Medium Effect On Enzymatic Reactions In Surfactant Self-Assembly Systems

Biocatalysis&biotransformation is nowadays a very important research field in organic synthetic chemistry because of its high efficiency,good selectivity and environmental friendliness.The key to the biocatalysis&biotransformation is the biocatalyst enzyme.Tuning the surrounding microenvironment of an enzyme,i.e.,enzyme medium engineering,is one of the main strategies to enhance the catalytic performance of the enzyme and expand its application scope.Compared with unstructured solvents,the self-assembly systems(e.g.micelle,microemulsion,etc.)formed by the spontaneous aggregation of surfactant molecules in solvents have many unique properties,such as nanoscaled microstructure and microenvironment,which not only provide compatible media for hydrophilic enzymes and hydrophobic substrates,but also regulate different types of enzymatic reactions through their microstructure and microenvironment.In order to further meet the demands for the green development of biocatalysis&biotransformation processes,the emerging "green solvent"hydrophobic ionic liquid is used in this dissertation to replace the traditional volatile molecular organic solvent to construct environmentally friendly and biocompatible surfactant selfassembly systems,and the systems are tried as media for some enzymatic reactions to understand the influences of medium microstructure and microenvironment on the enzymatic reactions and the related mechanism,the details are as follows:1.2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt(ABTS)mediated laccase-catalyzed oxidative polymerization of aniline(ANI)in sodium dodecylbenzene sulfonate(SDBS)micellar aqueous solutionStrong acid-type anionic micelles play an important role in the enzymatic synthesis of conductive polyaniline(PANI).In order to solve the compatibility problem between the efficient conversion of ANI catalyzed by laccase and the acidic environment required for ANI polymerization,a micellar aqueous solution formed by the anionic surfactant SDBS was used as the medium for laccase-catalyzed oxidative polymerization of ANI,and for the first time,the mediator ABTS was tried to enhance the ANI conversion rate under acidic conditions(pH=3.5).The results show that at high acidity,the presence of ABTS could improve the laccasecatalyzed ANI oxidative polymerization reaction rate in SDBS micellar solution without changing the ANI polymerization mechanism.During the reaction,the SDBS micelle as template can spatially confine the reaction in a microenvironment,thereby enhancing the ANI conversion rate and lowering the optimum pH of the reaction,which is favorable for the improvement of the linearity of the synthesized PANI and its conductivity.By use of the template role of SDBS micelles,and with the help of the mediator ABTS,laccase can efficiently catalyze the oxidative polymerization of ANI at low pH to obtain high ANI conversion rate and high-quality conductive PANI.The above study expands the application scope of anionic micellar templates,and provides rational guidance for the efficient synthesis of conductive PANI catalyzed by laccase.2.Novel hydrophobic ionic liquid(HIL)-based bicontinuous microemulsions(BMEs)as media for enzymatic polymerization of ANI2.1 Enzyme-catalyzed ANI polymerization reaction in HIL-based BMECompared to anionic surfactant micelles,HIL-based BME is more advantageous as an enzymatic reaction medium.To construct an HIL-based BME system stabilized by anionic surfactant AOT,an ionic liquid-type surfactant[C4mim][AOT](1-butyl-3-methylimidazolium bis(2-ethylhexyl)succinate sulfonate)was synthesized in this work,and the phase behavior of the[C4mim][AOT]/phosphate buffer(pH=4.3)/[C8mim][PF6](1-octyl-3-methylimidazolium hexafluorophosphate)/n-butanol pseudo-ternary system was investigated using the method of T-y fishlike phase diagram.It is shown that the system has a moderate phase inversion temperature(T～20℃)and is suitable as a medium for the horseradish peroxidase(HRP)triggered oxidative polymerization of ANI.The study on the composition-dependence of HRPcatalyzed ANI polymerization in this system shows that the important compositional parameters α(the mass ratio of HIL-to-water)and y(the surfactant content)affect the microdomain size of HIL-based BME and the