Aim Of The Project: To study the effect ionic strength in the the reverse micellar extraction of Enzymes.

Materials used: Method: Reagent grade sodium di-2-ethylhexyl sulfosuccinate (AOT) and N,N,N,N, Cetyl Trimethyl Ammonium Bromide were used as the surfactant, the organic solvent used for the reversed micelles was analytical grade isooctane in case of AOT, since CTAB is insoluble in isooctane it was directly dissolved in the enzyme sample, ternary system of 20% 1-Butanol in isooctane(v/v) was tried as well. Enzymes used were Lipase and Phenol Oxidase. Lipase Sodium Chloride was used to change the ionic strength of enzyme in Forward extraction. Backward extraction was carried out with NaCl, KCl , Et-OH,MgCl2 and H2SO4 in 50 mM Tris-Cl Buffer pH 7 in various concentrations. AOT concentration of 10 mM and pH 7 in Back extraction were already optimized. Lipase activity was assayed spectrophotometrically using p-nitrophenyl palmitate as substrate. Forward extraction was carried out by stirring 15ml of enzyme sample with 10 ml of surfactant for 30 min at 500rpm. Organic phase was separated from the microemulsion formed by centrifugation at 8000 rpm for 10 min. Back extraction was carried out by mixing 1ml of organic phase with 1 ml of new aqueous phase containing various concentrations of stripping agents in micro-centrifuge tubes. Mixing was done manually as well as on a gel rocker for 10 min. Phases were separated by centrifugation at 5000 rpm for 5 min. Results Most of the lipase was found to move from aqueous phase to organic phase during forward extraction. Back extraction of lipase from organic phase to new aqueous phase was found to be difficult. Recovery of lipase activity in new aqueous phase was at maximum 10%. Recovery of activity with NaCl as stripping agent was found to decrease from 10 mM to 50mM. and at higher concentrations no activity was recovered. At concentrations below 10 mM recovery almost remained constant. Addition of NaCl in forward made no difference to this pattern , except that there was no recovery with 50 mM NaCl stripping agent. Stripping with KCl showed decrease in recovery from 1mM to 50 mM with almost no activity at 50 mM. Addition of NaCl to enzyme in forward extraction did not affect the pattern.

Stripping with MgCl2 showed recovery only at 1mM. Stripping was done with Et-OH in Tris-Cl