Enzyme Active Site Engineering Service

Enzyme Active Site Engineering is a highly specialized service aimed at precisely modifying the enzyme's catalytic core to alter its functional properties, such as changing substrate specificity, enhancing catalytic efficiency, or reversing stereoselectivity. This rational approach utilizes data from structural biology, molecular modeling, and computational chemistry to pinpoint critical amino acid residues near the binding pocket. By strategically redesigning the active site geometry and electrostatic environment, we can generate bespoke biocatalysts with superior performance for specific industrial, chemical, or pharmaceutical applications.

CD Biosynsis offers end-to-end Active Site Engineering services, combining advanced in silico design with efficient wet-lab execution. We start by modeling the enzyme-substrate complex to identify residues governing binding and turnover. We then employ computational tools to propose and screen targeted mutations, followed by gene synthesis, expression, and functional characterization of the variants. Our service minimizes the reliance on random mutagenesis and high-throughput screening by focusing on high-probability mutations, significantly reducing project timelines and costs while achieving unparalleled control over enzyme function.

Highlights

We provide a highly rational and efficient route to engineered enzymes with custom-defined properties.

• Rational Mutation Design: Utilize predictive modeling (docking, MD) to focus mutagenesis efforts on residues most likely to impact the desired function.
• Targeted Specificity Switch: Precisely redesign binding pockets to switch recognition from a native substrate to a bulkier or chemically distinct industrial substrate.
• Stereoselectivity Reversal: Engineer active site residues to force the substrate into an inverted binding orientation, achieving high enantioselectivity (R to S, or vice versa).
• Enhanced Catalytic Rate: Introduce mutations that optimize transition state stabilization or improve the geometry of catalytic residues for higher turnover (kcat).

Applications

Active site engineering is vital for developing high-performance biocatalysts for industrial and therapeutic uses:

Novel Biocatalyst Development

Creating enzymes capable of catalyzing non-natural or chemically challenging reactions with high efficiency and purity.

Drug Metabolite Synthesis

Engineering cytochrome P450s or other enzymes to selectively produce specific drug metabolites for preclinical studies.

Biosensor Enhancement

Tailoring enzyme recognition for highly specific, high-sensitivity detection of target molecules (e.g., toxins, glucose) in diagnostic devices.

Reduced Product Inhibition

Modifying the active site to allow efficient product release, thereby preventing inhibition and enabling high-yield continuous reactions.

Platform

Our platform integrates computational design and experimental validation for a comprehensive engineering solution.

Computational Saturation Mutagenesis

In silico scanning of all 20 amino acids at targeted active site positions, predicting the binding affinity and stability of each mutant.

Targeted Library Design

Design of small, highly enriched mutant libraries (e.g., NNS/NNK) based on promising in silico predictions, reducing screening burden.

High-Fidelity Gene Synthesis

Synthesis and assembly of single or multiple discrete point mutations with 100% sequence accuracy, ready for expression.

High-Throughput Functional Screening

Rapid screening of the engineered variants using custom-designed assays to quantify the improvement in activity or selectivity.

Kinetic and Stability Characterization

Full characterization of successful variants, including Km, kcat, kcat/Km, and thermal denaturation temperature (Tm).

Workflow

Our Active Site Engineering process is a cyclical workflow ensuring optimal design and functional success:

• Computational Design and Analysis: Perform Enzyme-Substrate Interaction Modeling to identify key residues for mutation (e.g., residues within 5 Å of the substrate).
• In Silico Prediction: Use stability and binding energy prediction tools (e.g., Rosetta, MM/GBSA) to select the highest-scoring single and double mutants.
• Gene Synthesis and Cloning: Synthesize the DNA sequence encoding the selected mutant enzymes and clone them into a suitable expression vector.
• Expression and Purification: Express the enzyme variants in the chosen host system (E. coli, yeast, etc.) and purify the recombinant protein.
• Functional Characterization and Validation: Test the purified variants using high-resolution kinetic assays to quantify the improvement in the target property (e.g., kcat/Km for the new substrate).
• Next Round Iteration (Optional): Use data from characterization to inform the next round of rational design for further optimization (multi-site mutagenesis).

CD Biosynsis delivers functionally verified, engineered enzymes with comprehensive data packages to support commercialization. Every project includes:

• Design Report: Detailed rationale for all selected mutations based on structural and computational data.
• Functional Clone: Delivery of the plasmid containing the optimized enzyme sequence, verified by Sanger sequencing.
• Full Kinetic Data: Quantitative comparison of the wild-type and engineered enzyme's performance (Km, kcat, selectivity ratio).
• Structural Models: Predicted 3D models of the mutant enzyme bound to the target substrate, ready for publication.