catalytic performance of the solubilized HRP,thereby altering the resulting PANI conductivity.Lower values of a and y are favorable for the biosynthesis of conductive PANI.In conclusion,the AOT-stabilized[C8mim][PF6]-based BME acts as a template in the biosynthesis of PANI.The above study not only broadens the green microemulsion system as medium,but also opens up a new pathway for the biosynthesis of the conducting polymer.2.2 Study on the improvement of the quality of biosynthetic conductive PANI in HILbased BME and its mechanismTo improve the efficiency of AOT used for formulating HIL-based BME,to reduce its negative effect on the solubilized HRP,and to further enhance the quality of PANI obtained,a new BME system consisting of[C2mim][AOT]/[N1888][NTf2](trioctylmethylammonium bis(trifluoromethylsulfonyl)imide)/buffer(NaMES(2-(N-morpholino)ethanesuIfonic acid sodium salt),pH=4.3)/n-butanol was constructed.Compared with the[C4mim][AOT]stabilized[C8mim][PF6]-based BME reported above,the present system results in higher surfactant efficiency(ca.25%increase),and moreover it becomes more compatible with HRP.Not only the catalytic activity of HRP enhances(ca.245-fold increase),but also the HRP stability improves greatly.A study on the HRP-catalyzed oxidative polymerization of ANI in the present system shows that a conductive PANI with a majority of structural linearity and a high degree of delocalization of polarons is obtained.To elucidate the template-effect mechanism of the system,s-RHG(spiro-rhodamine-glucose derivative)was used as a pH probe to characterize the interfacial acidity of the BME system.It is shown that the important parameter α,which can regulate the template microstructure,has a significant effect on the PANI conductivity.This effect is mainly attributed to the change in microenvironmental acidity caused by the change in microdomain size(interfacial curvature),and is no longer dependent on the HRP activity.The final product PANI was characterized using various techniques,all indicate that PANI with high conductivity is obtained.The above studies not only shed light on the template role of anionic surfactant aggregates in the biosynthesis of conductive PANI,but also provide scientific support for the large-scale green synthesis of processable high-quality PANI.3.Construction of a simple and efficient HIL-based BME and its application as lipasecatalyzed reaction mediumTo demonstrate the potential advantages of HIL-based BME as medium for enzymatic reaction,it is necessary to construct biocompatible HIL-based BME systems with less components(ions)but high surfactant efficiency.To this end,the biocompatible sulfobetainetype zwittcrionic surfactant SB-n was tried for the first time to construct HIL-based BME.The phase behavior of the SB-n/[Cmim]X/buffer pseudo-ternary system was systematically investigated by using the T-γ fishlike phase diagram method.The results show that the T decreases markedly with decreasing n of SB-n,but the surfactant efficiency changes little.In addition,the HIL greatly affects the phase behavior,depending on the ion structure.The above results are quite different from that observed in the traditional oil/water microemulsion system,which should be attributed to the unique nanostructure of neat IL.Compared to the reported HIL-based BME stabilized by conventional surfactants(γ≈0.45～0.60),the present SB-n stabilized system could be formed at moderate T without the addition of co-alcohols,and its surfactant efficiency is high(γ≈0.25).For the optimized SB-12-stabilized[C8min][PF6]based BME,the composition-dependent microstructure and its effect on the catalytic performance of lipase were investigated.As increasing α,the correlation length(ξ)changes slightly,but the microdomain size(d)decreases steadily.Unlike α,with increasing γ,the d changes little,but the ξ increases markedly,i.e.,the interfacial rigidity increases.The increase in the microdomain size,the interfacial area and the interfacial rigidity of this BME all lead to an increase in the catalytic efficiency kcat/Km of solubilized lipase.A study on the selective hydrolysis of racemic 1-phenylethylbutyrate catalyzed by lipase was made in the above HILbased BME,and the preliminary result indicates that this novel medium has application potential in the field of biocatalysis&biotransformation.