Study Of The β-cyclodextrin/F88 Supramolecular Self-assembly Constructing Hollow Microspheres To Encapsulate The Alkaline Protease

The alkaline protease is a protease, which generally the optimum pH is between 9 and 11, the optimum temperatures ranges from 50℃ to 70℃ and the molecular weight distribution of alkaline proteases ranges from 15 kDa to 30 kDa. Recently, the alkaline protease has been widely applied in detergent industry, food and feed industry, pharmaceutical industry, leather industry and other fields. However, the alkaline protease has poor stability for practical industrial applications, due to the influence of enzyme denaturation and autolysis. We can add borax, polyols, surfactants, metal ions, polys accharides or organic compounds to improve the stability of alkaline protease enzymes as traditional methods. In addition, the alkaline protease also treated with microwave radiation or made into microcapsules, etc. Unfortunately, the above methods are limited to application for the various their own problems.In order to solve this problem, our research group proposed using β-cyclodextrin molecules and non-ionic surfactants F88 supramolecular self-assembly constructed hollow microspheres to encapsulated alkaline protease. The β-cyclodextrin molecule is commonly used to constructed supramolecular host molecules, including a hydrophobic cavity and hydrophilic surface. and it can occur self-assemble reaction with object molecules, which match size, polarity, shape and properties, to form stable inclusion complexes. F88 is anon-ionic surfactant, belonging to the Pluronic series of products, which consists of hydrophilic PEO chains and hydrophobic PPO chains. The self-assembly or self-aggregation ability of F88 is stronger in aqueous solution.In this paper, the β-cyclodextrin was selected as host molecules and the non-ionic surfactants F88 selected as guest molecules builded hollow microspheres by supramolecular self-assembly which alkaline protease wrapped inside hollow microspheres, using host-guest molecular recognition in the hydrogen bonding, van der Waals forces, hydrophobic interaction and so on. We carried out the following studies on supramolecular self-assembly to encapsulated alkaline protease. The main conclusions were summarized as follows.-Firstly, we used self-assembly of β-cyclodextrin and F88 supramolecular to encapsulated alkaline protease, and we also verified the feasibility of the Encapsulation through a series of characterization methods.Secondly, we selected self-assembly time, the amount of alkaline protease,β-cyclodextrin, F88, Ca2+,1,2-propanediol and F127as variables to study their effects on the encapsulated alkaline protease. The studies had shown that:Since the self-assembled for 1h at 25℃, the effect of encapsulated alkaline protease is better, the enzyme activity of the products could up to 398700 U/g and ratio of encapsulation was 41.23%, and the ratio of enzyme inactivation was 8.65%. Since the concentration of alkaline protease enzyme was 10 mg/mL, the enzyme activity of the products could up to 1725600 U/g and the ratio of enzyme inactivation was 7.56%. Since the concentration of β-cyclodextrin was 10mg/mL, the enzyme activity of the products could up to 503600 U/g. Since the concentration of F88 was 20 mg/mL, the enzyme activity of the products can up to 386000U/g. At the same time, we also found that Ca2+,1,2-propanediol and F127 can also improve the stability of alkaline protease. In addition, we also explore the temperature resistance and incrassation of alkaline protease. The result shows, Alkaline protease original enzyme is completely inactivated after it is placed at 60℃ less than 10min. However, the relative activity of alkaline protease remained 46.56% after the encapsulated alkaline protease is placed at 80℃ about 30min. The original protein content of alkaline protease enzyme is36.8 μg/mL, but the protein content of the products is 86.62 mg/mL. It is clear that the alkaline protease had the better temperature resistance and incrassation than original enzyme.Finally, we studied the kinetics and thermodynamics of supramolecular self-assembly process to encapsulated alkaline protease. The self-assembly process will reaches an equilibrium state 1h at 25℃. We also studied the material ratio of reactants consumed after 1h, and found that the final material ratio and the original material ratio were almost unanimous. Next, we studied the entropy and enthalpy of the process of encapsulated alkaline protease, and made a reasonable evaluation of encapsulated alkaline protease from the point of thermodynamic.