-Ieucine was produced at 123-180 g L-1 d 1 in the same reactor set-up1821. The dehydroamino acid substrates can be prepared conveniently, either from 2-halogen carboxylic acid esters1841, or, specifically in the case of ACA, via the acetamidomalonic ester route by reaction with benzyl halogenides[851. 

Cephalosporin acylases include two types of enzymes cephalosporin C acylase (CC ac-ylase), which can hydrolyze cephalosporin C and 7-p-(4 Carboxybutanamtdo)cephal( po ranic acid GL-7-ACA) to 7-ACA and GL-7-ACA acylase, which can hydrolyze GL-7-ACA to 7-ACA (Figure 1). GL-7-ACA is generated from ccphalo rtn C by the action... 

The reaction scheme for enzymatic synthesis of 7-ACA from c halosportn C is shown in Figure 1. The pathway involves the oxidative deamination of cephalosporin C into 7 P (5cephalosporanic acid (keto-AD-7-ACA) by DAO. A portion of the product further reacts nonenzymatically with hydrogen peroxide, which is formed as a by-product in the above reaction, to give GL-7-ACA. GL-7-ACA is hydrolyzed by GL-7-ACA acylase to 7-ACA. C7 side chains of cephalosporin C, keto-AD-7-ACA, and GL-7-ACA ate hydrolyzed by GC acylase to 7-ACA. However, the CC acylase activity for cephalo xirin C is low compared with that for GL-7-ACA. 

An immobilized GL-T-ACA acylase of Pseudomoruis sp. GK16 has been utilized for industrial production of 7-ACA by the chemical and enzymatic method from cephalosporin C via GL 7 ACA by the Asahi Chemical Company (4,21). Several additional cephalosporin acylase genes have been reported (22—28). Enzymatic properties of various cephalosporin acylases are summarized in Table 1, and amino acid sequences of these are shown in Rgure 3. 

Molecular sizes of eniyroes were determined by SDS-PAOE or amino acid sequences deduced from DMA sequences, and the subunit structures were designated as a (small subunit) and p (large subunit). However. J1 GL-7-ACA acylase is a single polypeptide. 

GL-7-ACA acylase from Pseudomonas sp. C427 (C427) (23), and penicillin G acylase from E. cot ATCX ilI05 (PAC) (30). Amino aci of c ihalosporin and penicillin acylases identical with CX acylase from P. diminuta V22 are shovim as. ... 

Aramori et al. (24) cloned an another type of GL-7-ACA acylase gene from Bacillus kuerosporus Ji. The DMA sequence revealed an open reading frame of 1902 bp coding for 634 amino acid residues, and the acylase was composed of a single polypeptide with the molecular size of 70 kDa analyzed by SDS-PAGE. This acylase has a unique molecular structure compared with those of cephalosporin acylases of Pseudomojuis. The J1 GL-7-ACA acylase hydrolyzed GL-7-ACA to 7-ACA, but it could not hydrolyze cephalosporin C and keto-AD 7 ACA (24). 

---