Effect Of The Interaction Between Surfactant And BLC On The Enzymatic Activity And Its Conformations

Comparing with many synthetic catalysts,the enzymatic reaction has the advantages of higher selectivity and catalytic efficiency and less biotoxicity,and can take place in mild conditions.Enzymes are necessary for various biochemical processes in vivo.For the applications of any enzymes,two basic problems should always be considered,the enzymatic activity in the reaction environment and its dynamic stability.Surfactants are often used additives in enzyme-catalyzed reaction systems,and surfactant molecules can control the enzymatic activity and the construct stability.In this paper,the isothermal titration calorimetry(ITC),fluorescence spectroscopy,circular dichroism(CD)and UV-vis were used to characterize the interactions of cationic,anionic and zwitterionic surfactants with catalase and thus the resulting effects on the activity and structure of the enzyme were studied.The mechanism in the effect of the surfactants on the enzymatic activity was discussed in detail from the perspective of thermodynamics and molecular structure.The main research contents and results were summarized as follows:1.The effect of surfactant on enzyme activity was studied by UV-vis.The results showed that anionic surfactants(SDS,SDSo)can inhibit BLC activity even in a very dilute concentration range,and further can denaturate the enzyme near their individual critical micelle concentration(cmc).When the cationic surfactant(DTAB)was titrated into BLC solution,the mixed system below the cmc value of DTAB goes through a process from aggregation to redispersion.The relative activity of BLC associated with DTAB decreases till about 80%,while the kinetic stability is significantly improved.The zwitterionic betaine surfactant(SB3-12)had no significant influence on the activity of BLC,and can protect the natural structure of BLC in aqueous solution for a longer duration.SB3-12 can weaken and even avoid the effect of SDS on BLC activity in the presence of the mixture of SDS and SB3-12.It was found that BLC could retain its high activity if x SB3-12 >0.5,whereas xSB3-12 < 0.5,the activity of BLC decreases rapidly,so the activity of BLC should be controlled by their molar fractions.When the BLC was incubated in the mixed DTAB/SB3-12 solutions,the enzymatic activity was controld by DTAB concentration.2.The interaction between surfactant and enzyme was studied by ITC.The results showed the thermodynamics of interaction is related to the charges of the head groups.The ionic surfactant prefers tobind to the counter-charged sites on an enzyme molecule.The electrostatic interaction between enzyme and SDS is followed immediately by hydrophobic interaction between the alkyl chains of SDS and the hydrophobic region of BLC,so that excess SDS molecules should form clusters on the surface of enzyme molecule,and thus the BLC structure is unfolded.In the case of the electrostatic interaction between DTAB and BLC,the clusters fomed by the excess DTAB on the surface of different enzymes can assemble into the shared micelles due to the absence of the big hydrophobic region around the negatively charged sites,which promotes BLC molecules closing to each other and coacervating.As the concentration of DTAB increases,the shared micelles dissociate into semi-micelles binding the BLC and the enhanced electrostatic repulsion made the coacervation redispersion.The interaction between SB3-12 and the enzyme molecule is much weaker than between DTAB or SDS and the enzyme molecule.The interaction of the mixed SB3-12/SDS surfactants with BLC is controlled by their mole fraction.When xSDS < 0.50,SDS interacted with enzyme surface formed clusters,and SDS interacted with SB3-12 micelles formed mixed micelles,which can protect from the denaturation of BLC caused by SDS.When xSDS > 0.50,the excess SDS can make BLC structure unfolding,which results from both the electrostatic and hydrophobic interactions.For the system of SB3-12/ DTAB/BLC,the electrostatic interaction between DTAB and BLC is independent on the presence of SB3-12.However,SB3-12 can promote the shared micelles formed together by the mixed DTAB/SB3-12 surfactants or both the individual surfactants,leading to decreaseing the DTAB concentration to form coacervation of BLC.3.The effects of different types of surfactants on the conformation of the enzyme were studied by fluorescence spectroscopy and circular dichroism(CD).In the presence of anionic surfactants(SDS,SDSo),the tertiary structure of BLC varies progressively from relaxing to unfolding,meanwhile the α-helix andβ-sheet fractions in the secondary structure also vary,and the fraction of random coil component increases until the enzyme is fully unfolded.In the presence of SB3-12,the BLC’s secondary and tertiary structures don’t change significantly.The tertiary structure of the enzyme changes obviously when it is incubated in DTAB solution on the shift of the fluoresence emission wavelength,but CD spectrum is unvailable because of the appearence of the enzyme aggregates.When SDS was added into the BLC incubated in SB3-12 solution,after the excess SDS the secondary and tertiary structures show the similar changes to the profile presented in the absence of SB3-12.Combining the results from enzymatic activities,thermodynamics ofinteractions and enzyme structures,the correlation of the various properties with the concentration of respective surfactant was confirmed,and the mechanism of the interaction between the studied surfactants and BLC was drawn out.4.Previous studies in our research group have shown that α-chymotrypsin(α-CT)has superactivity in the presence of cationic gemini surfactants(12-10-12).In this paper,some studies on the system of 12-10-12 and α-CT were further extended.The interaction enthalpy between α-CT and 12-10-12 was systematically measured by ITC before the cmc value of 12-10-12.Thus the superactivity and low stability of α-CT incubated in 12-10-12 solution were discussed from the perspective of interaction enthalpy...