The Refolding Of Denatured Lysozyme Assisted By Temperature-Sensitive Gel And Trehalose

Based on the relevant experiment and the pre-designed research method, thisdissertation has briefly investigated that PNIPAAm and trehalose have refolding effecton denatured-reductive lysozyme. Firstly, the optimal condition for making andpreparing for gelation has been studied as well as the gel's physical character. Afterselecting the most suitable gel, the research has focused on various factors includinggel making temperature, enzyme concentration, and gradually temperature rising onrefolding yield. Secondly, the trehalose influence on refolding yield of lysozyme hasalso been addressed under various refolding conditions such as temperature, pH valueand salt concentration. In addition, with the application of dynamics model, the wholedynamics process of lysozyme refolding under the help of trehalose and guanidiniumchloride(GdmCl) with different concentration has been deeply analyzed and theprinciple of how trehalose accelerates the lysozyme refolding has been fully discussedeventually.According to the result, the PNIPAAm has the optimal swelling ratio anddeswelling ratio and has better temperature-sensitive character with monomerconcertation (T) of 10% and crosslinking density (C) of 5%, which gel-makingtemperature is 22℃. The swelling ratio of PNIPAAm selected in the experiment canreach more than 16 times under the condition of 4 ℃. While under the condition of37℃ , its swelling ratio is only 0.69 after shrinking for 48 hours. And this kind ofswelling and shrinking character can be used repeatedly.Owing to the temperature-sensitive absorb-release character of PNIPAAm, it hascompleted the gradually release and gradually refolding on denatured lysozymesolution in the whole refolding process, it will finally reach the refolding effect whichis similar to fed-batch operation and will improve the refolding yield eventually.Based on the research, the trehalose can effectively facilitate denatured lysozymerefolding. Furthermore, the best concentration of trehalose can make the highestrefolding yield from denatured trehalose. Its best manipulation condition fordenatured lysozyme refolding is: pH 8-8.5, refolding temperature is 35-37℃ . Thethree-level reaction dynamics equation has been further employed to help develop thedynamics process of lysozyme refolding by mixing trehalose and guanidiniumchloride with different concentration. It is found that at the presence of trehalose,aggregation generating speed constant KA has decreased obviously, but it has littleeffect on refolding speed constant KN. Under a proper concentration of guanidiniumchloride, KA will decrease first and then move up as the trehalose concentration isincreasing, especially under the optimal trehalose concentration, the aggregationgenerating speed constant KA is the lowest, while the KN/KA has biggest value, andrefolding yield (r) appears to be the most. Simultaneously, under the given lysozymeconcentration, the optimal concentration of trehalose assistant refolding will increasealong with the increase of guanidinium chloride concentration. In short, the factindicates that trehalose can restrain the protein inter-molecule folding, whereas it cannot restrain the intro-molecule refolding, then at a proper score, if we increasedtrehalose concentration, the denatured lysozyme could be well refolded, and therenaturation yield could be improved.