Continuous laccase concentration in an aqueous two-phase system.

Area of Science:

• Biochemical Engineering
• Enzyme Technology
• Separation Science

Background:

• Laccase enzymes are valuable biocatalysts with diverse industrial applications.
• Efficient separation and purification of laccase from microbial cultures are crucial for its economic viability.
• Aqueous two-phase systems (ATPS) offer a promising, scalable method for biomolecule partitioning.

Purpose of the Study:

• To develop and validate a continuous partitioning approach for Cerrena unicolor laccase.
• To establish an empirical short-cut method for predicting ATPS performance in laccase extraction.
• To assess the efficiency of a mixer-settler unit for one-step laccase concentration and recovery.

Main Methods:

• Batch experiments were conducted to determine phase equilibrium and laccase activity in a PEG 6000-phosphate ATPS.
• An empirical short-cut method was developed for calculating key partitioning parameters (mass/volume fractions, concentration factor, recovery).
• Continuous extraction experiments were performed using a mixer-settler unit, with automatic substrate feed and phase collection.

Main Results:

• The developed short-cut method accurately predicted phase compositions, volume fractions, concentration factors, and enzymatic yields within a 10% error range.
• A one-step extraction process in the mixer-settler unit successfully achieved the required laccase concentration.
• Continuous operation demonstrated good agreement between predicted and experimentally measured partitioning parameters at steady state.

Conclusions:

• The proposed approach provides an effective and predictable method for continuous laccase partitioning in ATPS.
• The empirical short-cut method simplifies the design and operation of ATPS for enzyme purification.
• The mixer-settler unit is suitable for scalable, one-step extraction of concentrated laccase.