Evaluation of Different Glutaryl Acylase Mutants to Improve the Hydolysis of Cephalosporin C in the Absence of Hydrogen Peroxide

<p>This article <span>describes the development of a more efficient and environmentally friendly method for producing </span><strong>7-aminocephalosporanic acid (7-ACA). This com</strong><span>pound is the vital core nucleus used to manufacture semi-synthetic cephalosporin antibiotics.</span></p> <div><span>The key aspects of the research include:</span></div> <ul> <li><strong>The Tri-Enzymatic System:</strong><span> The study proposes an entirely enzymatic </span><strong>"one-pot"</strong><span> process using three enzymes: </span><strong>D-amino acid oxidase (DAAO)</strong><span>, </span><strong>catalase (CAT)</strong><span>, and </span><strong>glutaryl acylase (GAC). T</strong><span>his 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 (</span><span><span><span><span><span><span>H</span><span><span><span><span><span><span><span>2</span></span></span></span><span></span></span></span></span></span><span><span>O</span><span><span><span><span><span><span><span>2</span></span></span></span><span></span></span></span></span></span></span></span></span></span><span>), which inactivates the enzymes.</span></li> <li><strong>The Role of Catalase:</strong><span> By co-immobilizing catalase with DAAO, the researchers were able to eliminate </span><span><span><span><span><span><span>H</span><span><span><span><span><span><span><span>2</span></span></span></span><span></span></span></span></span></span><span><span>O</span><span><span><span><span><span><span><span>2</span></span></span></span><span></span></span></span></span></span></span></span></span></span> <em>in situ.</em><span> This </span><span> prevented enzyme inactivation and allowed the reaction to proceed in a single reactor.</span></li> <li><strong>Engineering Better Enzymes:</strong><span> A significant challenge was the low activity of the native (wild-type) GAC towards the intermediate product, </span><strong>2-oxoadipoyl-7-ACA. </strong><span>To </span><span>solve this, the authors evaluated several mutants of glutaryl acylase from </span><em>Pseudomonas</em><span> SY-77.</span></li> <li><strong>The Y178F+F375H Mutant:</strong><span> The research identified a specific double mutant, </span><strong>Y178F+F375H</strong><span>, as the most effective. </span><span>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.</span></li> <li><strong>Performance Improvements:</strong><span> When implemented in the tri-enzymatic system, this mutant achieved a </span><strong>95% conversion yield of 7-ACA</strong><span>, whereas the wild-type enzyme only reached about 60%. </span><span>Furthermore, the mutant reduced the reaction time required to reach a 50% yield by </span><strong>3.5 times.</strong></li> </ul> <div><span>Overall, the work demonstrates how </span><strong>protein engineering</strong><span> and the use of engineered mutants can significantly optimize industrial biocatalytic processes, making them faster, more efficient, and cleaner.</span></div>