Improving enzymatic production of diglycerides by engineering binary ionic liquid medium system

New Biotechnology  Volume 26, Numbers 1/2  October 2009

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Research Paper

Improving enzymatic production of diglycerides by engineering binary ionic liquid medium system ¨ zc¸elik2 and Xuebing Xu1 Zheng Guo1, Derya Kahveci1,2, Beraat O 1 2

Department of Molecular Biology, Aarhus University, Gustav Wieds Vej 10, 8000 Aarhus C, Denmark Department of Food Engineering, Istanbul Technical University, 34396 Maslak, Istanbul, Turkey

The tunable property of ionic liquids (ILs) offers tremendous opportunity to rethink the strategy of current efforts to resolve technical challenges that occurred in many production approaches. To establish an efficient glycerolysis approach for enzymatic production of diglycerides (DG), this work reported a novel concept to improve DG yield by applying a binary IL system that consisted of one IL with better DG production selectivity and another IL being able to achieve higher conversion of triglycerides (TG). The candidates for combination were determined by individually examining lipasecatalyzed glycerolysis in different ILs, as a result, promising ones are divided into two groups based on their reaction specificities. The effects of parametric variables were then preliminarily evaluated, following a further investigation of the reaction performance in different binary IL systems from crossgroup combinations. The combination of TOMA.Tf2N/Ammoeng 102 was employed for optimization by Response Surface Methodology. Eighty to eighty-five percent (mol%) of oil conversion and up to 90% (mol%) of total DG yield (73%, wt%) were obtained, which are markedly higher than those previously reported. This work demonstrated the practical feasibility to couple the technical advantage (high TG conversion and high DG production selective in this work) of individual ILs into a binary system to overperform the reaction. It is believed that binary IL system could be also applicable to other enzymatic reaction systems for establishment of more efficient reaction protocols. Introduction Emerging as a different paradigm of solvents, ionic liquids have been growingly recognized as a promising replacement of conventional media to create environmentally benign processing techniques [1–3]. From the engineering point of view, the most distinguished feature of ILs is their ‘endless’ possibilities for property tuning, which leaves tremendous space to manipulate for a variety of applications [4,5]. There are two ways to do this: one is through judicious selection of anion and cation or appended substituents; another is proportionally mixing two property-differing ILs (construct binary IL system). In the effort toward the former respect, a number of novel ILs have been synthesized, and some new synthetic approaches have been developed [6–8]. HowCorresponding author: Xu, X. ([email protected]) 1871-6784/$ - see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.nbt.2009.04.001

ever, the interests in binary systems containing ILs are currently focusing on the investigation of mixing behaviors of ILs with organic solvents [9] or supercritical fluid (CO2), among others [10]. Little work is involved in the studies of the behavior of binary IL system [11], let alone examining the reaction specificity in binary IL system. Diglycerides (DG) are naturally occurring minor components of many edible oils, existing as two positional isomers: 1,3-DG and 1,2-DG, with a naturally isomeric ratio of 7:3 [12]. Traditionally, DGs are applied as food emulsifier; however, it has been recently developed as cooking oil and launched in Japan and U.S.A. market owing to newly uncovered beneficial effects and nutritional properties [13]. In general, there are two major approaches to produce diglycerides: one is re-esterification of glycerol with the free fatty acids released from the hydrolysis of natural plant oil; the other is www.elsevier.com/locate/nbt

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New Biotechnology  Volume 26, Numbers 1/2  October 2009

Materials and methods Ionic liquids and lipases

Research Paper

SCHEME 1

Possible pathways of enzymatic formation and degradation of diglycerides.

