Thermoresponsive Gigaporous Microspheres Facilitatethe Efficient Refolding of Recombinant Nitrilase Inclusion Bodies

In order to assist the refolding of recombinant nitrilase inclusion bodies, a series of thermoresponsive media were prepared by grafting poly­(N-isopropylacrylamide-co-butyl-methacrylate) [P­(NIPAM-co-BMA)] brushes onto PS microspheres with various particles and pore sizes via an atom transfer radical polymerization (ATRP) method. The effects of particle sizes, pore sizes, and brush grafting amounts of thermoresponsive microspheres on nitrilase refolding were investigated preliminarily. The results showed that the PS-P­(NIPAM-co-BMA) microspheres with the medium particle size (74 μm), gigapore size (320 nm), and high grafting amount (35.6 mg/m2) were the most effective candidates. The final nitrilase activity yield could be up to 84.5% with a high initial protein concentration (1 mg/mL) at 30 °C, which was 52.5% higher than that of a simple dilution refolding method at the initial protein concentration (0.1 mg/mL). After the refolding process, the PS-P­(NIPAM-co-BMA) microspheres can be easily separated by self-precipitation, and the activity yield of nitrilase still reached 74.5% after being reused for five batches. These results indicated that the thermoresponsive gigaporous medium was an ideal alternative as an artificial chaperone.