Cephalosporin C Acylase from Microbes for One-step Enzymatic Transformation of Cephalosporin C to 7-Aminocephalosporanic Acid

Cephalosporins are the βββββ -lactam antibiotics for the treatment of infection diseases caused by gram-positive and gram-negative bacteria. Fungus Cephalosporium acremonium produces cephalosporin C (CPC) that is not potent for clinical use. Its molecule can be transformed to 7-amino cephalosporanic acid (7-ACA) as the intermediate compound for making semi-synthetic cephalosporin derivatives. The first method for production of 7-aminocephalosporanic acid involves the chemical reaction using toxic reagents and laborious procedures. The second method uses two-step and one-step enzymatic transformation. In two-step method, the first step involves the conversion of Ceph-C to Glutaryl-7-amino cephalosporanic acid (GL-7-ACA) by D-amino acid oxidase (DAAO) and the second step, GL-7-ACA acylase (GA) hydrolyzes GL-7-ACA to produce 7-ACA. The two-step enzymatic transformation was used widely because of its safety and environmental friendship. The one-step enzymatic transformation is developed because of process simplification and cost reduction. This method uses cephalosporin C acylase for transformation CPC to 7-ACA. Same microbes can produce cephalosporin C acylase such as Achromobacter xylosoxidans , Aeromonas sp., Arthrobacter viscosus

Cephalosporin C (CPC) was the second β-lactam antibiotics to be discovered after penicillin from the fungus Cephalosporium acremonium by Giuseppe Brotzu 1,2 .CPC showed a moderate antibacterial activity: minimum inhibitory concentration values were in the range of 25-100 and 12-25 µg/mL for gram-positive and gramnegative bacteria, respectively 2 and is not potent for clinical use 3 .Robert Morin discovered a process by which the D-α-amino adipoyl side chain of CPC could be removed, generating 7aminocephalosporanic acid (7-ACA) in 1962 2 .7-ACA is the intermediate for semi-synthetic cephalosporin derivates.Some semi-synthetic cephalosporin such as ceftezole, cefotiam, cefazedone, and cefazolin were produced by 7-ACA 4 .
The cephalosporin antibiotics are divided into two groups; the first is derived from penicillin (G or V) and the second from CPC.The most products that have intrinsic oral activity are better made from penicillin, while the most products made from CPC are insufficiently absorbed from the human gut to be therapeutically adequate by the oral route, unless converted to pro-drugs by esterification 5 .The cephalosporins are clinically active against gram-positive as well as gramnegative organisms.Cephalosporins are broadspectrum, less toxic and resistant to degradation of β-lactamase as compared to penicillins 1,3 .