glycerolysis of oil. It is easier to obtain higher purity of DGs by enzymatic esterification, which is the method used by the manufacturer of the DG oil on the market. However, the processing undergoes multiple steps and is thus costly [14]. In consideration of time-space cost efficiency, glycerolysis of oil is a more straightforward approach, which directly produces DG by one step reaction [15]. Likewise, the answer is not that simple because glycerolysis of oil still remains to be a technical challenge to be resolved [16]. As indicated in Scheme 1, DG, as an intermediate product of triglyceride (TG) degradation, there are many different pathways to be formed or degraded; therefore, it is difficult to control the reaction evolution particularly stayed at DG stage. Currently industrial glycerolysis for emulsifier production is performed at high temperature (220–260 8C) with alkali as catalyst [17]. The obvious disadvantages of this method are lower desirable product yield (45–55% DG), energy consumption, bad product taste and unpreferable for the oil with unsaturated fatty acids [15]. Lipase-catalyzed glycerolysis, operated in mild conditions, avoids part of the disadvantages existed in the chemical approach; however, to increase the yield of DG remarkably still represents a challengeable topic to overcome, even though a number of efforts have been attempted [18,19]. Our group has particular interests in lipid (bulk oils and fats) processing by means of ionic liquids, which attempt to find better solutions to the technical limitations in current approaches [15,16]. We have successfully developed a new IL-based approach for enzymatic production monoglycerides (MG), revealing its unique reaction behaviors, distinguished technical advantages, and potential interest for industrial application [20,21]. The bottlenecks for maximizing DG yield are poor reaction selectivity to DG formation and lower TG conversion. However, in our attempts for enzymatic production of DG we found that these two tasks are difficult to be fulfilled by just using one type of ILs. Remembering the property of media can also be tuned by mixing two ILs, we therefore perceive that it may be possible to simultaneously achieve better selectivity and conversion by applying binary IL reaction systems. The preliminary results to be presented in this work have proved the correctness of this concept. Hence, this paper is organized as follows: envisage the association of the structure of ILs with reaction behaviors, select the IL candidates (classified into two groups according to their performance to give high TG conversion or high DG yield) for creating binary reaction systems, evaluate the reactions of cross-group coupling binary systems, and optimize the promising binary IL system. 38

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Except trioctylmethylammonium trifluoroacetate ([TOMA].[TFA]) from Merck KGaA (Darmstadt, Germany), all the other types of ILs used in this work were purchased from Solvent Innovation GmbH (Cologne, Germany). They are 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM].[BF4]), 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM].[PF6]), 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([BMIM].[CF3SO3]), 1,3-dimethylimidazolium dimethylphosphate ([DMIM].[DMP]), 1-ethyl-3-methylimidazolium 2(2-methoxyethoxy) ethylsulfate ([EMIM].[MDEGSO4]), 1-ethyl-3-methylimidazolium n-octylsulfate ([EMIM].[OctSO4]), 1methyl-3-octylimidazolium tetrafluoroborate ([OMIM].[BF4]), 1methyl-3-octylimidazolium hexafluorophosphate ([OMIM].[PF6]), trioctylmethylammonium bis(trifluoromethylsulfonyl)imide ([TOMA].[Tf 2 N]), 1-butyl-3-methylpyridinium dicyanamide ([BMPy][N(CN)2]), 1-butyl-1-methylpyrrolidinium dicyanamide ([BMPyo].[N(CN)2]), 1-ethyl-3-methylpyridinium perfluorobutanesulfonate ([MeEtPy].[C4F9SO3]), 3-methyl-1-octylpyridinium tetrafluoroborate ([MeOcPy].[BF4]), cocosalkyl pentaethoxi methyl ammonium methylsufate (Ammoeng 100), tetraalkyl ammonium sulfate (Ammoeng 102), and quaternary ammonium sulfate (Ammoeng 120). All ILs have minimum 98% purity and are used as received. Novozym 435 (Candida antarctica lipase B), Lipozyme RM IM (Rhizomucor miehei lipase), and Lipozyme TL IM (Thermomyces lanuginosus lipase) were provided by Novozymes A/S (Bagsvaerd, Denmark). Triolein with 90% purity, used as the model oil for glycerolysis, was purchased from Dr. Frischer GmbH (Bremen, Germany) and glycerol of minimum 99% purity was from Sigma–Aldrich Co. (St. Louis, MO, USA). Any other chemicals and solvents were of analytical grades.

