Studies On The Catalytic Performance Of Enzymes In Novel Reverse Micelles

Enzyme catalyzed biosynthesis and biotransformations are hot topics in the intersectant research area of biology and chemistry.It was the development trend of the chemistry in 21th Century.The maintenance and enhancement of the catalytic activity of an enzyme was the key problem of biosynthesis and biotransformations.To achieve the goal,the enzyme could be modified by molecular enzyme engineering,and the microenvironment of the enzyme located could also be changed,which is the so-called medium engineering.The development of medium engineering goes through three stages: water,organic solvent,reverse micelle.In the two phase system of water/organic solvent or the single phase system of organic solvent containing micro-water,the catalytic performance of enzyme usually not so good.Reverse micelle could simulate the natural environment of enzymes,so most of enzymes could retain its catalytic activity and stability in reverse micelle.However,there are several problems in the present study.Firstly,most of the study was based on the commercial ionic surfactants sodium bis(2-ethylhexyl) sulfosuccinate (AOT).In the AOT reverse micelle,there exists strong electrostatic interaction between the charged head group of surfactant and enzyme,which would cause a decrease of the catalytic activity of enzyme.Secondly,all the reverse micelle used for enzyme catalysis was based on traditional organic solvent,which would inactivate enzyme as well as pollute the environment.Therefore,the two key factors-surfactant and solvent were necessary to be improved.This thesis was based on the two sides.The formation of novel reverse micelle systems as well as the catalytic performance of several enzymes were described and discussed.1.Formation of novel reverse micelle composed of functional nonionic surfactant and enzyme catalysisAt present the reverse micelle used in enzyme catalyzed reactions is mostly composed of anionic surfactant sodium bis(2-ethylhexyl) sulfosuccinate (AOT).However,the activity of an enzyme in AOT reverse micelles is usually low because of the strong electrostatic interaction between the enzyme and the interface.Addition of nonionic surfactants to the AOT reverse micelle can to a certain extent decrease the electrostatic interaction,but can not eliminate it.A reverse micelle composed of nonionic surfactants may be a good choice to the end.However,at present,most of the commercially available nonionic surfactants need co-surfactants such as straight chain alcohols to form a water-in-oil microemulsion and the size of the reverse micelle can be tuned only in a narrow range.A nonionic surfactant N-gluconyl glutamic acid didecyl ester(GGDE) which has two hydrophobic hydrocarbon tails and one hydrophilic sugar head group was synthesized to formulate a novel reverse micelle.Based on the LiP catalyzed oxidation ofveratryl alcohol(VA) in this novel reverse micelle composed of GGDE and TritonX-100(TX-100),the effects of the size of the reverse micelle,the buffer pH,and the concentration of H₂O₂ on the catalytic activity of LiP were investigated.Under the optimized conditions, the catalytic efficiency of LiP in the GGDE/TritonX-100 reverse micelle was 40 times higher than that in the AOT reverse micelle.The full expression of catalytic activity of LiP in this medium was mainly due to the lack of electrostatic interaction between LiP and the head group of GGDE and TritonX-100.Study of the catalytic performance of yeast alcohol dyhydrogenase(YADH) in GGDE/TX-100 reverse micelle showed the advantages of this reverse micelle again.The oxidation of ethanol catalyzed by YADH at the presence of coenzyme nicotinamide adenine dinucleotide(NAD⁺) was selected as indicator reaction.The catalytic activity of YADH in GGDE/TX-100 reverse micelle was higher than that in AOT reverse micelle under respective optimum conditions. As far as the kinetic parameters were concerned,the turnover number in GGDE/TX-100 reverse micelle was 1.4 times as large as that in AOT reverse micelle,but