Enzyme Specificity & Selectivity Engineering Service

Enzyme Specificity & Selectivity Engineering focuses on modifying an enzyme's active site to precisely control which substrates are recognized (specificity) and which reaction pathway is favored (selectivity), particularly in the context of creating a single desired enantiomer, diastereomer, or regiomer. This service is crucial for high-value synthesis in the pharmaceutical, fine chemical, and agrochemical industries, where tight control over molecular structure is essential. We employ a combination of rational design based on crystal structure analysis and high-throughput directed evolution techniques to achieve precise molecular recognition.

CD Biosynsis offers comprehensive services for tailoring enzyme specificity and selectivity. Our approach involves redesigning the active site pocket—modifying residue size, charge, and polarity—to optimize non-covalent interactions with the desired substrate while disfavoring unwanted side reactions or non-target substrates. For improving enantioselectivity, we use advanced screening platforms like Yeast Surface Display (YSD) coupled with customized fluorescent probes that distinguish between different chiral products. This enables the efficient selection of enzyme variants that produce the required product with superior enantiomeric excess (ee) and regioselectivity.

Highlights

We deliver enzymes with optimized active sites for highly controlled and efficient biocatalysis.

• Enhanced Substrate Specificity: Increasing the catalytic efficiency (kcat/Km) for a target substrate while decreasing activity toward non-target or homologous substrates.
• Improved Enantioselectivity: Achieving high enantiomeric excess (ee) or diastereomeric ratio (dr) in the final product, crucial for chiral drug synthesis.
• Modified Regioselectivity: Controlling the exact position on a complex molecule where the catalytic reaction occurs (e.g., specific hydroxyl group in a multi-alcohol substrate).
• Reduced Byproducts: Minimizing side reactions and byproduct formation, leading to cleaner reaction mixtures and simpler purification processes.

Applications

Specificity and selectivity engineering drives efficiency in complex chemical manufacturing:

Asymmetric Synthesis

Developing highly selective enzymes (e.g., lipases, reductases) to catalyze the formation of single enantiomers for chiral drug molecules and precursors.

Non-Natural Substrate Utilization

Engineering enzymes to accept and efficiently catalyze reactions using structurally novel or non-natural amino acids, sugars, or metabolites.

Probing Reaction Mechanisms

Generating site-specific mutants to study how the active site structure dictates substrate orientation and catalytic trajectory.

Biosynthetic Pathway Optimization

Tuning the specificity of key enzymes in metabolic pathways to increase the titer and purity of a desired final product.

Platform

We integrate rational design, molecular modeling, and specialized high-throughput screening for precision engineering.

Active Site Molecular Modeling

Structural analysis (using PDB data) and docking simulations to identify key active site residues that interact with the substrate's stereocenters.

Saturation Mutagenesis (CAST/ISM)

Focused library generation at 2-5 key active site positions (e.g., residues defining the binding pocket volume) to systematically alter specificity.

Differential Fluorescent Screening

Using two fluorescent substrates or probes (one for the desired product, one for the undesired) in FACS to select for high selectivity ratio.

Negative Selection

Inclusion of the undesired substrate or a competing reaction in a high concentration during the selection round to actively discard mutants with broad specificity.

GC/HPLC Product Analysis

Final validation of the best-performing clones using advanced chromatography (GC/HPLC) to precisely determine the enantiomeric or regiomeric excess.

Workflow

Our engineering workflow focuses on the precise manipulation of the enzyme's active site:

• Structural and Mechanistic Analysis: Define the substrate binding pocket geometry and the proposed catalytic mechanism influencing selectivity.
• Rational Design and Library Construction: Design small, highly focused libraries targeting residues within 5-8 Å of the substrate (e.g., using Combinatorial Saturation Mutagenesis).
• High-Throughput Screening/Sorting: Screen the library using display platforms (YSD or Phage) and a customized selection assay that measures the desired selective reaction.
• Negative and Positive Selection: Alternate between positive selection (enriching desired activity) and negative selection (excluding undesired activity) to sharpen specificity.
• Product Analysis: Scale up the most enriched variants and perform detailed GC or HPLC analysis to measure the final enantiomeric excess (ee) or regiomeric purity.
• Iterative Refinement: Use the structural data from the best mutants to guide subsequent rounds of saturation or combinatorial mutagenesis for further improvement.

CD Biosynsis delivers enzymes with verified high selectivity, meeting stringent industry standards. Every project includes:

• Design and Selection Report: Detailed analysis of active site modifications and the rationale for the final selection strategy.
• Engineered Clone: Delivery of the plasmid containing the optimized enzyme sequence, verified by sequencing.
• Selectivity Data: Precise measurement of enantiomeric excess (ee), diastereomeric ratio (dr), or regiomeric purity achieved by the final enzyme.
• Kinetic Verification: Confirmation that the specificity modifications did not drastically compromise the overall catalytic rate (kcat/Km).