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Enzyme Kinetics Services

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Enzyme kinetics lies at the heart of enzymology, offering essential insights into catalytic efficiency, regulatory pathways, and inhibition patterns. At Creative Enzymes, we provide specialized enzyme kinetics services designed to accurately measure key parameters—including Km, Vmax, kcat, and inhibition constants. Leveraging advanced instrumentation and custom assay design, our team delivers reproducible, high-quality data for both research and industrial applications. Whether working with well-characterized enzymes or novel, low-activity systems, we provide precise kinetic analysis that empowers scientific discovery and product development.

Comprehensive Enzyme Kinetics Studies: Mechanisms, Parameters, and Applications in Research and Industry

Enzyme kinetics is the study of enzyme-catalyzed chemical reactions, focusing on how reaction rates change under varying conditions. This discipline reveals not only the catalytic mechanisms of enzymes but also their roles in metabolism, regulatory control, and drug interactions. By analyzing parameters such as Vmax, KD, Km, kcat, ka, and kb, researchers gain a comprehensive understanding of enzyme behavior.

A typical enzymatic reaction proceeds through a series of intermediate states:

E + S ↔ ES ↔ ES* ↔ EP ↔ E + P

These mechanisms are classified into single-substrate systems (e.g., triosephosphate isomerase, where studies reveal substrate affinity and turnover rates) and multi-substrate systems (e.g., dihydrofolate reductase, where kinetics can determine substrate binding order and product release sequence). Such detailed studies are crucial in fields ranging from drug discovery and diagnostics to metabolic engineering and industrial biocatalysis.

Our Comprehensive Enzyme Kinetics Service Offerings

Service Workflow

Workflow of enzyme kinetics services

Service Details

We offer a complete portfolio of kinetic analysis solutions:

Initial rate determination in enzyme kinetics

Initial Rate Determination

Measuring enzyme activity under defined conditions.

Michaelis-Menten analysis of enzyme activity

Michaelis–Menten Analysis

Calculation of Km, Vmax, and kcat.

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Enzyme inhibition studies for kinetic characterization

Inhibition Studies

Determination of competitive, non-competitive, uncompetitive, or mixed inhibition constants (Ki).

Multi-substrate enzyme kinetics analysis

Multi-Substrate Kinetics

Elucidating ordered, random, or ping-pong mechanisms.

Pre-steady-state kinetics of enzymes, adapted from Punekar 2018

Pre-Steady-State Kinetics

Characterizing rapid reaction phases using stopped-flow or quench-flow methods.

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Temperature and pH dependence of enzyme activity

Temperature and pH Dependence

Mapping stability and catalytic efficiency under varied conditions.

Allosteric regulation studies of enzymes, adapted from Herman and Lee 2012

Allosteric Regulation Studies

Investigate modulatory effects of allosteric activators or inhibitors to understand regulatory mechanisms of enzyme activity.

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Single-molecule enzyme kinetics analysis

Single-Molecule Kinetics

Study enzyme behavior at the single-molecule level for detailed insights into catalytic heterogeneity and dynamic mechanisms.

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Custom-designed enzyme kinetic assays

Custom Kinetic Assays

Tailored for novel or engineered enzymes.

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Advantages of Our Enzymes Kinetics Services

Comprehensive Expertise

Experience with enzymes from all major classes and diverse sources.

Advanced Instrumentation

Spectrophotometry, fluorometry, calorimetry, stopped-flow, and for high-precision measurements.

Custom Method Development

Adaptable to unusual substrates, cofactors, or environmental conditions.

Regulatory Compliance

Optional cGMP/cGLP-compliant testing for regulated industries.

End-to-End Support

From experimental design to data interpretation.

Efficient Delivery

Streamlined project management for timely results.

Case Studies and Real-World Applications

Case 1: Kinetic study of Liquid Lipase-Catalyzed Glycerolysis of Olive Oil Using Lipozyme TL 100L

This study explored the enzymatic synthesis of monoacylglycerol (MAG) and diacylglycerol (DAG) from olive oil via glycerolysis using the liquid lipase catalyst Lipozyme TL 100L, with Tween 80 and n-butanol as surfactants. Experimental design assessed enzyme load and temperature effects, identifying optimal conditions at 35 °C, 7.5 vol% enzyme, and a 2:1 glycerol-to-oil ratio, achieving ~98% triacylglycerol conversion in just 2 hours. Compared to conventional high-temperature chemical methods, this approach offers milder, efficient processing. Reaction kinetics were modeled with a Ping-Pong Bi-Bi mechanism, successfully describing system behavior and providing a basis for scaling enzymatic MAG and DAG production.

