Study On The Regulation Of The Catalytic Performance Of Laccase By Medium

Laccase is an important oxidase, and it has been widely used in the fields of the degradation & transformation of pollutants and the development of biofuel cells. Improving the activity and stability of laccase is one of the challenging topics in the above fields. Medium regulation is one of the strategies for the improvement, and the development of a medium or microenvironment suitable for laccase could promote the real application of laccase in biocatalysis & biotransformation.Aqueous solution is a significant medium for laccase catalyzed reactions. Ionic liquids as new designable solvents have the features of low volatility, good solubility for many substrate, etc. and some ionic liquids have been reported to be able to improve the catalytic performance of some enzymes in aqueous solution. For a given enzyme (laccase), a systematic study of the ions effects on the activity and the conformation of laccase is helpful to the rational selection of the ion pair compatible with laccase.Reversed micelle is an ideal medium for the enzymatic conversion of hydrophobic substrates. The catalytic activity of laccase in reversed micelles depends on the size and the interfacial properties of the micelles. Metal nanoparticle is rigid and its size and morphology are easily controlled. Using metal nanoparticles to change the interaction between the enzyme and the surfactant interface is helpful to tuning the catalytic performance of laccase in reversed micelles.Compared with drop-like reversed micelles, bicontinuous microemulsions as promising media for enzyme catalysis could provide larger interfacial area and lower mass transfer resistance. However, the related reports are very few and limit only to traditional oil/water/surfactant systems. It is necessary to construct some new bicontinuous microemulsion system for enzymatic reactions. Using hydrophobic ionic liquid (HIL) as a replacement of traditional oil to construct novel HIL-based bicontinuous microemulsion is a new attempt. Based on the above background, I carried out some works, which are summarized and presented as follows:1. Ionic liquids regulate the activity of laccase in aqueous solutionThe interaction between an enzyme and ionic liquids is rather complex. For a given enzyme, its activity and stability depends on the cation/anion combination. In an aqueous solution of 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([Bmim]TfO),1-butyl-1-methylpyrrolidinium trifluoromethanesulfonate ([Bmpyr]TfO) or tetramethylammonium trifluoromethanesulfonate ([TMA]TfO), the activity, stability and conformation of laccase were first investigated. Compared with the system without ionic liquids, high level of [Bmim]TfO or [Bmpyr]TfO destabilizes laccase while [TMA]TfO stabilizes laccase. These effects are more pronounced with the extension of the incubation time. The activity variations are well correlated with the changes of the conformation of laccase evidenced by fluorescence and circular dichroism spectra under specified conditions. The effects of the three ionic liquids on laccase are associated with the chaotropicity of the cations in Hofmeister series. For laccase, [TMA]TfO is not a good activating agent but it greatly enhances the stability of laccase in addition to maintaining the catalytic efficiency of laccase, showing its great potential in real application. The above research is helpful to the rational selection of the ion pair compatible with laccase.2. Gold nanoparticles regulate the activity of laccase in reversed micelleThe interfacial property of reversed micelles is an important factor affecting the catalytic activity of enzymes hosted in the micelles. The addition of metal nanoparticles in reversed micelles may change the interaction between the enzyme and the interface, and then regulate the catalytic performance of the enzyme. For reversed micelles with different electrical properties, the nanoparticle may have different effects, depending on the enzyme applied. In this section, the effect of gold nanoparticles (GNPs) on the laccase activity in AOT anionic reversed micelle was investigated. The monodispersed GNPs of different sizes were obtained by citrate reduction. Although the GNPs have little effect on the activity and on the conformation of laccase in aqueous solution, the catalytic activity of laccase in the reversed micelles was obviously varied by GNPs. It was found that laccase