Separation of enzymes by sequential macroaffinity ligand-facilitated three-phase partitioning

Area of Science:

• Biochemistry
• Enzyme technology

Background:

• Commercial enzyme preparations often contain multiple enzymatic activities.
• Efficient separation of specific enzymes is crucial for various industrial and research applications.
• Macroaffinity ligand-facilitated three-phase partitioning (MLFTPP) is a novel technique for enzyme purification.

Purpose of the Study:

• To demonstrate the efficacy of sequential MLFTPP for separating pectinase and cellulase from a commercial enzyme preparation.
• To achieve high purification factors and activity recovery for both enzymes.

Main Methods:

• Utilized macroaffinity ligand-facilitated three-phase partitioning (MLFTPP) for sequential enzyme separation.
• Employed alginate as a polymer for pectinase purification and chitosan for cellulase purification.
• Verified enzyme purity using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Main Results:

• Pectinase was purified 13-fold with 96% activity recovery using alginate in the first MLFTPP step.
• Cellulase was purified 16-fold with 92% activity recovery using chitosan in the second MLFTPP step.
• SDS-PAGE confirmed a single band for both purified enzyme preparations.

Conclusions:

• Sequential MLFTPP is a viable and efficient strategy for separating multiple enzymes from crude preparations.
• This method offers high specificity, purity, and recovery, making it suitable for valuable enzyme isolation.
• The study highlights the potential of MLFTPP in enzyme technology and bioprocessing.