Study On Biocatalysis Of Hemoglobin In Onic Liquid Based Microemulsion System

Compared to those traditional organic solvents,ionic liquid?IL?,a kind of novel green solvent,can enhance activity,stability,selectivity and recoverability of enzymes.However,the practical obstacle of using IL for biocatalysis is that many enzymes are not or seldom dissolved in most IL,limiting their further application in biocatalytic fields.Hence,it is key issue to develop an easy and effective way to combine enzyme solubility and activity in IL for the diverse application of IL-biocatalysis.In the microemulsion system including IL as hydrophobic phase,the nano/moicrometer-sized water domains can form in an IL continuous phase?named as W/IL microemulsions?stabilized by the surfactant or co-surfactant.Therefore,W/IL microemulsion system can be used as the most promising medium for simultaneous enhancement of solubility and activity of enzymes in IL.Accordingly,Hemoglobin was selected as enzyme template to study the spectral characteristics and biocatalysis of Hb in IL-microemulsion and surfactant-free IL-microemulsion systems,respectively.?i?Basing on the three phase diagram,the W/IL reverse phase microemulsion of the BmimPF₆/TX-100/H₂O system with the ratio of TX-100,BmimPF₆ and H₂O as 82:10:8 was chosen as a medium for study on the catalysis of enzyme.The UV-Vis and fluorescence spectral data indicates that Hb can be dissolved in the W/IL reverse phase microemulsion and retains their secondary structure.Using o-phenylenediamine?OPD?as the substrate,the catalytic activity of Hb in the W/IL medium was carefully investigated.In this microheterogeneous medium,enzyme molecules can be entrapped in tiny water pools and thus become stable against unfavorable contact with the surrounding organic solvent by layer of water and?co?-surfactant,thereby displaying desirable stability and activity.At the fixed concentration of substrate of OPD,reaction rate increases with increasing the concentration of Hb.H₂O₂ was optimized as 100?L for oxidation of OPD.Biocatalytic results demonstrate that Hb exhibits the obvious catalytic activity with V_maxax as 0.722 mM s^-11 and K_m as 7.034 mM.It was found that Hb-catalyzed oxidation of OPD by hydrogen peroxide in W/IL reverse microemulsion is effective.?ii?According to the three-phase diagram,the W/IL reverse phase microemulsion of the BmimPF₆/DMF/H₂O system with the ratio of BmimPF₆:DMF:H₂Oas 58:33:9 was selected as a medium for study on the catalysis of enzyme.UV-Vis and fluorescence spectra data reveals that Hb can be dissolved in W/IL reverse phase microemulsion of BmimPF₆/DMF/H₂O and their secondary structure was well maintained.The catalytic activity toward OPD of Hb in the W/IL medium was carefully investigated.The results show that there are a large number of small pools of water in numerous W/IL droplets in thisreversephasemicroemulsionsystem,whichprovidedacompatible microenvironment for Hb stable existence.Hb in this medium exhibits good biocatalytic activity to substrate OPD with V_maxax as 2.184 mM s^-1,K_m as 202.5 m M and V_max/K_m as10.79×10³ M^-11 s^-11 when changing the concentration of immobilized enzyme.It is also found that the reaction rate of the enzymatic reaction increases with the increase of water content in the w/oil region,and the reaction process is a dynamically controllable process.