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Background: Cephalosporins are the most widely used semisynthetic antibiotics, which acted on bacterial cell wall (peptidoglycan) synthesis. The key intermediate for fabricating about twothirds of cephalosporins in clinical use is 7-aminocephalosporanic acid (7-ACA), which is derived from chemical or enzymatic deacylation of the natural antibiotic cephalosporin C (CPC). The chemical deacylation process has been replaced by the enzymatic deacylation process because the chemical process required harsh conditions and released toxic waste. Methods: A two-step enzymatic process that utilized D-amino acid oxidase (DAAO) and 7-β-(4- carboxybutanamido)-cephalosporanic acid acylase (GLA) for two successive reactions has been applied for the conversion of CPC to 7-ACA in an industrial scale. Results: To simplify the process and lower costs, the one-pot enzymatic processes were developed by the application of the mono-enzymatic process (application of cephalosporin C acylase or the variants of GLA), di-enzymatic process (simultaneous action of DAAO and GLA) or the tri-enzymatic process (simultaneous action of DAAO, GLA and catalase) for direct conversion of CPC to 7-ACA. Conclusion: Here, we mainly focused on the description of these one-pot enzymatic processes and emphasized on the preparation of the involved biocatalysts.