Construction Of Novel Bicontinuous Microemulsions And Microstructure Dependent Activity Of Solubilized Enzymes

An enzyme is a protein with high catalytic activity and substrate specificity.Compared with chemically catalyzed reactions,enzyme-catalyzed reactions have the advantages of mild reaction conditions,high catalytic activity and low by-products.In the field of enzymatic biocatalysis and biotransformation,the maintenance of the catalytic activity and stability of an enzyme,which depends on its microenvironment,is a problem of crucial importance.Bicontinuous microemulsion is an intermediate phase during the transition between oil-in-water(O/W)and water-in-oil(W/O)type micromulsions.Compared with droplet-type microemulsions,bicontinuous microemulsions are more suitable media for enzymatic conversion of hydrophobic substrates,due to their unique advantages,such as high solubilization capacity,large interfacial area and so on.Ionic liquids(ILs)are a new type of solvents with low saturated vapor pressure,stable and adjustable physicochemical properties,and good ability to dissolve a variety of organic and inorganic substances.Compared with the traditional molecular solvents,ILs could be considered as "green solvents".The use of ionic liquid-based bicontinuous microemulsions will contribute to the greenization of biocatalysis and bioconversion process.At present,less attention has been paid to the construction of anionic surfactant-stabilized hydrophobic ionic liquid(HIL)-based bicontinuous microemulsions.In order to develop the anionic surfactant-stabilized bicontinuous microemulsions with the phase inversion temperature being close to the optimum temperature of an enzyme,it is necessary to study the composition-dependent phase behavior of the corresponding microemulsion system as well as the microstructure-dependent solubilized enzyme.So,in this thesis,two attempts have been made:1.Phase behavior of the anionic surfactant[Bmim][AOT]-stabilized hydrophobic ionic liquid-based microemulsions and the effect of n-alcoholsThe fishlike phase diagram of the H2O/[Bmim][AOT]/[Bmim][PF6]/n-alcohol as a function of temperature(T)and the mass fraction of[Bmim][AOT](with or without n-alcohol)in the total mixture(γ)has been observed for the first time at several mass ratios of[Bmim][PF6]to H2O(α)and with different n-alcohols.The larger area of the three-phase region occurs at a≤0.500,and the resulting fish-shapes are similar to each other.For a given a,a temperature scan(from lower to higher)at several y values reveals that the present system forms an upper phase microemulsion first and then a lower phase microemulsion.The formation of hydrophobic ionic liquid-in-water(HIL/W)microemulsion at low temperature and water-in-hydrophobic ionic liquid(W/HIL)microemulsion at high temperature was confirmed by DLS and SAXS techniques.Here,the phase sequence occurred during the temperature scan is opposite to that of a classic H2O/NaAOT/Oil system.At the lower temperature,the H-bonding interaction is considered to be the main driving force for the aggregation;at the higher temperature,however,the main driving force may be the hydrophobic interaction.n-Alcohols with medium/long alkyl chain have a great influence on the fish-tail coordinates of the present systems.Compared with the ternary system without alcohol,the addition of n-alcohols(C4～C8)decreases the phase inversion temperature(T)and the surfactant efficiency.With the increase of the alkyl chain length of n-alcohols,however,the decrement in T become smaller due to the increase of the interfacial rigidity.A comparison of these results with those obtained for the H2O/NaAOT/Oil system indicates that there are some similarities and also some differences,depending on the relative density,polarity or hydrophobicity among the HIL,Oil and n-alcohols.2.Construction of the anionic surfactant-stabilized hydrophobic ionic liquid-based bicontinuous microemulsion and the expression of the activity of solubilized enzymeBased on the preceding studies,the composition-dependent phase behavior of the[Cmmim][PF6]/buffer/[Cnmim][AOT]/1-butanol pseudoternary microemulsion system was further investigated.It is found that the phase inversion temperature and the surfactant efficiency decrease with the increase of the alkyl chain length on the surfactant counterion and the imidazolium cation of the hydrophobic ionic liquid.The[C2mim][AOT]-stabilized[Omim][PF6]-based pseudoternary microemulsion system was found to have high surfactant efficiency and moderate phase inversion temperature.The microstructure of bicontinuous microemulsion formed in the pseudoternary system was characterized by SAXS technique,and the influence of microstructure on the catalytic performance of solubilized laccase was further explored.As a increases,the microdomain size as well as the interfacial rigidity of the system decreases,and the catalytic efficiency(kcat/Km)of laccase also decreases.When the surfactant content gradually increases,the microdomain size of the system changes little,but its interfacial rigidity increases,and also the interaction between the interface and the solubilized enzyme increases accordingly,resulting in a decrease in the kcat/Km of the solubilized laccase.The variation of temperature over 35℃～55℃has little effect on the microdomain size of the system,but it has marked effect on the catalytic efficiency of the solubilized laccase.It is shown with the increase of the temperature,the kcat/Km increases first and then decreases,which is similar to that of the aqueous medium system.The optimal catalytic efficiency in the present microemulsion is estimated to be ca.50℃,which is significantly higher than that(40℃)in the aqueous medium system,indicating that the bicontinuous microstructure in the system is helpful to improve the thermal stability of laccase.The present studies is helpful for the rational construction of the anionic surfactant-stabilized hydrophobic ionic liquid-based bicontinuous microemulsion suitable for the expression of laccase activity,the latter will be a good template for the biosynthesis of conductive polymers.