Preparation and Characterization of a pH-responsive Polymer that Interacts with Microbial Transglutaminase during Affinity Precipitation

Abstract

Microbial transglutaminase (MTG) has been widely used in the food and pharmaceuticals industries. In this study, MTG was purified using affinity precipitation with an affinity polymer (PMMDN-T), which was synthesized using a pH-responsive polymer (PMMDN) coupled with L-thyroxin as an affinity ligand. Interactions between MTG and PMMDN-T were investigated using turbidimetric titration, zeta potential measurements, and low-field nuclear magnetic resonance (LF-NMR). We found different behaviors, architectures, and phase states of pH-dependent interactions between MTG and PMMDN-T interactions. Binding energetics between MTG and PMMDN-T were determined by isothermal titration calorimetry (ITC). The isoelectric point (pI) of the affinity polymer was 4.65 and was recovered with 96.7% efficiency after recycling the polymer three times. The optimal adsorption condition was 0.02 mol/L phosphate buffer (pH 6.0) with 1.0 mol/L NaCl at 30.0°C and a ligand density of 50.0 μmol/g. The maximum elution recoveries of total MTG were 98.44% (protein) with 92.19% (activity) using 0.02 mol/L pH 10.0 Gly-NaOH as the eluent.