This article describes the development of a more efficient and environmentally friendly method for producing 7-aminocephalosporanic acid (7-ACA). This compound is the vital core nucleus used to manufacture semi-synthetic cephalosporin antibiotics. The key aspects of the research include: The Tri-Enzymatic System: The study proposes an entirely enzymatic "one-pot" process using three enzymes: D-amino acid oxidase (DAAO), catalase (CAT), and glutaryl acylase (GAC). This system is designed to replace traditional industrial methods that rely on hazardous chemicals or "two-pot" enzymatic routes that are hindered by the presence of hydrogen peroxide (H2O2), which inactivates the enzymes. The Role of Catalase: By co-immobilizing catalase with DAAO, the researchers were able to eliminate H2O2 in situ. This prevented enzyme inactivation and allowed the reaction to proceed in a single reactor. Engineering Better Enzymes: A significant challenge was the low activity of the native (wild-type) GAC towards the intermediate product, 2-oxoadipoyl-7-ACA. To solve this, the authors evaluated several mutants of glutaryl acylase from Pseudomonas SY-77. The Y178F+F375H Mutant: The research identified a specific double mutant, Y178F+F375H, as the most effective. Molecular modeling revealed that these mutations created a more favorable spatial arrangement in the enzyme's binding pocket, allowing it to better accommodate the substrate's side-chain. Performance Improvements: When implemented in the tri-enzymatic system, this mutant achieved a 95% conversion yield of 7-ACA, whereas the wild-type enzyme only reached about 60%. Furthermore, the mutant reduced the reaction time required to reach a 50% yield by 3.5 times. Overall, the work demonstrates how protein engineering and the use of engineered mutants can significantly optimize industrial biocatalytic processes, making them faster, more efficient, and cleaner.
López‐Gallego et al. (Thu,) studied this question.