Enzyme Stability Engineering Service

Enzyme Stability Engineering is a critical protein design service focused on enhancing an enzyme's resilience against harsh operational conditions, including high temperatures, extreme pH levels, high concentrations of organic solvents, or proteolysis. Enzymes with improved stability are essential for industrial biocatalysis, therapeutic applications where longevity in vivo is required, and biomanufacturing processes where cost-efficiency depends on catalyst reuse. Our engineering approach combines rational design, based on structural analysis, with directed evolution techniques for high-throughput screening.

CD Biosynsis offers comprehensive solutions for Enzyme Stability Engineering, utilizing both computational modeling and experimental platforms like Yeast Surface Display (YSD) and Phage Display. We analyze the enzyme's three-dimensional structure to identify vulnerable regions, such as surface loops and hydrophobic core residues, guiding rational introduction of stabilizing disulfide bonds, optimized hydrogen bond networks, and improved core packing. For directed evolution, we design selection schemes that expose mutant libraries to the target stress condition, efficiently identifying variants with superior thermal or chemical resistance. Our goal is to deliver industrial-grade enzymes with significantly extended half-lives and operational robustness.

Highlights

We provide effective strategies to increase enzyme resilience without compromising catalytic function.

• Enhanced Thermostability: Significant increase in melting temperature (Tm) and activity half-life at high process temperatures.
• Chemical Resistance: Improved tolerance to denaturing agents like chaotropes, detergents, or high concentrations of organic solvents.
• Proteolysis Resistance: Rational redesign of surface loops and cleavage sites to prevent degradation by proteases in industrial or biological environments.
• Longer Shelf-Life: Increased stability during storage and formulation, reducing inventory costs and improving product reliability.

Applications

Stability engineering is crucial for enabling enzymes in harsh real-world applications:

Industrial Biocatalysis

Developing enzymes stable at high temperatures or in organic media required for efficient chemical synthesis and API manufacturing.

Therapeutic Enzyme Delivery

Enhancing stability for in vivo administration, such as resistance to blood proteases or stability in the low pH of the stomach.

Bioremediation

Engineering enzymes to remain active under the high pollutant concentrations and variable conditions found in waste streams or environmental clean-up sites.

Detergent and Food Enzymes

Improving resistance to high alkalinity, high heat, and the presence of surfactants or other ingredients in cleaning and food processing products.

Platform

Our platform employs a complementary mix of rational design and high-throughput screening techniques.

Computational Stability Prediction

Using algorithms (e.g., Rosetta, FoldX) to scan all possible single-point mutations and predict their stabilizing effect (delta delta G) on the protein structure.

Rational Design of Stabilizing Motifs

Targeted introduction of extra disulfide bonds, creation of stabilizing salt bridges, and optimization of hydrophobic core packing.

High-Throughput Stability Screening

Utilization of display technologies (Phage or Yeast) coupled with high-temperature or solvent stress steps before functional sorting.

Focused Consensus Design

Incorporating sequence patterns derived from highly thermostable homologous enzymes (consensus approach) into the target enzyme.

Biophysical Validation

Characterization of successful variants using Differential Scanning Fluorimetry (DSF) or Circular Dichroism (CD) to accurately measure the melting temperature (Tm).

Workflow

Our Stability Engineering workflow is a systematic approach to identify and implement stabilizing mutations:

• Structural Analysis and Vulnerability Mapping: Analyze the enzyme's 3D structure to locate regions prone to unfolding (e.g., terminal regions, surface loops).
• Computational Design and Prioritization: Perform in silico saturation mutagenesis or disulfide bond design to predict the most stabilizing mutations.
• Library Construction: Generate a focused mutant library based on rational predictions, or a random library for directed evolution screening.
• Stress Selection (High-Throughput): Subject the displayed or expressed library to the target stress condition (heat, solvent, pH) for a defined period.
• Functional Screening/Sorting: Screen the surviving variants for maintained activity or affinity, and amplify the best-performing clones.
• Biophysical Validation and Iteration: Purify the best hits, measure the increase in Tm or resistance, and combine synergistic mutations for further improvement.

CD Biosynsis delivers enzymes with validated, high stability profiles ready for harsh applications. Every project includes:

• Stability Design Report: Detailed analysis of stabilizing mutations and the rationale behind their selection.
• Engineered Clone: Delivery of the plasmid containing the optimized, stable enzyme sequence.
• Biophysical Data: Quantitative measurement of the enzyme's melting temperature (Tm) or half-life in the target solvent, showing the fold-increase in stability.
• Kinetic Verification: Confirmation that the stability mutations did not negatively impact the catalytic efficiency (kcat/Km).