Lipase-Catalyzed Synthesis and Characterization of 6-O-(11-Dodecenoic)-Glucose Ester in Ionic Liquids ()
1. Introduction
Fatty acid sugar esters have been widely used as sweeteners and non-ionic surfactants in pharmaceuticals, cosmetics, and food industries because they are biodegradable, non-toxic, and non-irritaing [1-2]. Currently, chemical synthesis is the major industrial method for sugar ester production. Existing chemical approaches have some obvious drawbacks, such as the low or non-selectivity for the site of estification, the requirement for protection and de-protection steps, and product purification difficulties. Since the discovery of lipase-catalyzed sugar esterification in organic solvents by the A. M. Klibanov [3] group in 1980’s, the bio-catalytic synthesis of sugar esters has been studied extensively. This method of synthesis has the advantage of producing regioand stereo-specific products under mild reaction condition [4-6]. However, the organic solvents used in the enzymatic synthesis (e.g. pyridine) are toxic, volatile, and non-reusable. Moreover, most enzymes are quickly inactivated in the organic solvents [7]. To overcome these limitations, researchers have been seeking a reaction medium that can dissolve both polar-sugars and nonpolar fatty acids, while at the mean time leaving the catalytic activity of the enzyme intact [8,9].
During the past decade, ionic liquids (ILs) have been found to be suitable (even improved) replacements for the organic solvents in many reactions, ILs consist of anions and cations that have liquid properties at room temperature [10]. Compared to organic solvents, ionic liquids are relatively non-toxic, and do a better job of maintaining the enzyme activity and good substrate solubility [11-14]. Today, ILs have becomes a more attractive possibility as a reaction medium for used in selective acylation of carbohydrates.
In this paper, Novozym-435 Lipase was used as the catalyst for the synthesis of dodecenoic-glucose ester in ILs, using glucose and 11-dodecenoic ethyl ester as the substrates. The effects of substrate ratio, lipase content, and temperature on reaction yield were studied. The purified product was characterized by HPLC, MS and NMR. The results demonstrated that ILs can be used as a “greener” solvent for sugar esterification, and illustrate the advantages of ILs vs. organic solvents for the specific lipase used for sugar ester synthesis.
2. Materials and Methods
2.1. Materials
Candida antarctica lipase B(CAL-B) were purchased from Sigma, lipase Novozym-435 and lipase Lipozyme TLIM were purchased from Novo Nordisk. TOKYO Chemical Industry Co. Ltd. was the source of 11-dodecenoic ethyl ester (99%). while the Silica gel G and H were obtained from Qingdao Haiyang Chemical Co. Ltd. Ethyl acetate, acetonitrile, ethyl ether, toluene, methanol, and petroleum ether were all of analytical grade from commercial sources.
2.2. Apparatus
Waters HPLC system (Waters 600E, USA) equipped with a ELSD detector (Alltech 2000, USA); Rotatory evaporator (Ya Rong, Shang Hai); Shaking Incubator (ZHWY211B, Shang Hai); Agilent 1100 LC/MSD System (Trap SL, USA); NMR spectrometer(Bruker Avance AV 500 MHz, Switzerland).
2.3. The Synthesis of ILs [Bmim][BF4]
87.3 g (0.5 mol) [Bmim][Cl] and 54.9 g (0.5 mol) NaBF4 were mixed in the flask with 300 ml acetone, the reaction was carried out with magnetic stirring for 24 h at room temperature, reaction liquids was vacuum filtrated, acetone was removed by reduced pressure distillation. Reaction liquids was extracted many times until no precipitation was appeared after dropping AgNO3, [Bmim][BF4] was collected. 78.3 g pale yellow ILs [Bmim][BF4] were collected after carbon decolourization and vacuum drying.
