Identification and Screening of Other Ligands

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The identification and characterization of non-substrate and non-inhibitor ligands play an increasingly significant role in enzyme engineering, drug discovery, and biochemical research. Beyond traditional substrate and inhibitor screening, these ligands—ranging from activators and cofactors to folding modulators and structural stabilizers—offer essential insights into enzyme function and regulation. At Creative Enzymes, our Identification and Screening of Other Ligands service provides comprehensive, data-driven solutions to discover and evaluate these critical molecular partners, supporting your research from hypothesis generation to validated results.

Understanding Enzyme "Secondary" Ligands

While substrate–enzyme and inhibitor–enzyme interactions have been extensively characterized, enzymes often rely on a diverse network of auxiliary ligands to function efficiently. These ligands—ranging from small-molecule activators and metal cofactors to folding facilitators, chaperones, and aggregation modulators—play crucial roles in maintaining enzyme structure, stability, and catalytic balance.

Illustration of an enzyme interacting with multiple cofactors that modulate catalytic activity Figure 1. The succinate dehydrogenase complex showing several cofactors, including flavin, iron–sulfur centers, and heme.

In both natural and engineered systems, such interactions can profoundly influence enzyme kinetics, conformational dynamics, and overall process performance. For example, an activator may enhance turnover rate by stabilizing a catalytically competent conformation, while a cofactor can enable entirely new reaction pathways. Conversely, the absence of supportive ligands can lead to reduced activity, aggregation, or loss of stability during industrial use.

Despite their importance, these "secondary" ligands are frequently underexplored in standard enzyme optimization workflows, which tend to prioritize substrates and inhibitors. Recognizing this gap, Creative Enzymes has developed a comprehensive platform that integrates biochemical assays, high-throughput screening, and computational modeling to systematically identify and evaluate such ligands. This approach ensures a holistic understanding of enzyme behavior, empowering clients to fine-tune enzyme performance, enhance formulation stability, and accelerate development in both biopharmaceutical and industrial biotechnology applications.

Our Services for Other Ligands Identification and Screening

Our Identification and Screening of Other Ligands service provides a flexible and data-driven platform designed to uncover a wide spectrum of molecular interactions influencing enzyme performance.

Service Details

Purpose and Scope

Identify ligands beyond substrates and inhibitors that impact enzyme activity, stability, and structure.
Support diverse research goals such as enhancing catalytic efficiency, improving folding behavior, and minimizing aggregation.

Core Approaches

High-throughput screening (HTS): Rapidly evaluate large numbers of potential ligands.
Biophysical characterization: Assess changes in enzyme conformation, binding affinity, and thermal stability.
Computational modeling: Predict ligand interactions and refine hit selection through in silico analysis.

Ligand Types Covered

Small organic molecules
Metal ions and cofactors
Peptides and protein-based modulators
Chemical additives and stabilizers

Analytical Focus

Monitor variations in enzyme kinetics, folding state, and aggregation profile under ligand influence.
Quantify ligand-induced effects using spectroscopic, calorimetric, and structural methods.

Deliverables and Outcomes

Comprehensive datasets summarizing ligand–enzyme interactions.
Mechanistic insights that guide rational enzyme engineering and formulation design.
Practical recommendations for optimizing enzymatic performance and process robustness.

Service Workflow

Workflow diagram of Creative Enzymes’ identification and screening process for enzyme-associated ligands

Contact Our Team

Our Comprehensive Services

Services	Description	Price
Screening of Enzyme Activators	This service focuses on identifying small molecules or ions that enhance enzyme activity through allosteric modulation, active site stabilization, or structural reinforcement. By integrating kinetic assays with computational analysis, we help pinpoint activators that boost catalytic performance and process efficiency.	Get a quote
Evaluation of Cofactors and Additives	Cofactors and additives often determine enzyme functionality and stability. This service systematically assesses cofactor preferences, analog efficiency, and formulation additives to optimize enzymatic performance under target conditions.
Analysis of Enzyme Aggregation and Oligomerization	Understanding enzyme aggregation and oligomerization is crucial for both formulation stability and biological activity. Our analysis employs biophysical and biochemical methods to evaluate how ligands, buffers, and additives influence enzyme assembly and aggregation behavior.
Identification of Enzyme Folding Facilitators	This service identifies molecular chaperones, osmolytes, and chemical folding aids that assist in correct enzyme folding and refolding. By enhancing folding efficiency, we help clients improve enzyme yield, activity, and stability during expression and purification.

Why Choose Creative Enzymes

Comprehensive Ligand Coverage

We go beyond conventional substrates and inhibitors to explore a broad range of chemical and biological entities that influence enzyme performance.