Experimental procedure of enzymatic glycerolysis in ILs and analysis by TLC-FID In a typical reaction, 1 mmol of triolein (0.885 g) and 0.5 mmol of glycerol (0.045 g) were mixed with 1 g of IL in 25 mL jacketed reactor. The substrates and the IL are pre-mixed by magnetic agitation at 700 rpm; thereafter, the reaction was initiated by the addition of lipase (10 wt% based on the oil mass). The reaction was thermostated at desired temperature by circulated water bath with magnetic agitation at 300 rpm. Aliquots of 20 mL were periodically withdrawn, and analyzed by thin layer chromatography coupled with a flame ionization detector (TLC-FID). A parallel reaction in tert-pentanol was conducted in 25 mL jacketed reactors with 5 mL solvent containing 4 mmol oil, 2 mmol glycerol and with other identical conditions. Samples were analyzed by thin layer chromatography coupled with a flame ionization detector (Iatroscan MK-6s, Bechenheim, Germany). Aliquots were dissolved in 0.8 mL of chloroform/ methanol mixture (2:1, v/v), and 1 mL of diluted sample was spotted onto silica-coated Chromarod1 quartz rods by a semiautomatic sample spotter. Samples were developed with the developing system of n-hexane, diethyl ether, and acetic acid (45:25:1, v/v/v). An exception in the procedure was [TOMA].[Tf2N], which gave an ion peak at the same time interval with MG. To separate MG precisely, reaction product was mixed with aqueous solution of ammonium acetate (10%, w/w) to exchange anions between

New Biotechnology  Volume 26, Numbers 1/2  October 2009

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TABLE 1

Dependency of the conversion and selectivity of enzymatic glycerolysis for diglyceride production on the property of mediuma Mediumc

Conversion of TG (mol%)

1,3-DG (mol%)

1,2-DG (mol%)

1,3-/1,2-DG (mol/mol)

Total DG (mol%)

DG/MG (mol/mol)

1

BMIM.BF4

34.9

34.2

9.7

3.5

43.9

2

BMIM.PF6

37.8

28.7

9.0

3.2

37.7

2.2

3

EMIM.MDEGSO4

9.2

6.8

6.3

1.1

13.2

30.9

4

OMIM.PF6

7.4

6.1

2.1

2.9

8.2

2.1

5

TOMA.Tf2N

49.5

41.3

15.3

2.7

56.6

7.4

7.3

6

MeEtPy.C4F9SO3

5.8

4.3

2.7

1.6

7.0

3.7

7

Ammoeng 100

90.9

14.1

8.1

1.7

22.2

0.4

8

Ammoeng 102

82.6

25.6

12.3

2.1

37.9

0.7

9

Ammoeng 120

58.1

33.8

16.5

2.1

50.3

2.4

10

tert-pentanol

61.7

40.7

15.9

2.6

56.6

2.6

Research Paper

Entryb

a

The data used are the means of duplicated determinations at the 95% confidence limit. Reaction conditions: 1 mmol oil (885 mg), 0.5 mmol glycerol (45 mg), 88.5 mg Novozym 435, 1 g IL, 60 8C, 700 rpm, and 24 h. c The Novozym 435 catalyzed glycerolysis with the same conditions was also conducted in [BMIM].[CF3SO3], [DMIM].[DMP], [EMIM].[OctSO4], [OMIM].[BF4], [TOMA].[TFA], [BMPy][N(CN)2], [BMPyO].[N(CN)2], and [MeOcPy].[BF4] respectively, neither which resulted in more than 5% conversion of TG. Data are not shown in this table. b

ammonium acetate and [TOMA].[Tf2N], following sample extraction by chloroform, before the TLC-FID analysis. The area percentages of TG, DG (1,3-isomers and 1,2-isomers separately), MG, and free fatty acids (FFA) were used as weight for results evaluation. The relative mole content of glycerides in reaction mixture was calculated by percent normalization of TG, DG, MG and FFA of respective quotient of area/M.W. In the calculation of mole conversion of TG, 1,2-DG, 1,3-DG, MG and FFA were treated as 2/3, 2/3, 1/3 and 1/3 TG unit, respectively [16]. All the reactions in this work were conducted in duplicates. The means of duplicated determinations are used for result evaluation. All analysis and measurements were also done in duplicate. The adopted values in figures and tables are the means at the 95% confidence limit.

