Related Experiment Video
Updated: Jul 17, 2025
08:37
Measuring Enzymatic Stability by Isothermal Titration Calorimetry
Published on: March 26, 2019
13.2K
Stabilization of Enzymes by Using Thermophiles.
Ana-Luisa Ribeiro1, Mercedes Sánchez1, Sandra Bosch1
1Centro de Biología Molecular Severo Ochoa (UAM-CSIC). Facultad de Ciencias. Universidad Autónoma de Madrid, Madrid, Spain.
Methods in Molecular Biology (Clifton, N.J.)
|August 29, 2023
Summary
Enzyme engineering enhances steroid bioconversions. Error-prone PCR (epPCR) creates mutant libraries for selecting thermostable steroid-processing enzymes, improving reaction yields.
Area of Science:
- Biotechnology
- Enzyme Engineering
- Biocatalysis
Background:
- Steroid compounds are vital in pharmaceuticals, driving demand for efficient bioconversion processes.
- Enzyme-based bioconversions offer advantages over chemical synthesis due to steroid complexity and chirality.
- Thermostable enzymes are crucial for improved reaction yields, enzyme half-life, and substrate/product solubility.
Approach:
- Explores enzyme engineering via error-prone PCR (epPCR) to generate variant libraries.
- Utilizes folding interference vectors for selection of thermostable enzyme variants.
- Employs Thermus thermophilus as a host for selecting enhanced enzyme folding and thermostability.
Key Points:
- Metagenomic screening for thermostable steroid-processing enzymes can yield low hit rates.
- Error-prone PCR (epPCR) introduces random mutations to create enzyme variant libraries.
- Folding interference vectors link enzyme folding to selectable traits (e.g., kanamycin resistance) for life-or-death selection.
Conclusions:
- This protocol details a method for selecting thermostable steroid-processing enzyme variants.
- The approach enhances enzyme folding independently of enzymatic activity.
- Improved thermostability of enzymes is key to advancing pharmaceutical bioconversion technologies.
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