Kinetics of lipase-catalyzed glycerolysis of olive oilFigure 1. Kinetics of glycerolysis of olive oil. Reaction conditions: 35 °C, 7.4 % of glycerol, 10 vol% of olive oil, 5 vol% of water and 7.5vol % enzyme. (Finco et al., 2022)

Case 2: Rapid Kinetic Profiling of Thermostable PETase Variants

This study introduces a rapid bulk absorbance method for assessing relative enzyme kinetics of PET-degrading enzymes (PETases). A thermostable Ideonella sakaiensis PETase variant (R280A S121E D186H N233C S282C) with a denaturation temperature of 69.4 ± 0.3 °C was engineered and tested. At 400 nM enzyme, measurements over 24–72 hours showed 5- to 7-fold higher catalytic rates compared to wild-type. Despite similar maximum rates, the thermostable mutant accumulated more product over time due to distinct reaction trajectories. These findings highlight the importance of kinetic profiling to understand structure–function relationships and to guide engineering of PETases and other PET hydrolases.

Kinetic profiles of thermostable PETase variantsFigure 2. Kinetic profiles of TS-PETase. Initial rates of reaction in terms of mA260 per minute at various enzyme concentrations of TS-PETase were determined at 30 °C, 48 °C, and 58 °C (n ≥ 4 per time point and concentration). (Zhong-Johnson et al., 2021)

FAQs

  • Q: What types of enzymes can you analyze in your kinetics services?

    A: We provide kinetic studies for virtually all enzyme classes, including oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases. Our expertise extends to both single-substrate systems and multi-substrate mechanisms, covering everything from well-characterized enzymes to novel or low-activity biocatalysts.

  • Q: What kinetic parameters can you determine?

    A: We routinely measure key kinetic parameters such as Km, Vmax, kcat, KD, ka, and kb, as well as inhibition constants. These data provide comprehensive insights into catalytic efficiency, substrate affinity, and inhibition modes, which are critical for drug discovery, diagnostics, and industrial enzyme applications.

  • Q: What techniques do you use for kinetic analysis?

    A: Depending on the enzyme and project requirements, we apply spectrophotometric, fluorometric, or luminescent assays, often combined with advanced tools such as microplate readers, HPLC, microfluidics, and computational modeling. Each method is chosen and optimized to ensure the highest precision and reproducibility.

  • Q: Can you customize kinetic assays for my specific enzyme or application?

    A: Yes. Every project is custom-designed to match the enzyme's properties and your research or industrial objectives. We carefully tailor reaction conditions (pH, temperature, buffer systems) and detection methods, ensuring that results are both scientifically accurate and commercially relevant.

  • Q: How do you ensure reproducibility and reliability in your results?

    A: Our workflows follow strict quality control, with validated protocols, state-of-the-art instrumentation, and expert data interpretation. We also offer cGMP/cGLP-compliant services upon request, making our data suitable for regulatory submissions and industrial standards.

  • Q: What industries typically use your enzyme kinetics services?

    A: We work with clients in pharmaceutical R&D (drug target validation, inhibitor screening), diagnostics (enzyme-based assays), biotechnology (enzyme engineering, biocatalysis), and academic research. Our services are flexible enough to support both fundamental studies and product development.

  • Q: What do I need to provide to start a project?

    A: Typically, clients provide enzyme samples (or expression systems), substrates, or inhibitors of interest, along with background information such as desired assay conditions or application goals. If needed, we can also source substrates and cofactors from our extensive library.

Whether for drug discovery, bioprocess optimization, or academic research, our enzyme kinetics services deliver precise, reproducible, and actionable data to advance your project. Contact us to discuss your specific enzyme kinetic study and receive a detailed proposal.

References:

  1. Finco GF, Fiametti KG, Lobo VDS, et al. Kinetic study of liquid lipase‐catalyzed glycerolysis of olive oil using Lipozyme TL 100L. J Americ Oil Chem Soc. 2022;99(7):559-568. doi:10.1002/aocs.12593
  2. Herman P, Lee JC. The advantage of global fitting of data involving complex linked reactions. In: Fenton AW, ed. Allostery. Vol 796. Springer New York; 2012:399-421. doi:10.1007/978-1-61779-334-9_22
  3. Punekar NS. ES complex and pre-steady-state kinetics. In: ENZYMES: Catalysis, Kinetics and Mechanisms. Springer Singapore; 2018:107-114. doi:10.1007/978-981-13-0785-0_11
  4. Zhong-Johnson EZL, Voigt CA, Sinskey AJ. An absorbance method for analysis of enzymatic degradation kinetics of poly(Ethylene terephthalate) films. Sci Rep. 2021;11(1):928. doi:10.1038/s41598-020-79031-5
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