Results and discussion IL-structural dependency of the reaction specificity of enzymatic production of DGs The results in Table 1 depicted that the reaction specificities of lipase-catalyzed glycerolysis are strongly dependent on the medium applied. With identical conditions, 17 types of ILs with varied cations and anions have been tested as solvents to host the reaction in this work and compared with tert-pentanol (one of the best conventional solvents). In the discussion of the reactions in BMIM.CF3SO3, among others, eight kinds of ILs were not detailed here, because all of them resulted in < 5% TG conversion. A few indexes have been used to evaluate the glycerolysis reaction system in terms of DG production, such as conversion of TG (indexing reaction degree), ratio of 1,3-/1,2-isomer, total DG yield and ratio of DG/MG (indicating selectivity). Therefore, by integrating consideration of reaction degree and selectivity, the reaction systems of TOMA.Tf2N, Ammoeng 120, and Ammoeng 100 and 102 deserve further investigation, in which around or higher than 50% TG conversions were obtained. BMIM.BF4 and BMIM.PF6 are intensively studied ILs, with the result of relatively higher total yield of DG but lower TG conversion. Other three types of ILs resulted only in < 10% conversion of TG.

However, if one considers the reaction selectivity, TOMA.Tf2N and Ammoeng 120 preferably facilitate the formation of DG, with the mole ratio of DG/MG up to 7.4 and 2.4, respectively (Table 1). Contrarily, the yields of MGs in Ammoeng 100 and 102 overweight DG, despite under the conditions used facilitating the equilibrium shift to DG generation. This reflected the profound influence of IL properties on reaction specificity. Figure 1 presented the time course of the enzymatic glycerolysis in TOMA.Tf2N (A) and Ammoeng 102 (B). As indicated, the glycerolysis in Ammoeng 102 is much faster with high reaction degree, which almost reaches equilibrium at 4 h and TG conversion amounts up to 90% after 8 h, while the reaction in TOMA.Tf2N takes around 12 h to achieve maximum TG conversion of 56%. Another distinct difference between the two systems is the ratio of DG and MG produced at equilibrium. TOMA.Tf2N produces over 50% (mol%) DG, at the same time over 50% products in Ammoeng 102 is MG (Figure 1). The relative content of MG in TOMA.Tf2Nbased system is always lower than 10%, with the values of DG/MG varied 5–8 after 12 h. Such a high selectivity to DG formation is much encouraging to be further investigated. Differing from typical lipases [22], Candida antarctica lipase B (used in this work) does not display any interfacial activation and usually adopts an ‘open’ conformation that makes the active site accessible to solvents [23]. It is well known that substrate specificity and catalytic efficiency depend upon the ability of the enzyme to utilize the free energy binding with the substrates [24]. This binding energy reflects the difference between binding energies of the substrate–enzyme and the substrate–solvent interactions [25]. Thus, the kinetic behaviors depend strongly on solvent medium. On the contrary, the specific interactions between solvent and solvated products and reactants influence their thermodynamic activities, as such affect the reaction equilibrium accordingly [20]. Thus, an elegant explanation accounting for high selectivity of MG formation in Ammoeng 100 and 102based systems, with the assistance of computation of quantum chemical model, has been delineated in previous work [21]. The results in this work again displayed how the structure of an ionic www.elsevier.com/locate/nbt

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New Biotechnology  Volume 26, Numbers 1/2  October 2009

Effects of parametric variables on conversion and selectivity

Glycerolysis in binary ionic liquid systems

To acquire optimum conditions, the effects of parameters on the conversion and selectivity of glycerolysis for DG-production in

Cross-combination of two DG-selective ILs (TOMA.Tf2N and Ammoeng 120) and two MG-preferable ILs (Ammoeng 100 and