activity was dependent on the size of the particle and its concentration as well as on the water content and the concentration of AOT. It was shown that there existed several types of micelles in the present reversed micellar system in the presence of GNPs. The population of the various micelles depends on the concentrations of both GNPs and AOT. Fluorescent-probe and dynamic light scattering techniques proved the existence of GNP-doped reverse micelles. Moreover, fluorescence and circular dichroism spectra of laccase at different water contents and GNP concentrations indicated that the conformation of laccase and its activity were tuned by GNPs via changing the structure of the reversed micelles. Analysis showed that changes in the thickness of the water layer and in the apparent occupied area of individual AOT molecules caused by GNPs were the main parameters affecting the activity of laccase. The present work extends and deepens the understanding of the tuning mechanism of GNPs on enzymatic performance in reversed micelles and provides guidance for rational design of the optimal microenvironment of laccase.3. Medium effect of HIL-based bicontinuous microemulsion on the catalytic performance of laccase3.1 Constructing novel bicontinuous microemulsion and kinetics study of laccaseAn HIL-based microemulsion is a promising medium for enzymatic reactions. It has the features of both ionic liquid and bicontinuous microemulsion. In this section, the fish-shaped phase diagram method was applied to investigate the effects of surfactants, hydrocarbon alcohols (C5～C10), and HILs on the phase behavior of the microemulsion stabilized by polyoxyethylene-type nonionic surfactant (CnEm). It was found that small head group of the surfactant, high concentration of the alcohol and low cohesive energy density of the HIL result in low phase inversion temperature (T). The phenomena are primarily connected with the dissolution and aggregation of surfactants in HIL as well as with the interface effect and the co-solvent effect of alcohols. Based on the above understanding on the phase behavior, we chose some appropriate microemulsion systems to study the composition dependence and temperature effect on the kinetic parameters of laccase. The result showed that temperature and alcohol are the key parameter affecting the catalytic performance of laccase. At ambient temperature, the optimum alcohol depends on the type of HIL as an oil phase. All the kinetic parameters indicate that [Omim]NTf2/buffer/CnEm/1-hexanol bicontinuous microemulsion is a suitable medium for the laccase-catalyzed reaction; in the C16E10 stabilized microemulsion the catalytic efficiency of laccase is as high as 95.3 mM-1 min-1, which is 156 times higher than that in the water saturated [Omim]NTf2 (0.607 mM-1 min-1) at 35.0 ℃3.2 Comparative study on the hydrocarbon & fluorocarbon alcohols effect on the phase behavior and on the catalytic performance of laccaseAs an important additive, an alcohol plays a key role in the formation and structural regulation of microemulsions.The effects of both hydrocarbon and fluorocarbon alcohols with short/medium alkyl chain length (C2-C5) on the phase behavior of the hydrophobic ionic liquid (HIL)-based nonionic surfactant (CnEm) stabilized bicontinuous microemulsion as well as on the catalytic performance of the solubilized laccase were reported. The partially plotted fish-cut phase diagram, which shows the position of the intersection of the two critical lines (X-point) indicates that high levels of short chain fluorocarbon alcohols (C2-C5) result in a low T; however, high levels of short chain hydrocarbon alcohols (C2-C3) leads to an increase in T. It is also found that the distinct effects of the two kinds of alcohols on the phase behavior of the microemulsion depend on the composition of HIL. Studies on the laccase kinetics at 35.0 ℃ indicates that in the hydrocarbon alcohol systems, the bicontinuous microemulsion consisting of 1-pentanol/C12E10/buffer/[Omim]PF6 gives the highest catalytic efficiency (kcat/Km) of laccase (124.3 mM-1 min-1); while in the fluorocarbon alcohol systems, the microemulsion consisting of 1H,1H-trifluoroethanol/C12E10/buffer/[Omim]PF6 is the most suitable one for the expression of the laccase activity with kcat/Km being as high as 169.0 mM-1 min-1. On the whole, the catalytic performance of laccase in C12E10-stabilized [Omim]PF6-based bicontinuous microemulsion can be tuned by adjusting the structure of alcohols.