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A monomeric L-aspartase obtained by in vitro selection
Xiangduo Kong1, Zhengqiang Li, Xiaojun Gou
1Key Lab for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun 130023, Peoples Republic of China.
The Journal of Biological Chemistry
|May 2, 2002
Summary
Researchers engineered a stable, active monomeric l-aspartase enzyme by mimicking its natural structure. This engineered enzyme exhibits enhanced activity and reversibility compared to the native tetrameric form, opening new applications for protein engineering.
Area of Science:
- Protein Engineering and Design
- Enzymology
- Biocatalysis
Background:
- Native l-aspartase is a tetrameric enzyme crucial for fumarate-amination.
- Oligomeric proteins often present challenges in stability and application.
- Mimicking structural motifs can guide the design of novel protein variants.
Purpose of the Study:
- To design and obtain a functional monomeric variant of l-aspartase.
- To investigate the structural and functional properties of the engineered monomeric enzyme.
- To explore the potential applications of converting oligomeric proteins into functional monomers.
Main Methods:
- Design of l-aspartase variants by ligating domains (D1D2, D2D3) with random hexapeptide loops.
- In vitro selection to identify variants with highest enzymatic activity.
- Biophysical characterization including size-exclusion chromatography, activity assays, and denaturation/renaturation studies.
Main Results:
- A monomeric l-aspartase variant (drAsp017) with 21.3% relative activity of the native enzyme was obtained.
- drAsp017 demonstrated significantly higher activity-reversibility and stability at elevated temperatures compared to native l-aspartase.
- Reactivation of denatured drAsp017 was independent of protein concentration, unlike the native enzyme.
Conclusions:
- Successful engineering of a stable and active monomeric l-aspartase through domain ligation and in vitro selection.
- The monomeric variant exhibits improved stability and reactivation properties.
- Conversion of oligomeric proteins into functional monomers holds significant potential for biotechnological applications.