Research Paper

liquid affect the specificity of its mediated reaction, as well as part of molecular characteristics as a MG-selective IL (like Ammoeng 102) as previously described: cation bonded with long chain alkyl or acyl and free OH groups and strong polar anion (Scheme 2) [21]. As such, the structure of TOMA.Tf2N seems to facilitate the accumulation of DG, because three octyl groups in the cation increase hydrophobicity of TOMA.Tf2N and then increase oil dissolution for reaction, at the same time the weak polar anion of tf2N may not act as polar anion, promote further degradation of DG into MG [21]. Both interactions facilitate the accumulation of DG at equilibrium.

TOMA.Tf2N and Ammoeng 120 were examined (Table 2). Firstly, we examined effects of substrate concentration (entries 1–6). For the glycerolysis in TOMA.Tf2N, no evident change of TG conversion is observed but the reaction selectivity to DG is significantly enhanced (DG/MG varied from 3.2 to 6.2), corresponding to total DG yield increased from 47.3 to 67.9; while in Ammoeng 120 TG conversion is achieved at 0.89 g/g IL, and the total DG yield shows little dependence on the variation of TG concentration (entries 4– 6). These results suggested that high substrate concentration could be loaded and thus resulted in high volumetric productivity. This also indicated that ILs herein play the role more like creating compatible environment for glycerol and TG interaction, and the substrates are not really dissolved in ILs. The influences of temperature were tested at 50–70 8C (entries 3, 6, and 7–10), in which generally positive effect was observed. Over 10% oil conversion enhancements in both IL systems were obtained with temperature increased from 50 to 60 8C. However, either TG conversion or total DG yield maximized around 60 8C, further increasing to 70 8C added nothing for the augment. The effects of the ratio variation of glycerol/TG were also investigated (entries 3, 6, and 11–14); the dosage increase in glycerol seems to generate little influence on TG conversion and total DG yield of TOMA.Tf2N-based system (entries 3, 11, and 12). However, for Ammoeng 120-based system the increase in glycerol dosage does result in a marked increase in conversion of TG (entries 6, 13, and 14), but the value of DG/MG is decreased instead of increasing, suggesting most of converted TG contributed to the increase in MG rather than DG. It is proposed here that the different consequences of the two IL systems resulted from different properties of respective anions as aforementioned. tf2N is a weak H-bonding acceptor; by contrast, MeOSO3 is able to form strong H-bonding with glycerol. Therefore, increase in glycerol in TOMA.Tf2N behaves simply as in organic solvent; however, the strong Hbonding interaction for glycerol in Ammoeng 120 greatly reduces the thermodynamic activity of glycerol. Thus, the increase in glycerol dosage actually increases the concentration of glycerol available for reaction, corresponding to the enhancement of TG conversion and total yield of DG. However, the corresponding increase in MG yield is slightly faster than that of DG, which leads to the decrease in the reaction selectivity (entries 6, 13, and 14). The results in Table 2 present us a general tendency of the effects of parameters on the reaction specificity in two DG-preferable IL systems. A higher conversion and total DG yield compared with in tpentanol are obtained (Tables 1 and 2). However, it should be pointed out that this result is not very satisfactory to the desired goal. This promotes us to rethink the strategy whether it is practical to reach the goal by just using single IL system. Inspection of Table 2 reveals that lower TG conversion in TOMA.Tf2N-based system seriously limits further increase in the total yield of DG, while high conversion of oil can be readily carried through by applying Ammoeng 100 or 102. We, therefore, perceive that it might be feasible to employ binary IL system combined with a high DGselective IL and another IL with low DG-selectivity but being able to achieve high TG conversion, in order to obtain high total DG yield.

FIGURE 1

Time course of Candida artarctica B catalyzed glycerolysis in TOMA.Tf2N (A) and Ammoeng 102 (B). Reaction conditions: 1 mmol of oil, 0.5 mmol of glycerol, Novozym 435 in the amount of 10 wt % based on the oil mass, 1 g IL, 60 8C, 48 h, 700 rpm. The data presented are the means of duplicated determinations at the 95% confidence limit.

