High-Throughput Screening & Activity Profiling of Designed Enzyme Libraries

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Creative Enzymes provides High-Throughput Screening & Activity Profiling of Designed Enzyme Libraries Services to evaluate and optimize the performance of rationally designed enzymes. These services bridge the gap between computational design and practical application, transforming in silico predictions into measurable catalytic outcomes.

Using advanced high-throughput screening platforms, precision activity assays, and detailed kinetic analyses, we identify enzyme variants that exhibit superior properties such as increased activity, selectivity, or stability. Our integrated approach ensures that every rationally designed enzyme undergoes rigorous functional validation and quantitative assessment before downstream optimization or scale-up.

By combining structural insight, biochemical expertise, and state-of-the-art instrumentation, Creative Enzymes delivers a reliable and efficient route to transforming rational enzyme designs into proven biocatalysts.

Why High-Throughput Screening and Activity Profiling Matter

Rational enzyme design enables targeted modification of protein structures to achieve improved catalytic efficiency, altered substrate specificity, or enhanced environmental resilience. However, computational predictions alone are not sufficient—experimental confirmation is essential to ensure that theoretical improvements translate into functional performance.

High-throughput screening and activity profiling play a critical role in verifying the success of rational design. Through activity-based assays, binding analyses, and kinetic profiling, researchers can identify the most promising enzyme variants and understand the mechanistic implications of introduced mutations.

High-throughput screening allows rapid evaluation of a large number of designed variants, using diverse analytical platforms such as spectrophotometric, fluorometric, and chromatographic assays. Once high-performing candidates are identified, activity profiling and kinetic characterization can be used to quantify their catalytic parameters (K_M, k_cat, and k_cat/K_M), providing a mechanistic understanding of how structural modifications affect enzyme function.

Creative Enzymes integrates both stages—high-throughput functional screening and detailed activity and kinetic profiling—into a single, seamless service platform. Our multidisciplinary team combines expertise in enzymology, structural biology, and computational design, ensuring every rationally engineered enzyme is thoroughly evaluated with scientific precision and industrial relevance.

Molecular docking results of the native active site (NAS) of E1 and engineered active site (EAS) of EE Figure 1. Natural active site (NAS) and engineered active site (EAS) (Adapted from Krishna et al, 2024)

What We Offer: High-Throughput Screening & Activity Profiling

Creative Enzymes offers comprehensive services covering the entire process of functional validation for rationally designed enzymes:

Functional Screening of Designed Variants

Development of customized, high-throughput assays to detect enzymatic activity across variant libraries.
Screening under multiple conditions (pH, temperature, substrate concentration) to identify performance optima.
Evaluation of substrate range and reaction selectivity.

High-Throughput Assay Development

Design and optimization of colorimetric, fluorometric, or coupled enzyme assays.
Miniaturization of reactions for 96-, 384-, or 1536-well formats.
Integration of automated liquid-handling systems for efficient throughput.

Kinetic Characterization

Determination of key catalytic parameters: K_M, V_max, k_cat, and k_cat/K_M.
Michaelis–Menten and Lineweaver–Burk analyses for reaction kinetics.
Assessment of catalytic efficiency across different substrates and cofactors.

Thermodynamic and Stability Testing

Evaluation of thermal denaturation (Tm), half-life (t_1/2), and residual activity.
pH and solvent tolerance assays for industrial enzyme optimization.
Stability profiling under process-relevant conditions.

Substrate Binding and Specificity Analysis

Determination of binding affinities via surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC).
Analysis of substrate discrimination and product distribution.

Mechanistic Insights and Data Reporting

Correlation of kinetic outcomes with structural and computational models.
Comprehensive data analysis with graphical representations of kinetic curves and parameter fitting.
Technical reporting and optional recommendations for further optimization.

Service Workflow

Workflow diagram for high-throughput screening and activity profiling of rationally designed enzymes

Service Outputs

Service Module	Deliverables
Assay Development	Customized activity assay, validation data
High-Throughput Screening	Activity profiles for all variants
Kinetic Characterization	K_M, k_cat, k_cat/K_M data, kinetic curves
Stability Evaluation	Thermal/pH stability data
Substrate Specificity Analysis	Binding and activity data for multiple substrates
Final Reporting	Full technical report with interpretation

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Why Partner With Us

Integrated Computational and Experimental Expertise

We combine rational enzyme design with precise experimental validation, ensuring seamless continuity from theory to function.

Customizable Screening and Assay Development

Our in-house enzymology specialists tailor each assay to your enzyme's unique catalytic mechanism and industrial application.

