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Related Experiment Videos

Immobilized D-amino acid oxidase.

M Naoi, M Naoi, K Yagi

    Biochimica Et Biophysica Acta
    |March 14, 1978
    PubMed
    Summary

    Immobilizing D-amino acid oxidase (DAO) on agarose supports maintained enzyme activity and stability. Spacer length influenced enzyme kinetics, enhancing molecular activity and decreasing the Michaelis constant for optimal biocatalysis.

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    Area of Science:

    • Biochemistry
    • Enzyme immobilization
    • Biocatalysis

    Background:

    • D-amino acid oxidase (DAO) is crucial for metabolizing D-amino acids.
    • Enzyme immobilization is vital for enhancing enzyme stability and reusability in biocatalytic processes.
    • Understanding the impact of immobilization matrices and linker lengths on enzyme kinetics is essential for optimizing biocatalyst performance.

    Purpose of the Study:

    • To investigate the stability and activity of immobilized D-amino acid oxidase (DAO) on various agarose supports.
    • To evaluate the effect of different immobilization methods and spacer lengths on DAO's kinetic parameters.
    • To determine the optimal conditions for immobilized DAO in biocatalytic applications.

    Main Methods:

    • Immobilization of DAO (apoenzyme, holoenzyme, and enzyme-benzoate complex) onto aminoalkyl, carboxyalkyl, and cyanogen bromide-activated agarose.
    • Characterization of immobilized enzyme activity, stability, and kinetic parameters (apparent Michaelis constant, substrate specificity).
    • Assessment of the influence of varying spacer lengths (3-5 methylene groups) on enzyme molecular activity and kinetics.

    Main Results:

    • Immobilized DAO, including apoenzyme, holoenzyme, and enzyme-benzoate complex, demonstrated significant activity and stability on tested agarose supports.
    • The immobilized enzyme-benzoate complex could be converted to holo- and apoenzyme forms without elution from the support.
    • Apparent Michaelis constants and substrate specificity remained comparable to the free enzyme, while the optimal pH shifted 1.0-2.0 units towards the acidic side.
    • Increasing the spacer length from 3 to 5 methylene groups enhanced molecular activity and decreased the apparent Michaelis constant.

    Conclusions:

    • Agarose-based immobilization is a viable strategy for enhancing the stability and activity of D-amino acid oxidase.
    • Enzyme-ligand interactions and spacer arm length significantly influence the kinetic properties of immobilized DAO.
    • Optimized immobilization conditions, including appropriate spacer length, can improve the efficiency of DAO-based biocatalytic systems.

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