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SCHEME 2

Molecular structures of TOMA.Tf2N and Ammoeng 102.

102) into binary IL systems have been examined for enzymatic production of DGs (Table 3, entries 1–4). Among the four combinations with equal weight mixing of two ILs, some observations can be generalized: the binary IL systems with the presence of Ammoeng 100 result in higher TG conversion (around 80%, entries 1 and 3), and Ammoeng 102 presented systems give lower conversion (entries 2 and 4); Ammoeng 120-based binary IL systems show fast reaction rate, arriving at the equilibrium at 8 h (enries 3 and 4). However, in terms of the selectivity toward DG formation, TOMA.Tf2N/Ammoeng 102 obtained maximum value 8.8 with total DG yield of 63.5% (mol%) (entry 2). Other binary systems like entries 1 and 3 achieved high TG conversion, but also generated a relatively high percentage of MG. Therefore, combination of TOMA.Tf2N/Ammoeng 102 was chosen for further optimization of variables. According to the results in Table 2, we selected ratio of glycerol/ oil and temperature, as well as the blend ratio of the two ILs as the parameters to optimize TOMA.Tf2N/Ammoeng 102 binary system. A statistic model of Response Surface Methodology (RSM) (Umetrics, Sweden) was used for optimization design, with TG conver-

sion, total DG yield and reaction selectivity (DG/MG) as response indexes (detail no shown). Part of the results has been shown in Table 3 (entries 5–8) for a conceptual demonstration. It is clear that both conversion of TG (78%–85%) and yield of desired product (total DG) (82–92% by mole, 70–74% by weight) are markedly improved by binary IL reaction systems, which are significantly higher than the results of DG production oriented glycerolysis reaction systems ever reported [26]. In the four attempts shown in Table 3 (entries 5–8) the selectivities are also remarkably high (DG/ MG > 20), indicating that the formation of MG was seriously suppressed. The yield of byproduct MG varied from 1.1% to 4.3% (wt%). If having a close examination of the parameters applied, we could find out that the results in TOMA.Tf2N/ Ammoeng 102 binary system seems not to be significantly affected by temperature variation. However, for glycerol dosage all the four entries with better performance appeared within the range of glycerol: TG > 1:2, which agree with aforementioned hypothesis that excessive glycerol amount (by stoichiometric ratio, glycerol:TG, 1:2) is needed to make up for the amount of glycerol available for reaction owing to the lower thermodynamic activity

TABLE 2

Effects of parametric variables on the conversion and selectivity of Novozym 435 catalyzed glycerolysis in ionic liquidsa Entryb

Ionic liquid

Concentration of oil (g/g IL)

Temperature (8C)

Mole ratio of glycerol/oil

Conversion of TG (mol%)

Total DG (mol%)

DG/MG (mol/mol)

1

TOMA.Tf2N

0.44

60

1:2

48.9

47.3

3.2

2

TOMA.Tf2N

0.89

60

1:2

48.8

57.5

7.4

3

TOMA.Tf2N

1.77

60

1:2

51.0

67.9

6.2

4

Ammoeng 120

0.44

60

1:2

54.8

49.4

3.4

5

Ammoeng 120

0.89

60

1:2

58.1

50.2

2.4

6

Ammoeng 120

1.77

60

1:2

51.3

49.3

2.6

7

TOMA.Tf2N

1.77

50

1:2

32.6

40.1

7.1

8

TOMA.Tf2N

1.77

70

1:2

41.4

56.5

15.2

9

Ammoeng 120

1.77

50

1:2

35.3

37.8

2.8

10

Ammoeng 120

1.77

70

1:2

50.7

51.5

2.9

11

TOMA.Tf2N

1.77

60

1.25:2

45.4

52.2

5.8

12

TOMA.Tf2N

1.77

60

1.50:2

50.9

61.0

3.5

13

Ammoeng 120

1.77

60

1.25:2

59.5

50.3

1.9

14

Ammoeng 120

1.77

60

1.50:2

65.0

53.1

1.6

a b

The data used are the means of duplicated determinations at the 95% confidence limit. The reaction was conducted in 1 g ionic liquid with Novozym 435 load of 10% of oil weight and agitation at 700 rpm for 24 h. www.elsevier.com/locate/nbt