Customizable Screening Platforms

Flexible assay designs accommodate enzymes from diverse classes and origins, supporting soluble, membrane-bound, and multimeric systems.

Integrated Analytical Technologies

We combine kinetic, thermodynamic, and structural data through advanced analytical instruments such as DSC, ITC, DSF, and SPR.

Computational Support

In silico modeling and molecular docking assist in ligand prediction, hit prioritization, and structure–function correlation.

Expert Scientific Team

Our enzymology specialists and biochemists possess extensive experience in assay development, enzymatic kinetics, and ligand interaction studies.

Reliable and Reproducible Results

Stringent quality control and validation steps ensure the accuracy, consistency, and scientific rigor of all experimental outcomes.

Case Studies and Real-World Applications

Case 1: Discovery of Selective Small-Molecule Activators of a Bacterial Glycoside Hydrolase

Fragment-based screening was applied to identify small-molecule activators of a bacterial glycoside hydrolase (O-GlcNAc hydrolase), a rare example of enzyme activation discovery. Using ligand-observed NMR, researchers found a fragment that increased catalytic efficiency (k_cat/K_M) by about 90%. Structural studies showed that the activator binds near the catalytic center, stabilizing the enzyme's active, "closed" conformation. Optimization of initial hits produced nonessential activators that modulate both K_M and V_max. These findings demonstrate that small molecules can enhance, rather than inhibit, enzyme activity—offering new tools for studying enzymatic regulation in cells and potential applications for boosting glycoside hydrolase performance in industrial bioprocesses.

Visualization of glycoside hydrolase activity across varying compound concentrations with structural representation of the identified small-molecule activator Figure 2. Graphic abstract. Small-molecule activators of a glycoside hydrolase are identified from a biophysical fragment-based screening approach. In the crystal structure, the activators (yellow molecule in picture) are seen to bind close to the catalytic center of the enzyme and behave as nonessential activators. Activation potentially occurs through stabilization of a substrate-bound form of the enzyme. (Darby et al., 2014)

Case 2: Optimized Enzymatic System for CO₂-to-Methanol Conversion

The enzymatic reduction of carbon dioxide to methanol was achieved using a cascade of three oxidoreductases dependent on NADH as a cofactor. The final step—conversion of formaldehyde to methanol by alcohol dehydrogenase—was optimized through kinetically synchronized cofactor regeneration using either glucose dehydrogenase (System I) or xylose dehydrogenase (System II). Mathematical modeling guided the design of balanced reaction conditions to maximize cofactor recycling and enzyme efficiency. Experimental validation confirmed enhanced methanol yields, turnover numbers, and productivity across repeated cycles. System II outperformed System I, yielding 8 mM methanol with a TTN of 160 and a BPR of 24 μmol CH₃OH/U·h over 6 hours.

Enzymatic systems I and II for formaldehyde-to-methanol conversion using cofactor recycling pathways Figure 3. Conversion of CHOH to CH₃OH coupled with in situ NADH cofactor regeneration featuring System I with glucose dehydrogenase (GDH) and System II with xylose dehydrogenase (XDH). (Marpani et al., 2017)

FAQs About Our Services

Q: What types of enzymes can be analyzed in this service?

A: Our platform supports a wide variety of enzymes, including oxidoreductases, hydrolases, transferases, and synthetases. Both soluble and membrane-associated forms can be evaluated.
Q: Can I provide my own ligand library for screening?

A: Yes. We can screen client-supplied libraries or utilize our in-house curated compound collections, which include cofactors, activators, additives, and other chemical modulators.
Q: How are promising ligands validated?

A: Hits identified during the initial screening are re-evaluated through secondary assays such as dose-response testing, enzyme kinetics, and biophysical binding studies to ensure reproducibility and functional relevance.
Q: What data do I receive after the study?

A: Clients receive a detailed report including raw data, statistical analyses, assay conditions, and mechanistic interpretations, along with recommendations for further development.
Q: How long does a typical screening project take?

A: The project duration depends on enzyme complexity and screening scale but typically ranges from 4 to 8 weeks from assay development to final reporting.

References:

Darby JF, Landström J, Roth C, He Y, Davies GJ, Hubbard RE. Discovery of selective small‐molecule activators of a bacterial glycoside hydrolase. Angew Chem Int Ed. 2014;53(49):13419-13423. doi:10.1002/anie.201407081
Marpani F, Sárossy Z, Pinelo M, Meyer AS. Kinetics based reaction optimization of enzyme catalyzed reduction of formaldehyde to methanol with synchronous cofactor regeneration. Biotech & Bioengineering. 2017;114(12):2762-2770. doi:10.1002/bit.26405

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