Chromobacterium viscosum lipase solubilized in microemulsion droplets of glycerol containing small amount of water stabilized by a surfactant could catalyze the glycerolysis of triolein. A sensitive and simple HPLC method of lipase assay for the glycerolysis reaction was developed.
Kinetic analysis of the lipase-catalyzed reaction was found to be possible in reversed micellar system. Among surfactants and organic solvents tested, bis(2-ethylhexyl) sodiumsulfosuccinate (AOT) and isooctane were most effective, respectively for the glycerolysis of triolein in reversed micelles. Temperature effect, pH profile, $k_{m,app}$ and $v_{max,app}$ were determined. Among various chemical compounds, $Fe^{3+}$, $Cu^{2+}$, and $Hg^{2+}$ inhibited the lipase-catalyzed glycerolysis severely. But the glycerolysis activity was partially restorable by adding histidine or glycine to the system containing these metal ions. The glycerolysis activity was dependent on water content and maximum activity was obtained at R value of 1.21.
Owing to the computerized trial-and error method (Runge-Kutta numerical integration method), we could present a general kinetic model for lipase-catalyzed glycerolysis in reversed micelles.
Chromobacterium viscosum lipase which was adsorbed on liposome and solubilized in microemulsion droplets of glycerol containing a little amount of water could catalyze the glycerolysis of olive oil. Studies on the continuous glycerolysis of olive oil by the immobilized enzyme was done at 37℃ in a continuous stirred vessel bioreactor with polysulfone membrane. The effect of the flow rate of substrate (olive oil) in isooctane on the conversion and composition of the outlet was investigated using high performance liquid chromatography. The conversion increased with decrease in the flow rate. And we studied the effect of water content in the glycerol-water-lipase solution on the glycerolysis reaction. The conversion to desirable products, mono-and di-olein, was improved without a substantial production of oleic acid at lower water concentrations i.e., below 8.0%(w/v) which corresponds to R value of 0.97. At water concentration higher than 8.0(w/v), the amount of free fatty acid was dramatically increased. Higher operational stability of the enzyme was obtained in this continuous stirred vessel reactor and the half-life of the enzyme during this continuous reaction was about 7 weeks.