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New Biotechnology  Volume 26, Numbers 1/2  October 2009

TABLE 3

Reaction specificity of enzymatic production of diglycerides in binary ionic liquid systemsa Entry

Binary ILs

IL 1/IL 2 (wt: wt)

Temperature (8C)

Mole ratio of glycerol/oil

Time (h)

Conversion of TG (mol%)

Total yield of DG (mol%)

DG/MG (mol/mol)

Total yield of DG (wt%)

Research Paper

1

TOMA.Tf2N/Ammoeng 100

50/50

60

1.0/2.0

24

80.7

58.8

1.8

2

TOMA.Tf2N/Ammoeng 102

50/50

60

1.0/2.0

24

54.7

63.5

8.8

51.0

3b

Ammoeng 120/Ammoeng 100

50/50

60

1.0/2.0

8

80.9

40.7

0.9

46.5

4b

Ammoeng 120/Ammoeng 102

50/50

60

1.0/2.0

8

62.5

48.0

1.6

45.5

5

TOMA.Tf2N/Ammoeng 102

25/75

45

1.75/2.0

24

82.6

89.1

34.4

73.9

6

TOMA.Tf2N/Ammoeng 102

25/75

75

1.75/2.0

24

79.7

91.4

22.9

70.6

7

TOMA.Tf2N/Ammoeng 102

50/50

60

1.95/2.0

24

78.7

82.3

9.0

70.8

8

TOMA.Tf2N/Ammoeng 102

8/92

60

1.45/2.0

24

84.9

87.9

22.0

71.9

57.6

a

The reaction was conducted in 1 g of two ionic liquid mixture with 1.77 g oil and 177 mg Novozym 435, with magnetic agitation at 700 rpm.

a b

The data used are the means of duplicated determinations at the 95% confidence limit. The equilibrium is reached at 8 h.

of glycerol in the environment of Ammoeng 102 resulted from its H-bonding with polar anion of EtOSO3 [21]. Last but not least to be emphasized is that Ammoeng 102 other than TOMA.Tf2N covers more fraction in an optimized composition of TOMA.Tf2N/Ammoeng 102 (within the range of 8/100–50/50), which suggested that an essential role Ammoeng 102 played to increase the conversion of TG.

Conclusions The basic objective of this study is to establish an efficient protocol for enzymatic production of DG by means of the tunable property of ionic liquids. To this end, different types of ILs have been tested, and a simply parameter optimization of those promising IL systems for DG production were also carried out. It proved that single IL system was insufficient to achieve remarkably higher yield of desired products than conventional solvents. The authors thus resort to binary IL systems based on the observations of the characteristics of enzymatic glycerolysis in single IL system. The binary IL systems are constructed with one IL with better DG generation selectivity and another IL being able to

achieve high conversion of oil. A preliminary parameter optimization was then conducted for selected binary IL system: TOMA.Tf2N/Ammoeng 102. Ninety percent (mol%) of total DG yield (73% by wt%) with up to 85% (mol%) of oil conversion were obtained, which are remarkably higher than any organic solventsbased glycerolysis systems ever reported (In the authors’ another effort that is not detailed in this work, about 65% (mol%) (60% by wt%) maximum yield of total DG with the maximum conversion of TG around 72% (mol%) is obtained under optimized conditions in t-pentanol system). From this point, the strategy adopted in this work is successful. To our best knowledge, this is also the first report by using binary IL system to improve the volumetric productivity and selectivity of an enzymatic reaction. It is believed that this strategy could be also applicable to other enzymatic reaction systems for setting up more efficient reaction protocols.

Acknowledgement Financial support from Danish Research Council for Technology and Production (FTP) (274-05-0286) is gratefully acknowledged.

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