One-pot conversion of cephalosporin C by using an optimized two-enzyme process

The main industrial process for producing 7-aminocephalosporanic acid (7-ACA), a precursor of semi-synthetic cephalosporin antibiotics, is the two-step enzymatic route based on d-amino acid oxidase and glutaryl acylase working in separate reactors. Taking advantage of the recently developed variants of cephalosporin C acylase from Pseudomonas N176 (named VAC) and the optimized recombinant overproduction of the two enzymes, we set up a one-pot system to directly convert cephalosporin C into 7-ACA. We optimized the process by identifying the most favorable operational conditions, substrate, and enzyme concentrations. Among the VAC variants employed, our results indicated that HS-HS-F72βR VAC is the best choice because of the high activity on both substrates (glutaryl-7-ACA and cephalosporin C) and the absence of substrate and product inhibition effects. Under optimized conditions and by adding further aliquots of the biocatalysts, >98% of cephalosporin C was converted, yielding 7-ACA as the main reaction product (oxo-7-ACA was below the detection level and glutaryl-7-ACA was <1 mM). At the 20 mL bioconversion scale, approximately 81 mg of 7-ACA are produced in 41 hours from 15 mM cephalosporin C. The low cost of the one-pot enzymatic production of 7-ACA is the main advantage of the proposed method, which is further strengthened by the good purity of the final product