Affinity fibre--a new support for rapid enzyme purification by high-performance liquid affinity chromatography

Area of Science:

• Biochemistry and Biotechnology
• Chromatography and Separation Science

Background:

• Development of specialized chromatographic supports is crucial for efficient biomolecule purification.
• Existing matrices may have limitations in capacity, speed, or suitability for large-scale processing of dilute samples.

Purpose of the Study:

• To develop a new affinity fiber support for chromatographic applications.
• To evaluate its performance for the large-scale purification of lactate dehydrogenase.
• To compare its efficacy against conventional chromatographic matrices.

Main Methods:

• Functionalization of nonporous quartz fiber (0.5 micron diameter) with mercaptopropyltrimethoxysilane.
• Covalent coupling of tresyl chloride-activated dextran to the fiber's SH groups.
• Immobilization of a nicotinamide adenine dinucleotide (NAD) derivative onto the dextran matrix.

Main Results:

• The developed affinity fiber demonstrated a binding capacity of 0.3 μmol NAD derivative per gram and 15 mg of lactate dehydrogenase per gram.
• Achieved an operational capacity of 1.0 g of pure lactate dehydrogenase per hour per 100 g of fiber material via adsorption and salt elution.
• The affinity fiber proved highly effective for rapid processing of large volumes of dilute enzyme solutions.

Conclusions:

• A novel, high-capacity affinity fiber support has been successfully synthesized.
• This support enables rapid and efficient large-scale purification of lactate dehydrogenase.
• The developed material is particularly advantageous for processing dilute enzyme solutions, outperforming common chromatographic matrices in specific applications.