Related Experiment Videos
alpha-lactalbumin mutant acting as lysozyme
1State Key Laboratory of Pharmaceutical Biotechnology, Department of Biochemistry, Nanjing University, Nanjing, People's Republic of China.
Proteins
|November 28, 2000
Summary
Researchers engineered a mutant alpha-lactalbumin with a lysozyme catalytic site. This protein mutant demonstrated glycosidic bond hydrolysis, though less efficiently than chicken lysozyme.
Area of Science:
- Biochemistry
- Protein Engineering
- Enzymology
Background:
- Alpha-lactalbumin normally lacks enzymatic activity.
- Lysozyme is a well-characterized glycoside hydrolase.
Purpose of the Study:
- To engineer alpha-lactalbumin with lysozyme-like catalytic activity.
- To characterize the enzymatic properties of the engineered mutant.
Main Methods:
- Site-directed mutagenesis was used to introduce six specific amino acid substitutions.
- Enzyme kinetics were measured using a synthetic substrate (pNP-(NAcGlc)(3)).
- Isothermal Titration Calorimetry (ITC) was used to determine binding enthalpy with chitopentaose.
Main Results:
- The engineered alpha-lactalbumin mutant catalyzed the hydrolysis of pNP-(NAcGlc)(3).
- Kinetic parameters (K(M) and k(cat)) were determined and compared to chicken lysozyme.
- Binding enthalpy with chitopentaose was measured, showing a lower value for the mutant compared to chicken lysozyme.
Conclusions:
- Six point mutations successfully converted alpha-lactalbumin into a functional enzyme.
- The mutant enzyme exhibited glycosidic bond hydrolysis activity, albeit with lower efficiency than chicken lysozyme.
- This study demonstrates the feasibility of engineering new enzymatic functions into existing proteins.