Conversion CPC to 7-ACA
Cephalosporins derivates are semisynthetic products that derive from the fermentative product CPC.CPC is initially converted to 7-ACA by either a chemical or an enzymatic catalysis removal of the 7-amino adipoyl side chain.The chemical catalysis was developed by Morin et al. and this process is used to 7-ACA production in an industrial scale (Scheme 1) 5 .Enzymatic transformation of CPC to 7-ACA is the best alternative and has industrial significance 3 .
The chemical method has been replaced by enzymatic method for producing 7-ACA because of (1) safety and environmental contamination: dangerous and toxic reagents (i.e., trimethylchlorosilane, phosphorus pentachloride, dichloromethane, dimethylaniline, etc.) are absent in the enzymatic transformation 1,2 ; (2) selectivity: owing to enzyme selectivity, the use of permanent or temporarily protective groups is avoided 2 ; (3) energy consumption: the chemical process uses low temperatures and exothermic steps, while the enzymatic approach is carried out at a fixed temperature of 20-30°C 2 ; (4) equipment: owing to the use of aggressive reagents, the chemical process requires glass-lined vessels and dedicated and expensive equipment to transport and charge these materials, to trap spilling or vapors, etc. 2 ; and (5) quality: in the enzymatic 7-ACA preparations, the levels of oligomers, 7desacetoxycephalosporanic acid, and desacetyl-7-ACA (or corresponding lacton) are considerably lower-the typical assay is 3-6 % higher than for the chemical process 2 .
GAs (glutaryl acylases; EC 3.5.1.93)have been classified into five classes on the basis of their sequence, molecular mass and enzymatic properties.Members of each class are very similar on the basis of substrate specificity and sequence conservation (they share >90%of nucleic acid or amino acid sequence identity).Members of class I (e.g.P130 from Pseudomonas sp.130) and class III (e.g.N176 from Pseudomonas sp.N176) show the highest activity to CPC (4%relative to Gl-7ACA) 10 .
Glutaryl acylase (GA) is a metal free heterodimer of 16 kDa+54 kDa subunits without any prosthetic group.This enzyme has been isolated from many organisms, but only the one from Pseudomonas diminuta was cloned and expressed in a recombinant E. coli and has been developed into an industrial biocatalyst 5 .GA activity has been reported in bacteria from Aeromonas 2 , Arthrobacter 2 , Bacillus 2 ,and Flavobacterium 2 , Pseudomonas 2, 5 , and from the fungus Paceilomyces 2, 3 .
The alternative enzymatic approach is a one-step enzymatic method process in which CPC is converted directly into 7-ACA by a CPC acylase (CA; Scheme 2).As shown in Scheme 2, acylases can cleave the amide bond through which the cephem nucleus is linked to the acyl side chain 2 .Cephalosporin acylase (CA, EC 3.5.1.11)is a member of the N-terminal nucleophile (Ntn) hydrolase superfamily, in which the precursor gene is translated into a single polypeptide chain and then folded into a self-activating pre-protein activated by intra-molecular double cleavages.The first cleavage is most important for generating the active center catalysis residue (Ser) of the enzyme at the N terminus of the β-subunit.The second cleavage is also essential for activating the enzyme, forming a mature heterodimer with the αand βsubunits and releasing a spacer peptide of different lengths (8-11 aa) in varying CAs.According to the diversity in substrate specificity, CAs can be divided into two classes, the cephalosporin C (CPC) acylase and the glutaryl-7aminocephalosporanic acid (GL-7-ACA) acylase classes.Both enzymes classes form 7aminocephalosporanic acid (7-ACA) 11 .Sonawane (2006) classified CA base on their substrate (CPC, GL-7-ACA, and GL-7-ADCA) specificity.The types of cephalosporin acylase are: type 1 is true cephalosporin C acylase.CPC acylase can hydrplyze only CPC and is not active for GL-7-ACA; type 2 GL-7-ACA acylase.This enzymes can hydrolyze GL-7-ACA to 7-ACA in high yield but have less activity against CPC; type 3 GL-7-ADCA acylase.Their enzymes have activity against GL-7-ADCA but their activity against GL-7-ACA is not known 12 .
The natural of cephalosporin C acylase catalyzed the CPC to 7-ACA directly in a very low efficiency 14,15,16 , significant substrate inhibition 14 , and product inhibition 14 .In recent years, protein engineering of cephalosporin C acylase was developed to overcome the above limitations 14,15 .Protein engineering approach based on combined use of error-prone PCR mutagenesis, molecular modeling, site saturation and site-directed mutagenesis 17 were used to enhance the catalytic efficiency of CPC acylase.Wang   14 ; Pollegiani et al. found two mutants of the CPC acylase from Pseudomonas N176, M31βF/H57βS/H70âS and A215αY/M31βF/H70βS showed 3.3-and 4.3 fold improvements in V max on CPC compared with its starting template (M31βF), respectively with eliminated substrate inhibition and reduced product inhibition 14 ; A mutant of the CPC acylase acyII from Pseudomonas SE83, V122αA/G140αS/F58βN/I75βT/I176βV/S471βC (S12) showed a 7.5-and 3.7-fold improvement in the specific activity and K ip , respectively, compared with the wild type 14 ; Ishii et al. mutated Met269 and Ala271 of cephalosporin C acylase can increase activity 1.6-fold and 1.7-fold respectively 15 ; Ren et al. found a mutant of the cephalosporin acylase from gene acyII in Pseudomonas sp., G139αS/ F58βN/I176βV/S471βC showed 2.86-fold improvement in K cat /K m compared with wild type 14 ; Oh et al. found that the deacylation activity of the mutation Q50βM-Y149αK-F177αG toward CPC improved by 7.9-fold 16 .

1.
The biotransformation of cephalosporin C to 7-ACA in one step enzymatic with cephalosporin acylase is a simple process with low cost and potential to applied in industrial production.

2.
The protein engineering of cephalosporin C acylase was developed to increase its activity and stability of CPC acylase.