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Hexose oxidase from Chondrus crispus: improved purification using perfusion chromatography(*)
B J. Savary1, K B. Hicks, J V. O'Connor
1Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 600 E. Mermaid Lane, 19038, Wyndmoor, PA, USA
Enzyme and Microbial Technology
|June 28, 2001
Summary
An improved method purifies hexose oxidase (HO) from Chondrus crispus algae. This enzyme is crucial for bioconverting lactose into lactobionic acid, with applications in structural studies and industrial processes.
Area of Science:
- Biochemistry
- Enzymology
- Marine Biotechnology
Background:
- Hexose oxidase (HO) is an enzyme found in marine algae.
- HO has potential applications in bioconversion and structural studies.
- Efficient purification methods are needed for HO characterization and use.
Purpose of the Study:
- To develop an improved purification method for hexose oxidase from Chondrus crispus.
- To obtain highly purified HO suitable for structural analysis.
- To prepare HO for bioconversion of lactose to lactobionic acid.
Main Methods:
- Enzyme extraction from lyophilized Chondrus crispus tissue using sodium phosphate buffer.
- Chromatographic separation using Poros perfusion media (DEAE-50, HP2, HQ) and Sephacryl S200.
- Enzyme activity assay using a peroxidase-coupled system with ABTS substrate.
Main Results:
- Achieved 100-fold purification of hexose oxidase with a specific activity of 69 U/mg.
- Obtained approximately 10% activity recovery.
- Determined native size of ~117,000 Da and identified polypeptide subunits of 38,000 and 29,000 Da via SDS-PAGE.
Conclusions:
- The developed method provides a rapid and high-resolution purification of hexose oxidase.
- The purified enzyme is suitable for structural characterization and bioconversion applications.
- Further characterization of hexose oxidase properties is described.