Enzyme Catalyzed Reactions In H₂O/[BMIM]PF₆ Microemulsion

With the ever-accelerating exhaustion of oil resource,transformation of the economical frame in world's main industrialized countries will occurs,from the irreproducible sources and raw materials-based,i.e.hydrocarbons-based,to the reproducible ones-based,i.e.carbohydrates-based.Microbe,for its powerful and diverse metabolic ability,will become the key strategic and technological sources to construct the sustainable economical frame.Enzymes are the metabolites from microbe.Because of their distinguished catalytic performance in biosynthesis and biotransformation as well as biodegradation of pollutants,enzymes studies and their applications have attracted scientists' attention.Room temperature ionic liquids(RTILs)are generally composed by organic cation and inorganic anions.And they have many good characteristics,such as almost no vapor pressure,great temperature range of stability,better chemical stability and electrochemical stability with wider potential windows,so RTILs are usually known as the "green solvents".Results indicated that ionic liquids would be used as medium for these enzymes that usually had catalytic activity in organic solvents.Moreover.the catalytic performance(including activity,stability and stereoselectivity)in ionic liquids is usually better than that in traditional organic solvents.Furthermore,ionic liquids as reaction media have many other advantages,such as the easy separation of products,reuse of ionic liquids and enzymes,etc.However,they also have some drawbacks(excluding their high cost),such as their high viscosity,poor ability to dissolve non-polar substrates,etc.For hydrophilic substrates,their solubility in ILs is also limited.In addition,it was reported that enzymes which were completely dissolved in ILs usually had no catalytic activity,for example,the soluble thermolysin in[BMIM]PF₆.As we all know,water-in-oil microemutsions have many advantages such as large interfacial area,enhanced solubility of substrates,and monodispersion of enzymes at a molecular level,etc.Furthermore,the protection of an enzyme by the interfacial film from the inactivation of ILs might enhance the activity of the enzyme. The shortcomings of ILs mentioned above may be overcome by the use of surfactant to create an IL-based water-containing microemulsion.Based on the phase behavior of the ternary system consisting of[BMIM]PF₆,Triton X-100,and water,we firstly studied the catalytic activity of several enzymes in H₂O-in-[BMIM]PF₆ microemulsion.This may provide a good theoretical basis for futher study,and it was also the main innovation of this paper.In the first section,we summarized the recent development of RTILs,micellar enzymology and LiP,laccase,lipase study.In the second section,we studied the catalytic activity of LiP in the H₂O/[BMIM]PF₆ microemulsion,and also studied the effect of[BMIM]PF₆ on LiP and its chemical center hemin.The results showed that LiP could express high catalytic activity in H₂O/[BMIM]PF₆ Microemulsion,and the best condition for LiP catalyzed oxidation of OPDA was as followed:ω₀=8.0,pH=3.2,[H₂O₂]= 0.225mM.In the water/ethanol/[BMIM]PF₆ homogeneous system with 1:1 molar ratio of ethanol and water,we found[BMIM]PF₆ had an inactivating effect on LiP. And the higher the concentration of[BMIM]PF₆ was,the stronger the inactivation of LiP was.In addition,in the aqueous solution with the concentration of TX-100 about 15wt%and 21wt%,we also found[BMIM]PF₆ had an inactivating effect on LiP by adding a small amount of[BMIM]PF₆.And in the pure[BMIM]PF₆,LiP could hardly express its catalytic activity.So we thought this might be due to existence of the surfactant membrane,which separated LiP from[BMIM]PF₆.But result also showed [BMIM]PF₆ could improve the activity of hemin.We speculated it might be due to [BMIM]PF₆ containing imidazole group which was electronic enriching group.The electronic enriching group could enhance the activity of hemin and alter the potential of hemin via coordination with hemin.Therefore,we thought that the reason for the inactivation of[BMIM]PF₆ on LiP was due to the interaction with its surrounding amino acids,which changed the conformation of LiP and lowered its catalytic activity. And what's more,the formation of[BMIM]PF₆ microemulsion also reduced the microviscosity of the reaction medium.In the third section,the mechanism of o-phenylenediamine(OPD)oxidation mediated by VA in H₂O/[BMIM]PF₆ microemulsion was studied.Experiments indicated that VA could mediate the oxidation of OPD obviously,and its mechanmism was same as that of phenols.That was VA and OPD were competitive substrates for LiE but LiP(â… )preferably oxidized VA due to the restriction of the geometry of the active site,forming a one-electron oxidation product VA⁺.The cation radical was a very active intermediate,but its C-H deprotonation from the benzylic carbon of the side chain was a slow step as compared with its one-electron transfer to OPD,thereby preventing the formation of veratraldehyde but stimulating the oxidation of OPD.At the same time,it was worth noting that the system OPD/LiP and OPD/VA/LiP were stable in aqueous media.But the reactions happened in H₂O/[BMIM]PF₆ microemulsion without the existence of H₂O₂,and also got the product as 2,3-diaminophenazine.Whether micellar catalysis or the presence of ethyl peroxide and other peroxide in Triton X-100 led the occurrence of the reaction has yet to be confirmed.In the forth section,the effect of[BMIM]PF₆ on Laccase and the catalytic activity of laccase in the H₂O/[BMIM]PF₆ microemulsion were studied.Experiments indicated that in the water/ethanol/[BMIM]PF₆ homogeneous system with 1:1 molar ratio of ethanol and water,we found[BMIM]PF₆ had an inactivating effect on laccase. And the higher the concentration of[BMIM]PF₆ was,the stronger the inactivation of laccase was.And in the aqueous solution with the concentration of TX-100 about 15wt%and 21wt%,we also found[BMIM]PF₆ had an inactivating effect on laccase by adding a small amount of[BMIM]PF₆.And in the pure[BMIM]PF₆,laccase could hardly expressed its express catalytic activity.But laccase could express high catalytic activity in H₂O/[BMIM]PF₆ Microemulsion.So we thought this might be due to existence of the surfactant membrane,which separated laccase from[BMIM]PF₆.The formation of microemulsion could greatly expand the potential application of ionic liquids,especially as enzyme-catalyzed reaction medium.In the fifth section,we studied the catalytic activity of lipase(Candida rugosa lipase)in the H₂O/[BMIM]PF₆ microemulsion.The best condition for the reaction was as followed:ω₀=10,pH=7.38,T=35℃.In addition,we studied the catalytic activity of Candida rugosa lipase in several organic solvents and[BMIM]PF₆. Experiments indicated the smaller polarity of the organic solvents were,the higher activity lipase could express,just as Candida rugosa lipase expressed high catalytic activity in hexane than in acetone and dichloromethane.And the activity of Candida rugosa lipase was higher in[BMIM]PF₆ than in hexane.But the lipase all suspended in these systerms,not dispersing at a molecular level.Though lipase expressed lower activity in H₂O/[BMIM]PF₆ microemulsion,it could disperse at a molecular level.So it also hinted the feasibility of using ionic liquids microemulsion as a lipase-catalyzed reaction medium,and it provided a reference value for the later research.