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Published on: July 11, 2015
[Various properties of immobilized terminal deoxynucleotidyl transferase from the cattle thymus]
Insights
This study optimized conditions for immobilized terminal deoxynucleotidyl transferase (TdT) from cattle thymus. Maximum enzyme activity was achieved using sodium cacodylate buffer at pH 7.5-7.9 and temperatures of 37-40 degrees Celsius.
Area of Science:
- Biochemistry
- Enzyme kinetics
- Biotechnology
Context:
- Terminal deoxynucleotidyl transferase (TdT) is a key enzyme in DNA synthesis.
- Immobilization of enzymes on solid supports like BrCN-Sepharose is crucial for their reusability and stability.
- Understanding optimal reaction conditions is vital for efficient enzymatic applications.
Purpose:
- To investigate the impact of temperature, pH, and buffer concentration on the activity of immobilized TdT.
- To determine the optimal conditions for maximum activity and stability of the enzyme.
- To characterize the behavior of partially purified TdT from cattle thymus.
Summary:
- Partially purified cattle thymus TdT immobilized on BrCN-Sepharose showed significant stability, retaining over 60% activity after 6 hours at 30°C in potassium phosphate buffer.
- Short-term activation of the immobilized enzyme was observed during incubation.
- Optimal activity for the immobilized TdT was achieved in 240-280 mM sodium cacodylate buffer at pH 7.5-7.9 and temperatures between 37-40°C.
Impact:
- Provides crucial data for optimizing the use of immobilized TdT in molecular biology and biotechnology.
- Facilitates the development of more efficient and cost-effective DNA synthesis and labeling techniques.
- Contributes to the understanding of enzyme immobilization and its effects on enzyme kinetics.
Abstract:
The effects of temperature, pH, and concentration of sodium cacodylate buffer on the activity of partially purified terminal deoxynucleotidyl transferase from cattle thymus immobilized on BrCN-Sepharose were studied. The enzyme retained at least 60% of the initial activity after 6 h of incubation at 30 degrees in 50 mM potassium phosphate buffer, pH 7.2 in the absence of substrate. Short-term activation of the enzyme during incubation was noticed. The maximum activity of the immobilized preparations was observed in 240-280 mM sodium cacodylate buffer in the reaction mixture, pH 7.5-7.9 at 37-40 degrees.
