Title: PEI-crosslinked lipase on the surface of magnetic microspheres and its characteristics Cao YP, Xia YP, Gu XF, Han L, Chen Q, Zhi GY, Zhang DH Ref: Colloids Surf B Biointerfaces, 189:110874, 2020 : PubMed
Here, PEI@PMMA microspheres were prepared by grafting polyethyleneimine (PEI) on poly(methyl methacrylate) (PMMA) magnetic microspheres and successfully used to immobilize lipase. The results showed that PEI@PMMA microspheres had strongly adsorbed lipase (49.1mg/g microsphere) via electrostatic attraction. To prevent lipase shedding, the adsorbed lipase was further crosslinked with PEI on microspheres using glutaraldehyde as crosslinker. Consequently, PEI-crosslinked lipase (2.14 U/mg) exhibited 2.6 times and 1.4 times higher activity respectively than the directly covalent lipase (0.82 U/mg) and the crosslinked lipase aggregates (1.57 U/mg), which was close to the activity of adsorbed lipase (2.20 U/mg). Conformational analysis from FTIR spectroscopy showed that PEI-crosslinked lipase retained its natural structure well. And the alpha-helix structure seemed to play a key role in enhancing lipase activity. Furthermore, the effects of various parameters on crosslinking reaction were investigated. Also, PEI-crosslinked lipase revealed higher pH and thermal stability. The Michaelis constant (Km) was increased and the optimum temperature of lipase was widened observably after crosslinking with PEI on PEI@PMMA magnetic microspheres.
Title: High-yield synthesis of bioactive ethyl cinnamate by enzymatic esterification of cinnamic acid Wang Y, Zhang DH, Zhang JY, Chen N, Zhi GY Ref: Food Chem, 190:629, 2016 : PubMed
In this paper, Lipozyme TLIM-catalyzed synthesis of ethyl cinnamate through esterification of cinnamic acid with ethanol was studied. In order to increase the yield of ethyl cinnamate, several media, including acetone, isooctane, DMSO and solvent-free medium, were investigated in this reaction. The reaction showed a high yield by using isooctane as reaction medium, which was found to be much higher than the yields reported previously. Furthermore, several parameters such as shaking rate, water activity, reaction temperature, substrate molar ratio and enzyme loading had important influences on this reaction. For instance, when temperature increased from 10 to 50 degrees C, the initial reaction rate increased by 18 times and the yield of ethyl cinnamate increased by 6.2 times. Under the optimum conditions, lipase-catalyzed synthesis of ethyl cinnamate gave a maximum yield of 99%, which was of general interest for developing industrial processes for the preparation of ethyl cinnamate.
Title: Synthesis of benzyl cinnamate by enzymatic esterification of cinnamic acid Wang Y, Zhang DH, Chen N, Zhi GY Ref: Bioresour Technol, 198:256, 2015 : PubMed
In this study, lipase catalysis was successfully applied in synthesis of benzyl cinnamate through esterification of cinnamic acid with benzyl alcohol. Lipozyme TLIM was found to be more efficient for catalyzing this reaction than Novozym 435. In order to increase the yield of benzyl cinnamate, several media, including acetone, trichloromethane, methylbenzene, and isooctane, were used in this reaction. The reaction showed a high yield using isooctane as medium. Furthermore, the effects of several parameters such as water activity, reaction temperature, etc, on this reaction were analyzed. It was pointed out that too much benzyl alcohol would inhibit lipase activity. Under the optimum conditions, lipase-catalyzed synthesis of benzyl cinnamate gave a maximum yield of 97.3%. Besides, reusable experiment of enzyme demonstrated that Lipozyme TLIM retained 63% of its initial activity after three cycles. These results were of general interest for developing industrial processes for the preparation of benzyl cinnamate.
Thermomyces lanuginosus lipase (Lipozyme TLIM)-catalyzed esterification of L-ascorbic acid was studied. It was suggested that Lipozyme TLIM was a suitable biocatalyst for enzymatic esterification of L-ascorbic acid. Three solvents were investigated for the reaction, and acetone was found to be a suitable reaction medium. Furthermore, it was found that water activity could notably affect the conversion. Moreover, pH memory of Lipozyme TLIM lipase for catalyzing L-ascorbic acid esterification in acetone was observed and the effect of pH on the reaction was estimated. In addition, the influences of other parameters such as substrate mole ratio, enzyme loading, and reaction temperature and reusability of lipase on esterification of L-ascorbic acid were also analyzed systematically and quantitatively. Kinetic characterization of Lipozyme TLIM showed that K(m,a) and V(max) were 80.085 mM and 0.747 mM min(-1), respectively. As a result, Lipozyme TLIM-catalyzed esterification of L: -ascorbic acid gave a maximum conversion of 99%.
Title: pH memory of immobilized lipase for (+/-)-menthol resolution in ionic liquid Ren MY, Bai S, Zhang DH, Sun Y Ref: Journal of Agricultural and Food Chemistry, 56:2388, 2008 : PubMed
Magnetic DEAE-GMA-EDMA microspheres were prepared via suspension polymerization and used for the immobilization of Candida rugosa lipase by ion exchange. The effect of pH values on the immobilization of lipase was investigated. Resolution of (+/-)-menthol in the hydrophobic ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate was performed by immobilized lipase-catalyzed enantioselective esterification with propionic anhydride as acyl donor. The effects of pH condition at lipase immobilization on the conversion and enantioselectivity were investigated. As a result, pH memory of the immobilized lipase for catalyzing (+/-)-menthol resolution in the ionic liquid was observed. Better conversion and the best enantioselectivity were obtained with the immobilized lipase prepared at pH 5.0. Under the condition, (-)-menthyl propionate with enantiomeric excess of >90% was obtained. Moreover, the enantioselectivity of the immobilized lipase decreased gradually with increasing pH value.
Title: Characterization of lipase in reversed micelles formulated with cibacron blue F-3GA modified span 85 Zhang DH, Guo Z, Dong XY, Sun Y Ref: Biotechnol Prog, 23:108, 2007 : PubMed
Sorbitan trioleate (Span 85) modified by Cibacron Blue F-3GA (CB) was prepared and used as an affinity surfactant to formulate a reversed micellar system for Candida rugosa lipase (CRL) solubilization. The system was characterized and evaluated by employing CRL-catalyzed hydrolysis of olive oil as a model reaction. The micellar hydrodynamic radius results reflected, to some extent, the redistribution of surfactant and water after enzyme addition, and the correlation between surfactant formulation, water content (W0), micellar size, and enzyme activity. An adequate modification density of CB was found to be important for the reversed micelles to retain enough hydration capacity and achieve high enzyme activity. Compared with the results in AOT-based reversed micelles, CRL in this micellar system exhibited a different activity behavior versus W0. The optimal pH and temperature of the encapsulated lipase remained unchanged, but the apparent activity was significantly higher than that of the native enzyme in bulk solution. Kinetic studies indicated that the encapsulated lipase in the reversed micelles of CB-formulated Span 85 followed the Michaelis-Menten equation. The Michaelis constant was found to decrease with increasing surfactant concentration, suggesting an increase of the enzyme affinity for the substrate. Stability of the lipase in the reversed micelles was negatively correlated to W0.
