Cutinase purification on poly(ethylene glycol)-hydroxypropyl starch aqueous two-phase systems

Area of Science:

• Biochemistry
• Separation Science
• Enzyme Purification

Background:

• Aqueous two-phase systems (ATPS) are utilized for biomolecule separation.
• Cutinase is an enzyme with potential industrial applications.
• Optimizing enzyme partitioning in ATPS is crucial for efficient purification.

Purpose of the Study:

• To characterize the partition behavior of cutinase in poly(ethylene glycol) (PEG)-hydroxypropyl starch ATPS.
• To investigate the influence of system parameters (PEG molecular mass, pH, tie-line length) on cutinase partitioning.
• To evaluate the effect of different salts on cutinase partition and yield.

Main Methods:

• Preparation and characterization of ATPS using purified (Reppal PES 100) and crude (HPS) hydroxypropyl starch.
• Systematic variation of PEG molecular mass, pH, and tie-line length.
• Addition of various salts (NaCl, Na2SO4, (NH4)2SO4) to assess their impact on cutinase partitioning.
• Determination of cutinase partition coefficients and yields.
• Construction of cross-partition graphs to determine the isoelectric point (pI).

Main Results:

• PEG-hydroxypropyl starch ATPS demonstrated low efficiency for cutinase purification, with partition coefficients near 1.
• System pH was the most influential factor affecting cutinase partition in the absence of salts.
• Salt addition significantly enhanced cutinase partition coefficients.
• Partition coefficients of 3.7 and 12 were achieved with PEG 4000/(NH4)2SO4 and PEG 4000/NaCl systems, respectively.
• The isoelectric point (pI) of cutinase was determined to be 7.8.

Conclusions:

• PEG-hydroxypropyl starch ATPS are not ideal for efficient cutinase purification.
• Salt addition is critical for improving cutinase partitioning in these systems.
• The study successfully determined the isoelectric point of cutinase, providing valuable data for further purification strategies.