the Michaelis constants of ethanol and coenzyme NAD⁺ in AOT reverse micelle were ca.twice and 5 times as large as the counterparts in GGDE/TX-100 reverse micelle.It indicated that the interface not only affect YADH,but also affect the positive charged NAD⁺.The strong electrostatic attraction between the head group of AOT and NAD⁺ reduced the ability of NAD⁺ to bind to YADH in AOT reverse micelle.For the conversion of ethanol, the catalytic efficiency of YADH GGDE/TX-100 reverse micelle was higher than that in AOT.2.The inactivation mechanism of ionic liquid on YADH and the use of ionic liquid-based microemulsion as a medium for enzyme catalysisThe use of room temperature ionic liquids(RTILs) as media for enzymatic catalysis has aroused the interest of many researchers. 1-butyl-3-methylimidazolium hexafluorophosphate([BMIM][PF₆]) is a hydrophobic ionic liquid which could be considered as a environmentally friendly solvent for biocatalysis.It was reported that YADH was catalytically active in the[BMIM][PF₆]-H₂O binary system.However,in pure[BMIM][PF₆] or a homogeneous solution formed by water and hydrophilic ionic liquids,such as 1-ethyl-3-methylimidazolium tetrafluoroborate([EMIM][BF₄]),etc.,it had no catalytic activity.To make it clear,the negative effect of[BMIM][PF₆]on the catalytic activity of YADH and the related mechanism was quantitatively studied,and an attempt was made to lessen the negative effect of[BMIM][PF₆] on YADH by microemulsifying[BMIM][PF₆].The activity of YADH in the homogeneous solution formed by H₂O, CH₃CH₂OH and[BMIM][PF₆]decreased rapidly with the increase of the molar fraction of[BMIM][PF₆].The inhibitory effect of[BMIM][PF₆]on YADH was probably caused by the competition of the imidazole group of[BMIM][PF₆] with the coenzyme NAD⁺ for the binding sites on YADH.In a water-in-[BMIM][PF₆]microemulsion,YADH was catalytically active due to the formation of the interfacial membrane of the nonionic surfactant TX-100, which separated YADH from[BMIM][PF₆]and avoided the direct inactivation of[BMIM][PF₆]on YADH.Under the optimum conditions,the activity of YADH was as high as 51μM min^-1.3.Study of novel RTIL-based microemulsion and enzyme catalysis Previous studies indicate that enzymes suspended in ionic liquids usually have catalytic activity,however,dissolved enzymes lose their catalytic activity. To enhance the catalytic efficiency of an enzyme,an IL-based microemulsion was therefore tried.In H₂O/TX-100/[BMIM][PF₆]microemulsion,alcohol dehydrogenase and oxidase were demonstrated to be catalytically active due to the protection of the enzyme from the direct inactivation of the ionic liquids as well as to the solubilization of the enzyme in a water pool at a molecular level. The shortcoming of this strategy is the use of large amounts of a surfactant (＞50%,W/W),which might inactivate enzyme and also make the downstream treatment difficult.So an IL-based microemulsion with low content of surfactant is urgently needed.AOT was a commercially available anionic surfactant with two tails. Because of its easy aggregation in organic solvents,it is widely used to formulate water-in-oil microemulsions.In the presence of nonionic surfactant TX-100,anionic surfactant AOT could be easily dissolved in a common hydrophobic ionic liquid[BMIM][PF₆]to formulate a[BMIM][PF₆]-based microemulsion.In this novel microemulsion,the optimumω₀ and pH were ca. 5.6 and 7.8 respectively for the model reaction of lipase-catalyzed transesterification.Under the respective optimum conditions,the catalytic activity of lipase in the present H₂O/AOT-TX-100/[BMIM][PF₆] microemulsion was comparable with that in the H₂O/TX-100/[BMIM][PF₆] microemulsion,though AOT was a stronger inhibitor than TX-100.This result indicated that the method was feasible for the reduction of surfactant dosage. Moreover,the activity of lipase tended to increase with the increase of carbon length of ionic liquid.This result not only enrich the traditional micellar enzymology,but also was important for the further study of RTILs-based microemulsion in the future.