Solvent-free enzymatic synthesis of 1, 3-Diacylglycerols by direct esterification of glycerol with saturated fatty acids
© Zhong et al.; licensee BioMed Central Ltd. 2013
Received: 14 March 2013
Accepted: 26 April 2013
Published: 8 May 2013
Pure 1, 3-diacylglycerols (1, 3-DAG) have been considered to be significant surfactants in food, cosmetics and pharmaceutical industries, as well as the effect on obesity prevention.
In this study, a vacuum-driven air bubbling operation mode was developed and evaluated for the enzymatic synthesis of 1, 3-DAG of saturated fatty acids, by direct esterification of glycerol with fatty acids in a solvent-free system. The employed vacuum-driven air bubbling operation mode was comparable to vacuum-driven N2 bubbling protocol, in terms of lauric acid conversion and 1, 3-dilaurin content.
Some operation parameters were optimized, and 95.3% of lauric acid conversion and 80.3% of 1, 3-dilaurin content was obtained after 3-h reaction at 50°C, with 5 wt% of Lipozyme RM IM (based on reactants) amount. Of the lipases studied, both Lipozyme RM IM and Novozym 435 exhibited good performance in terms of lauric acid conversion. Lipozyme TL IM, however, showed low activity. Lipozyme RM IM showed good operational stability in this operation protocol, 80.2% of the original catalytic activity remained after 10 consecutive batch applications. Some other 1, 3-DAG were prepared and high content was obtained after purification: 98.5% for 1, 3-dicaprylin, 99.2% for 1, 3-dicaprin, 99.1% for 1, 3-dilaurin, 99.5 for 1, 3-dipalmitin and 99.4% for 1, 3-disterin.
The established vacuum-driven air bubbling operation protocol had been demonstrated to be a simple-operating, cost-effective, application practical and efficient methodology for 1, 3-DAG preparation.
Keywords1, 3-diacylglycerol Air-bubbling Enzyme Synthesis
With attractive intermediates for synthetic application, pure 1, 3-diacylglycerols (1, 3-DAG) have been considered to be significant surfactants in food, cosmetics and pharmaceutical industries [1, 2], as well as the effect on obesity prevention. 1, 3-DAG may potentially function on building blocks for synthesis of lipid derivatives, such as phospholipids, glycolipids or lipoproteins, which have been shown to improve bioavailability and reduce side effects, so as to used as starting materials for the preparation of some drugs [3–5].
Despite its key role in industries, however, production of high yield 1, 3-DAG by chemical methods proves difficult and multi-step reaction sequences and tedious purification steps are still required, which remains the major obstacle for the broad application of 1, 3-DAG . As a promising chemical methodology, enzymatic approach had been commonly employed to obtain high yield of pure 1, 3-DAG [3, 7–9], and among these approaches, direct esterificaiton of glycerol with fatty acids had been widely used, where water removal remains the critical significance to shift the equilibrium toward the formation of 1, 3-DAG. Approaches to remove water generated include application of molecular sieves, nitrogen gas (N2) evaporation and vacuum evaporation [3, 4, 10, 11].
Glycerol with a purity of more than 99.0% was purchased from Guangzhou Chemical Reagent Factory (Guangzhou, China). Saturated fatty acids used in this experiment were all from Shanghai Reagent Co. Ltd with a purity of more than 99.0% (Shanghai, China). The sn-1, 3 specific Lipozyme RM IM (immobilized Rhizomucor miehei lipase), Lipozyme TL IM (immobilized Thermomyces lanuginosus lipase) and Novozym 435 (immobilized Candida antarctica B lipase) were obtained from Novozymes (Beijing, China). All other solvents and reagents were analytical or chromatographic grades.
1, 3-DAG synthesis
The reaction blends consisted of 10 mmol glycerol, 20 mmol fatty acid and 5 wt% of lipase based on reactants, were incubated in a 50 mL pear-shaped flask (Figure 1). Reaction temperature was controlled by water bath, with vacuum at 4 mm Hg applied throughout the reaction. Lauric acid was used as model fatty acid, and the reaction temperature was 50°C unless otherwise stated. The reaction was initiated by the application of vacuum, once the vacuum state of reactor formed via vacuum pump, air automatically inhaled into the reactor bottom, glycerol layer and solid lipase “blown” up to interact with fatty acid, thus the reaction proceeded. At approximate time intervals, 20 μL of samples were withdrawn for lipid profiles analysis.
