Determination of ascorbic acid with immobilized green zucchini ascorbate oxidase

Area of Science:

• Biochemistry
• Enzyme Technology
• Analytical Chemistry

Background:

• Ascorbate oxidase is crucial for vitamin C metabolism.
• Enzyme immobilization enhances stability and reusability for analytical applications.
• Accurate measurement of ascorbic acid is vital in biological and food science.

Purpose of the Study:

• To immobilize ascorbate oxidase from zucchini squash.
• To characterize the immobilized enzyme's properties.
• To develop a flow-through assay for rapid ascorbic acid quantification.

Main Methods:

• Immobilization of ascorbate oxidase onto CH-Sepharose using carbodiimide chemistry.
• Characterization of immobilized enzyme activity and stability.
• Development of a flow-through system with a polarographic detector for oxygen monitoring.

Main Results:

• Immobilized ascorbate oxidase retained properties similar to the free enzyme.
• The assay demonstrated linearity for ascorbic acid concentrations between 3 x 10(-7) M and 5 x 10(-4) M.
• The method enabled rapid analysis (approx. 60 determinations/hour) of ascorbic acid in human plasma and fruit juices with <5% standard deviation.

Conclusions:

• Enzyme immobilization is a viable strategy for creating stable and reusable ascorbate oxidase biocatalysts.
• The developed flow-through assay is efficient and accurate for determining ascorbic acid levels in complex biological matrices.
• This method offers a significant advancement for routine vitamin C analysis in clinical and food industry settings.