Enzymatic activity ofChromobacterium viscosum lipase in an AOT/Tween 85 mixed reverse micellar system

Abstract

To enhance the Chromobacterium viscosum lipase (glycerol-ester hydrolase; EC 3.1.1.3) activity for the reaction of water-insoluble substrates, the AOT/isooctane reverse micellar interface was modi®ed by co-adsorption of a non-ionic surfactant. Polyoxyethylene sorbitan trioleate (Tween 85) was used as the non-ionic surfactant and olive oil as a water-insoluble substrate. An appreciable increase of lipase activity was observed and at higher W o values (where W o = molar ratio of water to total surfactants of the micellar system) there was no sharp fall of the enzyme activity such as a typical bell-shaped pro®le. The kinetic model for the lipase-catalysed hydrolysis of olive oil in AOT/isooctane reverse micellar system was applied to the enzymatic reaction in this mixed reverse micellar system. It was found that the predictions of the model agree well with the experimental kinetic results and that the adsorption equilibrium constant of olive oil molecules between the micellar phase and the bulk phase of the organic solvent is smaller in AOT/Tween 85 mixed reverse micellar systems than in simple AOT reverse micellar systems.

Enhancement of the activity of papain in mixed reverse micellar systems in the presence of Tween80

“…When a non-ionic surfactant Tween80 was introduced into the ionic RMs, the interaction between the anionic AOT head groups and the enzyme at the micellar interface decreased, which might suppress the hydrophobic and electrostatic interactions of enzyme molecules with the AOT molecules. 3,4 This phenomenon is similar to those reported in other recent works. 3 In addition, it is worth mentioning that the optimum W 0 was practically unchanged before and after Tween80 was added into the AOT reverse micellar system, while a change from 22 to 18 occurred in the TTAB reverse micellar system.…”

“…This effect has been reported by many researchers. 3,6,15,29 In addition, the results of Fig 3 also suggest that a non-mixed Tween80 RMs such as Tween80/isooctane RMs, is probably not a suitable medium for papain. In other words, the roles of two surfactants in mixed RMs cannot be mutually replaced because of their difference in polarity.…”

“…15,26 This is in agreement with the results of Hossain et al in AOT/Tween85 mixed RMs. 3 In the systems studied here the optimum ratio of Tween80 to the ionic surfactant was 0.06 and 0.04, respectively for AOT and TTAB systems. When the Tween80 concentration was relatively low, enzyme activity increased with Tween80 concentration.…”

“…In this case the surface charge density of the reverse micelles decreased, causing a decrease of electrostatic and hydrophobic interactions between AOT molecules and enzyme molecules at the micellar interface. 3 When the Tween80 concentration was relatively high, enzyme activity decreased with increasing Tween80 concentration because in this case the effects of the partition of substrates between the micellar phase and the bulk phase may have become dominant. With increasing Tween80, the amount of substrate located at the organic interface could also increase causing the decrease of free substrate in the micellar core which could take part in the enzyme reaction thus decreasing the measured enzyme activity.…”

“…From Fig 6, the effects of pH on the synthesis activity in mixed RMs seem more evident than in nonmixed RMs, which is similar with the case of AOT/ Tween85 RMs. 3 In addition, extreme acidic or alkaline pH would probably exert a disadvantageous effect on the surfactant stability. In particular, when one of the substrates is an ester, like L-PheOMe, its hydrolysis would be accelerated, leading to a decrease in the yield of the peptide formed.…”