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Enzyme Activity Measurement for Glycosylases

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Creative Enzymes has been recognized as a leader in enzyme activity assays, with a proven reputation for accuracy, reproducibility, and reliability. Our expertise in testing glycosylases has been endorsed by tens of thousands of clients worldwide. Building upon the most up-to-date enzymology knowledge, we have developed innovative and specific assays that are unique to our services. With a dedicated team of scientists and advanced analytical facilities, Creative Enzymes continues to be the top choice for glycosylase activity measurement.

Understanding Glycosylases

Glycosylases (EC 3.2) are a class of hydrolases that catalyze the removal of glycosyl groups from substrates via hydrolysis reactions. They are crucial for numerous biological processes and are present in all human tissues. Glycosylases play central roles in cell function, signaling, and metabolism, underscored by the fact that more than half of all human proteins are glycoproteins.

Additionally, glycosylases are vital for the structural and functional integrity of nucleic acids and carbohydrates:

  • Nucleic Acids: Ribose and deoxyribose are the building blocks of DNA and RNA, the carriers of genetic information.
  • Carbohydrates: Sugars and polysaccharides are essential for energy storage and structural support in living organisms.

Glycosylases are broadly classified into two groups:

Glycosidases (EC 3.2.1)

  • Hydrolyze glycosidic bonds in carbohydrates (e.g., starch, cellulose, glycoproteins).
  • Play key roles in digestion, lysosomal degradation, and biomass processing.
  • Examples: α-Amylase (starch breakdown), β-glucosidase (cellulose digestion), lysozyme (bacterial cell wall lysis).

DNA Glycosylases (EC 3.2.2)

  • Remove damaged or mismatched nucleobases from DNA via base excision repair (BER).
  • Critical for maintaining genomic integrity by repairing oxidative, alkylated, or deaminated bases.
  • Examples: Uracil DNA glycosylase (removes uracil), OGG1 (repairs 8-oxoguanine), TDG (excises thymine mismatches).

Comprehensive Service Offerings

Service Workflow

Workflow of enzyme activity measurement for glycosylases

Service Details

Creative Enzymes provides comprehensive solutions for glycosylase activity measurement, including:

Service Details
Standard Activity Assays Highly reproducible assays using spectrophotometric, fluorometric, and chromatographic methods.
Custom Assay Development Tailored assays designed for unique substrates and research conditions.
Substrate Supply Access to a wide range of natural and synthetic substrates, including proprietary molecules available only through our services.
Kinetic Studies Determination of Km, Vmax, and kcat to elucidate enzyme mechanisms.
Substrate Specificity Profiling Detailed characterization of enzyme preferences and efficiency.
Comparative Enzyme Evaluation Benchmarking engineered variants or homologs to identify optimal candidates for industrial or therapeutic use.

Our Specialized Glycosylases Activity Measurement Services

Ribbon structure of Streptomyces lividans β-1,4-endoglucanase catalytic domain from glycoside hydrolase family 12 Activity Measurement for Glycosidases (EC 3.2.1)
Hydrolyze O- and S-glycosyl substrates
Structure of the UNG protein (PDB code: 1akz) Activity Measurement for DNA glycosylases (EC 3.2.2)
Hydrolyze N-glycosyl groups

Contact Our Team

Why Choose Creative Enzymes

Scientific Expertise

Decades of combined enzymology and carbohydrate chemistry knowledge.

Cutting-edge Technology

State-of-the-art analytical methods to overcome the challenges of glycosylase substrates.

Unique Resources

Proprietary substrates and innovative assay methods.

Reliability

Proven track record of reproducibility and precision in enzyme activity testing.

Flexibility

Customized approaches tailored to each client's research goal.

Accuracy and Reproducibility

Powered by advanced spectrophotometric systems.

Representative Case Studies

Case 1: Cation Effects on UNG2 Activity

Ions, particularly Mg2+, strongly influence DNA–protein interactions and enzymatic activity. This study shows that uracil DNA glycosylase (UNG2) exhibits a biphasic response to Mg2+: low concentrations stimulate activity, while high levels inhibit it. Using statistical modeling, researchers found that optimal activity occurs when DNA substrates are nearly saturated with cations, but deviations disrupt protein–DNA affinity due to electrostatic changes. The sensitivity of UNG2 also depends on DNA length, since ion occupancy varies with substrate size. Interestingly, Mg2+-induced changes in DNA base stacking had little effect. These findings highlight cation–DNA interactions as key regulators of base excision repair.

Ion-DNA interactions as a key determinant of uracil DNA glycosylase activityFigure 1. Biphasic activity of uracil DNA glycosylase. (Greenwood et al., 2025)

Case 2: Catalytic Mechanism of SMUG2 DNA Glycosylase

5-Methylcytosine (mC) demethylation involves oxidation to hmC, fC, or caC, followed by excision via thymine-DNA glycosylase (TDG) and base excision repair. Screening DNA glycosylase activity in the UDG superfamily revealed that Pedobacter heparinus SMUG2, a family 3 SMUG1 enzyme, excises not only uracil but also fC and caC. Mutational and kinetic analyses identified residues I62, N63, F76, and H205 as key for catalysis, with H205 critical for fC and caC removal. Molecular modeling and dynamics highlighted structural and catalytic differences between SMUG2 and human TDG, providing mechanistic insight into its unique DNA glycosylase activity.

Screening of glycosylase activity on mC and its oxidative derivativesFigure 2. DNA glycosylase activity from Phe-SMUG2 on U-, hmU-, fC- and CaC-containing DNA substrates with different opposite base. A. U-containing substrates. B. hmU-containing substrates. C. fC-containing substrates. D. caC-containing substrates. (Chang et al., 2022)

FAQs

  • Q: Why are glycosylase activity assays more challenging than other enzyme assays?

    A: Due to substrate specificity and the lack of inherent spectrometric properties in carbohydrates, glycosylase assays require advanced analytical techniques and unique substrates, many of which we provide in-house.

  • Q: Can you test novel or engineered glycosylases?

    A: Yes. We routinely test natural, mutant, and engineered glycosylases, offering comparative activity profiles to guide research and development.

  • Q: How do you measure DNA glycosylase activity?

    A: We use specialized labeled substrates and integrate spectrophotometric, chromatographic, or fluorometric analysis, depending on the enzyme and substrate requirements.

  • Q: What is the turnaround time?

    A: Typical projects range from two to four weeks. Custom assays requiring substrate development may take longer, depending on complexity.

  • Q: Do you provide assistance in interpreting the results?

    A: Yes, our reports include both raw data and expert analysis, ensuring clarity and practical guidance for your project.

References:

  1. Chang C, Yang Y, Li J, et al. Screening of glycosylase activity on oxidative derivatives of methylcytosine: Pedobacter heparinus SMUG2 as a formylcytosine- and carboxylcytosine-DNA glycosylase. DNA Repair. 2022;119:103408. doi:10.1016/j.dnarep.2022.103408
  2. Greenwood SN, Dispensa AN, Wang M, et al. Ion-DNA interactions as a key determinant of uracil DNA glycosylase activity. Biochemistry. 2025;64(10):2332-2344. doi:10.1021/acs.biochem.5c00067
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For research and industrial use only, not for personal medicinal use.