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Enzyme Activity Measurement for Ligases

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Ligases (EC 6) are a fundamental class of enzymes that catalyze the formation of new chemical bonds through the joining of two molecules, usually accompanied by the hydrolysis of ATP or other nucleoside triphosphates. They are indispensable in DNA repair, protein synthesis, and various biosynthetic pathways. At Creative Enzymes, we provide comprehensive and accurate enzyme activity measurement services for ligases, tailored to support both academic research and industrial applications. With advanced analytical platforms and an experienced scientific team, we ensure reliable results that enable clients to move their projects forward with confidence.

Background on Ligases and Their Enzymatic Activity

Ligases, often referred to as "joining enzymes," are unique in their ability to catalyze bond formation reactions. They are classified into several subclasses based on the type of bond they form, including carbon–oxygen, carbon–sulfur, carbon–nitrogen, and carbon–carbon bonds.

Biological importance of ligases includes:

  • DNA Ligases: Crucial in DNA replication and repair, used widely in molecular biology research and biotechnology.
  • Aminoacyl-tRNA Synthetases: Essential in protein biosynthesis by charging tRNAs with their respective amino acids.
  • Ubiquitin Ligases: Key regulators of protein degradation pathways.
  • Metabolic Pathway Ligases: Involved in complex biosynthetic processes, such as fatty acid metabolism and secondary metabolite synthesis.

Given their wide distribution and importance, ligases are increasingly studied not only for fundamental biological research but also for therapeutic development and industrial biotechnology. Accurate activity measurement is a critical first step in characterizing their functions, engineering improved variants, or screening inhibitors for pharmaceutical applications.

Comprehensive Service Offerings

At Creative Enzymes, our ligase activity measurement services are designed to provide highly accurate and reproducible data. We support both standardized protocols and fully customized solutions depending on the client's specific enzyme of interest.

How It Works

Step Procedure Details
1 Enzyme Preparation
  • Initial discussion to identify the ligase subclass, substrate(s), and research objectives.
  • Selection of appropriate assay format (standardized or customized).
2 Substrate Selection
  • Handling of purified enzymes, recombinant proteins, or cell extracts.
  • Optimization of storage and assay conditions to preserve enzyme activity.
3 Assay Development Choice of analytical platform:
  • Spectrophotometric assays to monitor nucleotide hydrolysis or product formation.
  • Fluorescent assays for enhanced sensitivity.
  • Chromatographic methods (HPLC, LC-MS) for precise product detection and quantification.
  • Radioisotope or isotope-labeled substrates for mechanistic or high-sensitivity studies.
4 Enzyme Activity Determination
  • Determination of kinetic parameters (Km, Vmax, kcat).
  • Identification of optimal cofactors, pH, and temperature conditions.
  • Inhibitor or activator screening upon request.
5 Data Analysis & Reporting
  • Comprehensive report including methodology, raw data, statistical validation, and expert interpretation.
  • Optional follow-up consultation for application or engineering strategies.

Service Details

  • Broad coverage of ligase subclasses (DNA ligases, aminoacyl-tRNA synthetases, ubiquitin ligases, metabolic ligases).
  • Support for both routine enzymatic assays and mechanistic studies.
  • High-throughput options available for screening large enzyme libraries or inhibitor panels.
  • Flexible project scales, from small exploratory studies to industrial-scale applications.

Contact Our Team

Why Choose Creative Enzymes

Extensive Expertise

Decades of experience in enzymology and assay development.

Advanced Instrumentation

Access to cutting-edge technologies including LC-MS, fluorescence spectroscopy, and isotope-based methods.

Customizable Assays

Tailored solutions to match the complexity of each ligase system.

High Accuracy and Reproducibility

Rigorously validated methods ensuring dependable results.

Fast Turnaround Time

Efficient workflows without compromising data quality.

Comprehensive Client Support

Dedicated scientific team providing project consultation and post-analysis guidance.

