Synthesis of stimuli-responsive support material for pectinase immobilization and investigation of its controllable tailoring of enzymatic activity

Abstract

In this study, a novel type of stimuli-responsive support materials was synthesized successfully by atom transfer radical polymerization (ATRP) and ring opening polymerization (ROP) of N-isopropylacrylamide (NIPAAm), tert-butyl methacrylate (tBMA) and ε-caprolactone (CL). β-Cyclodextrin (β-CD) and azobenzene (Azo) were employed as the starting groups to form PCL-hv-PNIPAAm by host–guest inclusion complexation, while PCL-PMAA was converted by simple hydrolysis of PCL-PtBMA with trifluoroacetic acid. Both PCL-PMAA and PCL-hv-PNIPAAm formed the microspheres by self-assembly. Their characteristics were confirmed by NMR, TEM, GPC, DLS, etc. Then, the microspheres were used for the immobilization of pectinase by electrostatic interaction. The optimal immobilization reaction parameters were 10U/mL for pectinase at 25°C for 4h at pH=6. Compared with free pectinase, the immobilized enzymes were found to exhibit better tolerance of variations in pH and temperature, as well as improved storage stability. Furthermore, the relative activity of the immobilized pectinase was controlled at approximately 13% when the temperature was higher than the LCST of PNIPAAm. With different light irradiation, there is approximately a 15% influence on the relative activity of the immobilized pectinase. This new enzyme support material has good environmental adaptability and can make the activity of immobilized enzymes be tailored predictably and enable the enzyme to be used as a biocatalyst for various applications in the food industry.