Inductive Effect Of Cationic Surfactants On The Superactivity And Structure Of ?-Chymotrypsin

The catalysis of enzyme ensures the functionality and integrity of living system.Improving the activity and stability of enzyme is a hot topic in the fields of life sciences,pharmaceutical chemistry,biology,and medicine,etc.A large number of literatures have shown that surfactants can improve the catalytic efficiency and the stability of some enzymes.Therefore,it is of great significance to use more comprehensive means to study the mechanism of interaction between surfactant and enzyme.In this work,we used gemini surfactants with better physicochemical properties such as low critical micelle concentration to study their interactions with?-chymotrypsin??-CT?in monomers or worm-like micelles of surfactants.The main research contents were as follows:1.The effect of surfactant on the activity of?-chymotrypsin in dilute solutionThe effects of cationic gemini surfactants?12-s-12,s=2,6 and 10?with different spacer lengths on the activity of?-chymotrypsin were investigated by UV-vis spectrometry,meanwhile the conventional single-chain cationic surfactant DTAB was also used as a comparative study.All the studied cationic surfactants were able to induce the superactivity of?-CT and it's relative activity was related to the spacer chain length of the gemini surfactant.On the other hand,the gemini surfactant with longer spacer might have a much stronger influence on the enzyme activity due tolarger size and its stronger hydrophobicity of its head group..2.Thermodynamic study on the interaction between surfactant and?-CT in dilute solutionThe thermodynamic characterization of the interaction between surfactant and?-CT was studied by isothermal titration calorimetry?ITC?.The positive enthalpy change(?(?H_obs))demonstrate the conformation of?-CT changed after incubating in 12-s-12 solution.Under the same condition,the values of?(?H_obs)for the studied mixed systems–surfactants/?-CT are 12-10-12/?-CT>12-6-12/?-CT>12-2-12/?-CT>DTAB/?-CT in descending order.This sequence is consistent with one of maximum relative activity that occours in the same systems,indicating that moderate conformational change of?-CT is favorable to stimulate its superactivity.3.The inductive effect of surfactant on the conformation of?-chymotrypsin in dilute solutionThe effect of surfactants on the conformation of?-CT was studied by fluorescence spectroscopy and circular dichroism.The tertiary structure of?-CT in 12-s-12 or DTAB solution can change while the secondary structure of?-CT cannot change.Further,differential scanning calorimetry?DSC?was used to study the effect of surfactant on the thermal denaturation of?-CT.With the increase of 12-s-12concentration,the transition temperature?T_m?and transition enthalpy??H?of?-CT decrease gradually,which means that the structure of?-CT become slack and the thermal stability was reduced.4.The effect of worm-like micelles of surfactants on the activity of?-chymotrypsinIn a mixed system of gemini surfactant and uridine-5'-monophosphate?UMP?at a molar ratio of 1:2,we obtained a stable transparent hydrogel by changing the system concentration,adjusting temperature or introducing various additives.Pure gemini surfactants can form worm-like micelles within a certain concentration range.The worm-like micelles formed by 20 mmol·L^–1 14-2-14 were selected as the carrier of?-CT.The activity measurement show that the relative activity of?-CT in the gel is almost constant before 30 h?v_r=0.6?and begin to decrease at 48 h.In several methods for further increasing the activity of?-CT,the additives n-Dodecyl?-D-maltoside and Decyl?-D-glucopyranoside have the most significant effect.