Representative Case Studies

Case 1: TNA Enzymes and the RNA World Hypothesis

Threose nucleic acid (TNA), a simpler genetic polymer, is considered a plausible evolutionary precursor to RNA due to its base-pairing ability. In this study, researchers isolated catalytic TNA sequences with RNA ligase activity, highlighting their potential role in the RNA world transition. The enzyme T8-6 was identified, catalyzing 2′–5′ phosphoester bond formation between a 2′,3′-diol and a 5′-triphosphate group with measurable efficiency. T8-6 requires a UA|GA junction but tolerates other sequence variations. It can even generate functional RNAs, such as hammerhead ribozymes, with site-specific 2′–5′ linkages, underscoring TNA's evolutionary significance and biotechnological potential.

A threose nucleic acid enzyme with RNA ligase activityFigure 1. Biochemical characterization of TNA enzyme T8-6-catalyzed reaction. (A and B) Kinetics of T8-6-catalyzed RNA ligation reactions in the presence of 40 mM Mg2+ at pH 9.0 and 7.5, respectively. (C) pH dependence of the T8-6-catalyzed reaction rate constant. (D) Dependence of T8-6-catalyzed reaction yield (pH 9.0 for 3 h and pH 7.5 for 6 h) on Mg2+ concentration. (Wang et al., 2021)

Case 2: Growth Rates and Specific Aminoacyl-tRNA Synthetases Activities in Clupea harengus Larvae

This study investigated the growth of Atlantic herring (Clupea harengus) larvae under controlled laboratory conditions, testing three temperatures (7, 12, and 17 °C) and varying prey concentrations of Acartia tonsa. Growth rates were compared with aminoacyl-tRNA synthetase (AARS) activity as a potential proxy. A strong linear relationship (r2 = 0.71) was observed in larvae fed ad libitum, but the correlation weakened (r2 = 0.42) when food-limited larvae were included. High AARS activity in small, early-stage larvae indicated possible protein degradation, suggesting this process influences growth assessment. Further research on protein turnover is needed to refine AARS as a reliable field growth proxy.

Graphs showing how temperature and food concentration affect larval fish growth rate (SGR) and AARS activityFigure 2. (Left) Effect of temperature on SGR (day-1) and spAARS activity (nmol PPi·mg prot-1·h-1) and (right) effect of food concentration on SGR (day-1) and spAARS activity (nmol PPi·mg prot-1·h-1) at 12 °C. (Herrera et al., 2024)

FAQs

  • Q: What types of ligases can you analyze?

    A: We cover all major subclasses, including DNA ligases, aminoacyl-tRNA synthetases, ubiquitin ligases, and ligases involved in metabolic or biosynthetic pathways.

  • Q: Do you provide assay customization?

    A: Yes. We tailor assay conditions, substrates, and detection methods to suit the client's specific ligase and project goals.

  • Q: What level of sample purity is required?

    A: We accept purified enzymes, recombinant proteins, or crude extracts. Assay design will be adapted to the sample type to ensure accurate results.

  • Q: Can you provide kinetic parameters in addition to activity data?

    A: Absolutely. We routinely determine kinetic constants (Km, Vmax, kcat) as well as optimal conditions for enzyme activity.

  • Q: How long does a typical project take?

    A: Turnaround time depends on the complexity of the ligase and assay design. Standard projects are typically completed within 2–4 weeks, while more complex or custom studies may require additional time.

References:

  1. Herrera I, Yebra L, Santana-del-Pino Á, Hernández-León S. Growth rates and specific aminoacyl-tRNA synthetases activities in Clupea harengus larvae. Oceans. 2024;5(4):951-964. doi:10.3390/oceans5040054
  2. Wang Y, Wang Y, Song D, et al. A threose nucleic acid enzyme with RNA ligase activity. J Am Chem Soc. 2021;143(21):8154-8163. doi:10.1021/jacs.1c02895
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For research and industrial use only, not for personal medicinal use.

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For research and industrial use only, not for personal medicinal use.