Determination of lipid profiles
Content of free fatty acid was determined by KOH titration according to the standard method [13, 14]. The conversion of fatty acid was defined as the esterified fatty acid amount to the initial used fatty acid amount. Meanwhile, lipid profile was analyzed by a normal-phase high-performance liquid chromatography (NP-HPLC). The chromatography apparatus equipped with a binary waters 515 HPLC pump and a Waters 2410 differential refractive index detector. The separation of the compounds was performed on a phenomenex normal phase luna silica column (250 × 4.6 mm i.d., particle size 5 μm) and the column temperature was hold constant at 35°C. The mobile phase was n-hexane–2-propanol (15:1) at flow rate of 1.0 mL/min. Samples were dissolved in mobile phase (5 mg/mL) and 20 μL aliquots were injected for HPLC analysis with double determinations.
Reusability of lipase
Purification of products
After reaction, the mixtures were filtrated to remove the lipase (for solid products, petroleum ether was added to the mixtures to help the filtration and then evaporated the petroleum ether), solid 1, 3-DAG was purified by recrystallization from dry methanol and liquid 1, 3-DAG purification was achieved by a short column of silica gel. The liquid reaction mixture was dissolved in a mixture of n-hexane and diethyl ether (1:1, v/v) and then filtered over the column .
An analysis of variance (ANOVA) was performed using the SPSS 13.0 statistical analysis system, significance of differences was defined at P < 0.05 with Tukey’s test.
Esterification time courses under air-bubbling and N2-bubbling protocols
Effect of lipase on the conversion of lauric acid
In terms of lauric acid conversion, no significant difference was observed between Novozym 435 and RM IM after 3-h reaction, suggesting that Novozym 435 and RM IM may possess similar specificity towards the lauric acid, which had also been supported by a previous study . However, for Lipozyme TL IM, dramatically lower conversion of lauric acid was obtained. To further test the activity of TL IM in esterification reaction, oleic acid was employed (in the case of unsaturated fatty acids, other than air bubbling, N2 bubbling was adopted), while conversion of oleic acid turned out low (35.2 ± 0.8%), indicating that Lipozyme TL IM was unlikely suitable for esterification reaction, which was agreed with some previous reports demonstrating Lipozyme TL IM to be less active in esterification reaction [12, 16–18]. As consequence, Lipozyme RM IM was selected for subsequent experiments.
Effect of enzyme concentration on the conversion of lauric acid and content of 1, 3-dilaurin
Content of 1, 3-dilaurin was raised with enzyme concentration increasing from 3 to 5 wt%. However, with further increasing the enzyme concentration, a slight decrease of 1, 3-dilaurin content was observed. The decline of 1, 3-dilaurin content was ascribed to the acylmigration which led to an increase of 1, 2-dilaurin content accordingly (data not shown in detail), and this was supported by previous report .
Reusability of lipase
Synthesis of 1, 3-DAG under different reaction conditions
Synthesis of 1, 3-DAG a under different reaction conditions.
Fatty acid speciesb
In this study, a vacuum-driven air bubbling operation for 1, 3-DAG preparation had been developed and evaluated. Regarded as a significant surfactants in food, cosmetics and pharmaceutical industries, as well as its effect on obesity prevention and potential function on building blocks for synthesis of lipid derivatives, synthesis of 1, 3-DAG has been become one of the leading concerns. As resources were concerned, comparing with unsaturated fatty acids, saturated fatty acids exhibited additional advantages on its stability in air during synthesis of 1, 3-DAG.
With Lipozyme RM IM as catalysts, 95.3% of lauric acid conversion and 80.3% of 1, 3-dilaurin content was detected, with high content of 1, 3-DAG. In addition, 80.2% of the original catalytic activity of Lipozyme RM IM remained after 10 consecutive batch applications. The established vacuum-driven air bubbling operation protocol had been demonstrated to be a simple-operating, cost-effective, application practical and efficient methodology for 1, 3-DAG preparation.
3-DAG: 1, 3-Diacylglycerols
Nitrogen Gas (N2)
Normal-Phase High-Performance Liquid Chromatography.
This work was supported by the National Natural Science Foundation of China (31130042, 31101278, 31201362), 973-Plan (2012CB720800), the National Science and Technology Support Program (2012BAD37B01), GDPU Startup Foundation for Doctors, the Open Project Program of Process of Starch and Vegetable Protein Engineering Research Center of Ministry of Education (Dr. Zhenbo Xu, 2012–2013) and the Fundamental Research Funds for the Central Universities (2012ZB0022).
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