Improved activity of lipase immobilized in microemulsion-based organogels for (R, S)-ketoprofen ester resolution: Long-term stability and reusability.

Microemulsion-based organogels (MBGs) were effectively employed for the immobilization of four commonly used lipases. During the asymmetric hydrolysis of ketoprofen vinyl ester at 30 °C for 24 h, lipase from Rhizomucor miehei and Mucor javanicus immobilized in microemulsion-based organogels (RML MBGs and MJL MBGs) maintained good enantioselectivities (eep were 86.2% and 99.2%, respectively), and their activities increased 12.8-fold and 7.8-fold, respectively, compared with their free forms. They gave higher yields compared with other lipase MBGs and exhibited better enantioselectivity than commercial immobilized lipases. Immobilization considerably increased the tolerance to organic solvents and high temperature. Both MJL MBGs and RML MBGs showed excellent reusability during 30 cycles of repeated 24 h reactions at 30 °C (over 40 days). The system maintained yields of greater than 50%, while the ees values of RML MBGs and MJL MBGs remained nearly constant at 95% and 88%, respectively.

Effects of additives on lipase immobilization in microemulsion-based organogels.

An inexpensive, facile, and environmentally benign method was developed to improve the activity and stability of Candida rugosa lipase (triacylglycerol acylhydrolase) immobilized on microemulsion-based organogels (CRL MBGs) via the addition of additives during immobilization. The additives used were polyethylene glycol (PEG) or polysaccharides. This study is the first report on the effect of additives in CRL MBGs. Among the tested additives, PEG produced the most improvement in the immobilized CRL, enhancing its stability in organic solvents (specifically polar solvents). The results of circular dichroism and fluorescence spectra experiments indicated that exposure of the acidic CRL to electronegative additives in the buffer, such as polyethylenimine and the electropositive surfactant cetyltrimethylammonium bromide, may change the lipase secondary structure, ultimately causing enzyme inactivation. However, sodium bis(2-ethylhexyl)sulfosuccinate and PEG 2000 had minimal effects on the secondary structure of CRL. The CRL MBGs containing PEG 2000 demonstrated remarkable retention of their catalytic activity during the recycling test. No significant changes in enzymatic activity were observed, even after nine runs, and 90% of the original yield was maintained after 15 cycles.

Purification of porcine pancreatic lipase by aqueous two-phase systems of polyethylene glycol and potassium phosphate.

An aqueous two-phase system (ATPS) was applied for the purification of porcine pancreatic lipase (PPL) from crude PPL using polyethylene glycol (PEG) and potassium phosphate. Phase diagrams for polyethylene glycol (PEG) and potassium phosphate dibasic were determined at room temperature to find an operating region to first form the ATPS. The PPL was preferentially partitioned into the PEG-rich phase in systems with molecular weights of 1000 and 1500 and concentrated in the phosphate-rich phase in systems with PEG of 4000. Moreover, instead of tie line length (TLL), we used a stability ratio without NaCl in the system, and we first applied fluorescence spectroscopy for the protein conformational analysis of the ATPS. The molecular weight of the purified lipase was determined to be approximately 52 kDa by SDS-PAGE. The enzyme was efficiently purified in PEG 1500/potassium phosphate (17/13, %) at a pH of 7.0 at 4 °C. This system obtained an enzyme partition coefficient of 12.7, an extraction efficiency of 94.7% and a purification factor of approximately 4. These results demonstrate that the aqueous two-phase system is a highly efficient method for PPL purification.