Stability comparison of four lipases and catalytic mechanism during the synthesis of 1,3-di-oleic-2-medium chain triacylglycerols in a trace water-in-oil system: Experimental analyses and computational simulations.

In the present study, a kind of structured lipids, namely 1,3-di-oleic-2-medium chain (OMO) triacylglycerols, were synthesized through lipase-catalyzed reactions using coconut oil and rapeseed acid as materials in a trace water-in-oil system. Experimental analysis and computational simulations were undertaken to compare the stability of four lipases including Lipozyme RMIM, Lipozyme TLIM, Novozym 435, and Aspergillus oryzae immobilized lipase (AOIM), and illustrate catalytic mechanism of Novozym 435 during the synthesis of OMO. Fourier transform infrared and molecular dynamics simulation results demonstrated that a decrease in ordered structure (α-helix and ß-sheet) led to a reduction in enzyme activity. Compared with Lipozyme RMIM and Novozym 435, Lipozyme TLIM and AOIM exhibited better stability due to a short-chain lid in TLIM, which covers activity sites, and hydrogen bonds formed between activity center of AOIM and water. Among four lipases, AOIM exhibited best catalytic performance: a OMO yield of 30.7% at 3 hr and a good stability of long term (48 hr). Density functional theory results demonstrated that specifically, during the synthesis of OMO triacylglycerol, the addition of Novozym 435 (derived from Candida antarctica lipase B, CALB) substantially lowered reaction barriers (64.4 KJ/mol with CALB vs. 332.7 KJ/mol with no lipase), aiding in the generation of OMO because of the formations of transitional tetrahedral intermediates. A trace water-in-oil system was a green and efficient alternative for lipase-catalyzed production of OMO, and this study provided crucial insights into the stability/instability and catalytic mechanisms of lipase in the synthesis of structured lipids. PRACTICAL APPLICATIONS: We compared the stability of Lipozyme RMIM, Lipozyme 435, Lipozyme TLIM, and AOIM during the synthesis of the OMO triacylglycerols in a trace water-in-oil system, where exhibited a high catalytic activity of lipase in water-oil interface. AOIM had the highest stability and showed the best catalytic performance due to the formation of hydrogen bonds. Besides, for the first time, the transition tetrahedral structure was revealed in the enzymatic synthesis of medium- and long-chain triacylglycerols. This study provides a rational approach to compare lipase stability and a possible hint to choose appropriate enzyme in a specific catalytic condition.

The immunomodulatory effects of ginsenoside derivative Rh2-O on splenic lymphocytes in H22 tumor-bearing mice is partially mediated by TLR4.

PURPOSE: Previously, we reported the octyl ester derivative of ginsenoside Rh2 (Rh2-O) had better antitumor and immunomodulatory effects than Rh2 in H22 tumor-bearing mice. Therefore, this study further explored the effects of Rh2-O on splenic lymphocytes in H22 tumor-bearing mice and the underlying mechanism. METHODS: Wild type and Tlr4-/- mice were selected to establish the H22 tumor-bearing mice model. After the treatment of Rh2-O (10 mg/kg by gavage) for 15 days, the sizes of tumor were measured. Subsequently, the splenic lymphocytes were isolated and the activities (eg. cell proliferation, cytotoxicity and cytokine secretion) were evaluated. Then, the proteins and mRNA expression levels of TRAF6 and NF-ĸB p65 in splenic lymphocytes were examined. RESULTS: The results showed that Rh2-O administration enhanced the proliferative capacity and cytotoxicity of splenic lymphocytes, and the effects were Tlr4-associated. Compared to WT mice, the up-regulation of cytokines secretion (eg. IFN-γ, IL-2 and IL-4) in isolated splenic lymphocytes after Rh2-O administration was lower in Tlr4-/- mice. Moreover, the results showed Rh2-O increased the expression of TRAF6 and the level of endonuclear NF-ĸB p65, which was inhibited in Tlr4-/- mice (P < 0.05). CONCLUSION: Rh2-O could exert immunomodulatory effects on splenic lymphocytes with the partial participation of TLR4 in H22 tumor-bearing mice.

Trace water activity could improve the formation of 1,3-oleic-2-medium chain-rich triacylglycerols by promoting acyl migration in the lipase RM IM catalyzed interesterification.

New structured lipids with 1,3-oleic-2-medium chain (OMO) triacylglycerols were synthesized by promoting acyl migration in Lipozyme RM IM catalyzed interesterification between coconut oil (CO) and high oleic rapeseed oil (HORO). Results from an orthogonal design L25(55) showed that the maximal yield of OMO-structured triacylglycerols was 45.65% under the following conditions: the molar ratio of CO to HORO, 50:50; enzyme dosage, 12 wt%; reaction temperature, 60 °C; reaction time, 2 h; water activity, 0.07. Low water activity showed a high rate of acyl migration (10.86% vs 5.07% no water system), which promoted OMO synthesis due to medium-chain fatty acid migration to the sn-2 position. In a low water content (5%) system of the molecular dynamics simulation, water molecules stabilized the whole structure of RM IM through hydrogen bonding, which helped fix lipase-catalyzed active sites, making substrates more easily inserted into active sites, resulting in increased enzyme activity.