Engineering Highly Active and Enantioselective d-Lactate Hydrolase (D-Lac) Through Rational Design

d-lactate hydrolase (D-Lac) is an important enzyme for the production of chiral compounds, however, its low activity and enantioselectivity often limit its practical applications. To overcome these limitations, we employed a rational design approach to enhance the enzyme's catalytic properties. Our strategy involved a series of protein engineering steps, including:

1. Computational Analysis and Target Identification: Employing computational tools, we performed in-depth analysis of the D-Lac structure to identify amino acid residues potentially crucial for substrate binding and catalysis.
2. Site-Directed Mutagenesis: Based on the computational predictions, we introduced specific mutations at the identified target sites using site-directed mutagenesis techniques.
3. Screening and Characterization: Mutant D-Lac variants were expressed, purified, and subsequently screened for their activity and enantioselectivity towards d-lactate.
4. Evaluation and Optimization: The catalytic performance of promising D-Lac variants was further evaluated under various reaction conditions to optimize their performance.

By systematically implementing these steps, we aim to develop a D-Lac variant with significantly improved activity and enantioselectivity, thereby enhancing its potential for diverse biotechnological applications.