Comprehensive Kinetic Evaluation

Detailed kinetic profiling provides a quantitative understanding of catalytic behavior, supporting informed decisions for further engineering.

High-Throughput Screening Capacity

Using automated platforms, we screen thousands of enzyme variants rapidly, reducing project timelines while maintaining accuracy.

Robust Data Interpretation and Mechanistic Insights

Each project includes correlation of kinetic data with enzyme structure and computational predictions, offering deeper functional insights.

Trusted Quality and Confidentiality

We maintain strict confidentiality agreements and adhere to rigorous quality standards to ensure reliability and data integrity.

Success Stories

Case 1: Screening Rationally Designed Hydrolase Variants for Improved Activity

Client Need:

An industrial biocatalysis company sought to enhance the efficiency of a hydrolase used in ester bond cleavage for fine chemical synthesis. The native enzyme showed moderate activity but poor turnover with bulky substrates, limiting its commercial viability for broader substrate classes.

Our Approach:

We applied computational rational design to generate a hydrolase variant library, targeting residues near the catalytic triad to stabilize the transition state and improve substrate accommodation. Using a miniaturized fluorometric screening assay, we rapidly evaluated catalytic turnover across hundreds of variants. Structural modeling and Rosetta-based energy scoring were integrated to prioritize designs with predicted lower activation barriers and increased binding flexibility.

Outcome:

A top-performing variant was identified with a 3.5-fold increase in catalytic efficiency (k_cat/K_M) and significantly improved tolerance toward larger ester substrates. The results validated our design hypothesis of enhanced transition-state stabilization and provided a robust candidate for process optimization in industrial synthesis workflows.

Case 2: Kinetic Characterization of a Thermostable Oxidoreductase

Client Need:

A biotechnology client developing thermostable enzymes for biofuel production required an oxidoreductase capable of maintaining high catalytic activity under elevated process temperatures. The wild-type enzyme denatured rapidly above 60°C, reducing yield and stability in bioreactor conditions.

Our Approach:

Through structure-guided rational design, we engineered specific mutations predicted to reinforce the enzyme's hydrophobic core and improve hydrogen-bonding networks at critical loop regions. Following expression and purification, the variant and wild type were subjected to detailed kinetic and thermodynamic characterization across a 25–85°C temperature range. Differential scanning fluorimetry (DSF) and steady-state kinetics were used to quantify thermostability and catalytic parameters.

Outcome:

The engineered oxidoreductase exhibited a 9°C increase in melting temperature (Tm) and twofold higher catalytic efficiency at 65°C compared to the wild type. These improvements confirmed successful structural stabilization and enabled consistent enzyme performance in high-temperature bioprocessing, helping the client reduce enzyme loading and overall production costs.

Frequently Asked Questions

Q: What types of enzymes can be screened and characterized?

A: We can evaluate all major enzyme classes—hydrolases, oxidoreductases, transferases, lyases, isomerases, and ligases—using customized activity assays appropriate for each mechanism.
Q: How many enzyme variants can be screened in a single project?

A: Our high-throughput systems can efficiently analyze hundreds to thousands of variants, depending on assay complexity and substrate requirements.
Q: Can you design a custom assay for a novel or proprietary enzyme?

A: Yes. Our enzymologists can develop fully customized detection systems—colorimetric, fluorometric, or chromatographic—tailored to your enzyme's reaction type.
Q: What kinetic parameters do you provide?

A: We report K_M, k_cat, V_max, and k_cat/K_M, along with temperature, pH profiles, and detailed kinetic curve fitting for Michaelis–Menten or alternative models.
Q: How do you ensure reproducibility of results?

A: All assays are performed with multiple replicates under tightly controlled conditions, with statistical validation and error analysis included in final reports.
Q: Can you integrate these results into further enzyme engineering steps?

A: Absolutely. Functional and kinetic data can directly guide site-directed mutagenesis, loop optimization, or directed evolution for next-stage enhancement.
Q: What information do you require to start the project?

A: We typically need the enzyme sequence, structural model or PDB file (if available), and any known activity or assay data. Our team will then design a detailed experimental plan.
Q: What is the expected project timeline?

A: Depending on project complexity and the number of variants, the full workflow typically requires 6–10 weeks from assay setup to final reporting.

Reference:

Krishna NB, Roopa L, Pravin Kumar R, S GT. Computational studies on the catalytic potential of the double active site for enzyme engineering. Sci Rep. 2024;14(1):17892. doi:10.1038/s41598-024-60824-x

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Project Description:

For research and industrial use only. Not intended for personal medicinal use. Certain food-grade products are suitable for formulation development in food and related applications.

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