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Journal of Biotechnology 133 (2008) 486489

Short communication

Lipase-catalyzed synthesis of glucose fatty acid ester using ionic liquids mixtures
Sang Hyun Lee a , Sung Ho Ha a , Nguyen Minh Hiep b , Woo-Jin Chang a , Yoon-Mo Koo a,b,
a

ERC for Advanced Bioseparation Technology, Inha University, Incheon 402-751, Republic of Korea b Department of Biological Engineering, Inha University, Incheon 402-751, Republic of Korea Received 18 June 2007; received in revised form 29 October 2007; accepted 12 November 2007

Abstract Novozym 435-catalyzed synthesis of 6-O-lauroyl-d-glucose in ionic liquids (ILs) was investigated. The highest lipase activity was obtained in water-miscible [Bmim][TfO] which can dissolve high concentration of glucose, while the highest stability of lipase was shown in hydrophobic [Bmim][Tf2 N]. The optimal activity and stability of lipase could be obtained in [Bmim][TfO] and [Bmim][Tf2 N] mixture (1:1, v/v). Specically, the activity of lipase was increased from 1.1 to 2.9 mol min1 g1 by using supersaturated glucose solution in this mixture, compared with reaction using saturated solution. After 5 times reuse of lipase, 86% of initial activity was remained in this mixture, while the residual activity in pure [Bmim][TfO] was 36%. Therefore, the productivity obtained by using ILs mixtures was higher than those in pure ILs. 2007 Elsevier B.V. All rights reserved.
Keywords: Ionic liquids mixtures; Glucose ester; Lipase; Supersaturation

Fatty acid sugar esters offer a wide range of commercial applications such as pharmaceuticals, cosmetics, and food industry due to their surfactant properties (Flores et al., 2002). While most of these compounds are chemically synthesized, the enzymatic synthesis of sugar esters has garnered considerable interest with regard to the creation of environment-friendly processes (Kennedy et al., 2006; Liu et al., 2005). However, the choice of solvent for the enzymatic synthesis of sugar esters is very difcult, because one reactant is polar (sugar), the other is nonpolar (fatty acid or fatty acid vinyl ester), and resulting product is amphiphilic (sugar ester). In addition, most enzymes are quickly inactivated under hydrophilic organic solvents (e.g., pyridine or dimethylformamide) which are able to dissolve high concentration of both sugars and fatty acids (Ganske and Bornscheuer,

Abbreviations: [Bmim][PF6 ], 1-butyl-3-methylimidazolium hexauorophosphate; [Bmim][TfO], 1-butyl-3-methylimidazolium triuoromethanesulfonate; [Bmim][Tf2 N], 1-butyl-3-methylimidazolium bis[(triuoromethyl) sulfonyl]amide. Corresponding author at: Department of Biological Engineering, Inha University, Incheon 402-751, Republic of Korea. Tel.: +82 32 860 7513; fax: +82 32 872 4046. E-mail address: ymkoo@inha.ac.kr (Y.-M. Koo). 0168-1656/$ see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2007.11.001

2005a). Therefore, a partially dissolved or a solid-phase system or a metastable supersaturated solution can be used for the enzymatic reaction in less harmful organic solvents such as acetonitrile, acetone, t-butanol, and 2-methyl 2-butanol (Cao et al., 1997; Flores et al., 2002; Degn and Zimmermann, 2001). Alternatively, the solubility can be increased by using protected glucoses or alkyl glycosides. However, the use of these substrates requires extra steps; otherwise the products show different properties compared to non-derivatized glucose esters (Ganske and Bornscheuer, 2005b). Ionic liquids (ILs) are organic salts that melt below 100 C. Interest in ILs stems from their potential as green solvents (Sheldon et al., 2002). Specically, their non-volatile character and thermal stability make them attractive alternatives for volatile organic solvents. In chemical processes, ILs exhibit excellent physicochemical characteristics including the ability to dissolve polar and non-polar organic, inorganic, and polymeric compounds. Several groups (Park and Kazlauskas, 2001; Kim et al., 2003; Ganske and Bornscheuer, 2005b) have reported that anhydrous ILs containing [BF4 ] and [PF6 ] anions can be used as an alternative reaction media for biotransformation of sugars, but the solubilities of sugars in these ILs are very low. It was also observed that the use of ILs as reaction media enhanced

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the reactivity, selectivity, and stability of enzymes. However, most enzymes irreversibly deactivated in ILs containing chloride or dicyanamide ([dca]) anions which have been reported to be good solvents for sugar dissolution (Lee et al., 2006; van Rantwijk et al., 2006). Firstly, the lipase-catalyzed synthesis of 6-O-lauroyl-dglucose using supersaturated glucose solution in pure ILs was investigated. All ILs were synthesized and puried by C-TRI (Suwon, Korea) and had a residual chloride content of less than 30 ppm. The supersaturated solution, a solution which contains more dissolved solute than the solubility limit (Holtzclaw and Robinson, 1988), is usually prepared by dissolving excess solute at high temperature and then slowly cooling to low temperature. However, considerable time is required to dissolve sugars in viscous ILs and high temperature can cause degradation of sugars, and thus an alternative dissolution process that uses water as a mediator was recently developed to overcome this drawback (Lee et al., 2008). The supersaturated glucose solutions in water-miscible [Bmim][TfO] and [Bmim][BF4 ] had 12 and 7 times higher concentrations than solubility at 40 C, respectively (Table 1). Although, the dissolved glucose concentrations in water-immiscible ILs containing [Bmim][Tf2 N] and [Bmim][PF6 ] were very small, supersaturated solutions which contain 3 times higher concentration of glucose were obtained. To investigate the inuence of the dissolved glucose concentration on the reaction rate and conversion, lipase-catalyzed transesterication was carried out with the same content of glucose (222 mM) (Table 1). The initial rate and conversion for the reaction with supersaturated glucose solution in hydrophilic [Bmim][TfO] and [Bmim][BF4 ] were much higher than those of the reaction with partially dissolved glucose solution. The highest enzyme activity was obtained in the reaction with supersaturated glucose solution in [Bmim][TfO]. However, the activities of Novozym 435 in [Bmim][TfO] was signicantly

