Preparation Of Thermosensitive Gigaporous Microspheres And Their Effects On Lysozyme Renaturation

With the development of genetic engineering and fermentation engineering technology,heterologous expression of recombinant protein has become an important way to produce protein drugs.However,over-expressed proteins often form inclusion bodies(non-water-soluble protein aggregates without biological activity),so the refolding technology of inclusion bodies has become one of the technical difficulties in large-scale production of proteins.Refolding additives have attracted considerable attention in recent years due to their ability to promote protein refolding in vitro.Thermosensitive polymer N-Isopropyl acrylamide(NIPAM)has been proved to be an effective folding aid.However,the reported linear,cross-linked and grafted NIPAM onto the surface of hydrogels have many problems,such as molecular weight distribution is not easy to control,swelling rate is slow,protein loss is serious,specific surface area is small,and separation is difficult.Therefore,this paper proposes to prepare thermosensitive gigaporous polystyrene microspheres by grafting polymer brushes containing NIPAM on the surface of gigaporous polystyrene microspheres.Then,using the microspheres as refolding additives,the effects of size effect,pore size effect and hydrophobic and hydrophobic effect on the recovery of lysozyme were investigated,and the refolding process was systematically optimized.Firstly,gigaporous polystyrene microspheres were prepared by reverse micelle swelling of surfactants.Then,polymer brushes were grafted onto the surface of the microspheres,which were polymerized by NIPAM and Butyl Methacrylate(BMA).Finally,temperature-sensitive gigaporous PS-P(NIPAM-co-BMA)microspheres were obtained.On this basis,seven microspheres with different particle sizes(91μm,76μm and 61μm),different pore sizes(320 nm,94 nm and 62 nm),different grafting amounts(35.6 mg/m~2,15.9mg/m~2and 1.3 mg/m~2)were prepared by adjusting the amount of surfactant added,stirring rate and grafting amount.Secondly,lysozyme was selected as model protein,and the effect of seven microspheres on lysozyme refolding was investigated according to its activity yield.The optimum microspheres were determined as follows:microspheres with 74μm diameter,320 nm pore diameter and 35.6 mg/m~2grafting amount;the optimum addition amount of lysozyme was 5g/L,and the activity recovery of lysozyme refolding was still 63.6%after five times of use.Finally,the process parameters of microsphere-assisted lysozyme refolding were optimized by single factor.The optimal process conditions were as follows:initial lysozyme concentration 1.0 g/L,urea concentration 2.0 M,refolding temperature 30~oC,p H of refolding solution 8.5,shaking speed 150 rpm,refolding time 14 h.On this basis,the interaction of three significant factors(refolding temperature,urea concentration and initial lysozyme concentration)was analyzed by Plackett-Burman design and response surface methodology.The optimal conditions were as follows:refolding temperature 30 ~oC,urea concentration 1.96M and initial lysozyme concentration 1.02 g/L.Under these conditions,the activity yield of lysozyme refolding was 72.30%,which was very close to the theoretical predicted value of73.59%,indicating that the model could be used to optimize the refolding conditions of lysozyme.