2.4. Enzymatic Reaction in ILs [Bmim][BF4]
5 mmol (0.991 g) glucose and a certain amount of 11- dodecenoic ethyl ester were dissolved in 10 ml ILs, respectively. A certain mass concentration of lipase was added to the liquids which was mixed in the shaking incubator for 1h,the reaction was carried out at various temperature, 220 rpm for 48 h, The course of the reaction was confirmed by TLC. Sample was analyzed by HPLC for determining the yield of glucose ester at the end of reaction. Various factors such as the reaction temperature, mole ratio of glucose/vinyl palmitate, water content of the system, enzyme and concentration were investigated for the yield of glucose ester.
The unreacted glucose was removed by vacuum filtration and enzyme was recycled. ILs and glucose ester were separated by extraction, when [Bmim][BF4] was the reaction solution, toluene was the extractant. Glucose ester was separated by silicagel column chromatography with chloroform/methanol (8/1, V/V) as eluant. The white dodecenoic-glucose ester was obtain after lyophilization.
2.5. The Yield of Dodecenoic-Glucose Ester
Purified 11-dodecenoic glucose ester was weighed exactly and dissolved in methanol (CP), the standard solution of 20 mg·mL−1, 40 mg·mL−1, 60 mg·mL−1, 80 mg·mL−1, 100 mg·mL−1 were obtained, the peak area of standard solution were determined by HPLC, the standard curve between peak area and concentration was obtained. The peak area of synthesis production was determined on the same condition, the concentration of sample was obtained from standard curve. The yield of synthesis production was calculated as flow:
2.6. Methods of Instrumental Analysis
The product was analyzed by both TLC and HPLC methods. In TLC analysis, silica gel G recoated thin-layer chromatography plates (Qingdao fine chemical factory, China) was used, Chloroform/methanol (8/1, each by volume) was used as the developing system. TLC bands were visualized by spraying with reagent (10% sulfuric acid aqueous solution).
The HPLC analysis was performed using a HPLC system (Waters 600E, Waters, USA) equipped with a Diamonsil C18 column, a mobile phase consisting of methanol: water (90:10, v/v) at a flow rate of 1 ml/min. the injection volume was 10 μL The eluant was analyzed with a light scattering detector (Alltech ELSD 2000) at 100˚C.
The electrospray ionization mass spectrometry (ESIMS) analysis was carried out on a Agilent 1100LC/MSD instrument (Agilent, Trap SL USA) with N2 gas flow-rate of 10 L/min, and at a temperature of 350˚C.
1H NMR, 13C NMR, 1H-1H COSY and 13C-1H HSQC spectra of the product were recorded on a Bruker Avance 500 spectrometer, using samples dissolved in CD3OD.
3. Results
3.1. Three Lipase Activities in Different ILs
Three enzymes were tested in our reaction system, using [Bmim][BF4] as the reaction media. Figure 1 showed the final yield of the sugar ester with each of the enzymes. It is clear that lipase Novozym-435 gave the highest yield
Figure 1. Effect of enzymes to the yield of glucose ester.
(51.6%), with lipase CAL-B and lipase Lipozyme TLIM yielding significantly less (43.5% and 36.1%, respectively). Therefore, the lipase Novozym-435 was used in the subsequent reactions.
Three ionic liquids were tested in our reaction system as the reaction media, using Novozym-435 as the enzymes. Figure 2 showed the final yield of the sugar ester with each of the ionic liquids. It is clear that ILs [Bmim] [BF4] gave the highest yield (53%), with [Bmim][Cl] and [Bmim][PF6] yielding significantly less (31% and 20%, respectively). Therefore, the ILs [Bmim] [BF4] was used in the subsequent reactions as the reaction media.
3.2. Optimization of Reaction Conditions
The effects of system water content, temperature, substrate ratio, lipase content and re-use of the ionic liquid on the activity and stability of Novozym-435 lipase were studied.
In our research, the effect of water content was investtigated, the results in figure 3 showed that the water content of 2% was the best condition for synthesizing glucose ester, thus, the water content of 2% was confirmed in the reaction system.
Figure 4 shows the effect of temperature on the reactions yield, showing that the yield of dodecenoic-glucose ester was highest when the temperature was at 55˚C. These data suggest that the enzyme loses its activity at