decreased after reuse and the residual activity was 71%. On the other hand, the activities of Novozym 435 in hydrophobic [Bmim][Tf2 N] and [Bmim][PF6 ] were much lower than that in [Bmim][TfO], although the activities were enhanced around 10% by using supersaturated glucose solution. However, the residual activities after reuse of Novozym 435 in [Bmim][Tf2 N] and [Bmim][PF6 ] were higher than 95%. The enzyme in organic solvents or ILs usually shows higher activity and stability in the more hydrophobic solvents (Zhao, 2005). However, a hydrophobic solvent is inappropriate choice if the solubility of a hydrophilic substrate has to be taken into account (Degn and Zimmermann, 2001). In our work, the Novozym 435 showed higher activity in the hydrophilic and water-miscible ILs, while enzyme stability in these ILs was lower than those in hydrophobic ILs. Therefore, mixtures of ILs were used as reaction media for the lipase-catalyzed synthesis of glucose ester in order to obtain optimized activity and stability of enzyme, simultaneously. Mixing of two different ILs which show different physicochemical properties can easily make new reaction media, because hydrophobic ILs and hydrophilic ILs are generally miscible. The reaction rate of Novozym 435-catalyzed esterication of glucose with vinyl laurate and residual activity of the enzyme after reuse were determined in a range of ILs mixtures (Table 1). The dissolved glucose concentration of supersaturated solution prepared by water-mediated method in ILs mixtures was much higher than solubility. The enzyme activity was well correlated with initially dissolved glucose concentration in pure ILs and ILs mixtures (Fig. 1). These results indicate that homogeneously dissolved glucose in a supersaturated solution can be easily transported to the active site of lipase, while the low dissolution rate of partially dissolved glucose in ILs and ILs mixtures may limit the activity of the enzyme. In addition, the increase of dissolved glucose concentration yielded better conversion because the equilibrium was forced toward

Table 1 Dissolved glucose concentration, enzyme activity, and residual enzyme activity in ILs and ILs mixtures Solvents Dissolved concentration of glucose at 40 C (mM) Saturated solutionc [Bmim][BF4 ] [Bmim][TfO] [Bmim][TfO]:[Bmim][Tf2 N], 25% [Bmim][TfO]:[Bmim][Tf2 N], 50% [Bmim][TfO]:[Bmim][Tf2 N], 75% [Bmim][Tf2 N] [Bmim][TfO]:[Bmim][PF6 ], 50% [Bmim][PF6 ]
a

Enzyme activitya ( mol/min/g) Saturated solution 0.65 1.31 1.27 1.08 0.98 0.69 0.92 0.67 Supersaturated solution 1.29 4.21 3.75 2.85 1.89 0.77 1.94 0.76

Residual enzyme activitya,b (%) Supersaturated solution 66.1 70.5 87.9 96.0 95.9 95.7 95.0 96.0

Supersaturated solutiond 43.4 363.3 127.2 50.0 18.3 1.7 50.0 1.7

6.3 30.6 11.1 6.1 1.7 0.6 6.1 0.6

Reaction conditions: 222 mM glucose, 444 mM vinyl laurate, 0.5 mL IL, 50 mg Novozym 435, 40 C. The water contents in ILs were less than 0.1%. At the end of the reaction, deionized water (1 mL) was added to the reaction vials in order to remove unreacted glucose. The concentration of unreacted glucose in the supernatant after centrifugation was determined by the dinitrosalicylic acid (DNS) method with a glucose standard. Enzyme activity was determined from the glucose content consumed after 6 h reaction. The precipitated product (6-O-lauroyl-d-glucose) was dissolved in tetrahydrofuran and the concentration was determined by HPLC (Lee et al., 2008). b After reaction for 6 h, Novozym 435 was washed with water and tetrahydrofuran and then dried in desiccator before reuse. The residual activity was determined by the relative glucose conversion in subsequent reaction. c Ionic liquids and glucose crystal were directly mixed and incubated for 12 h at 40 C. d Ionic liquids and glucose solution in water were mixed. The mixture was then evaporated to remove water at 60 C and slowly cooled to 40 C.

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Fig. 1. Dependence of the enzyme activity in glucose ester synthesis on the initially dissolved glucose concentration in ILs or ILs mixtures. Data from Table 1.

synthesis. On the other hand, the residual activities of enzyme in the ILs mixtures of [Bmim][TfO] with more than 50% of [Bmim][Tf2 N] or [Bmim][PF6 ] were similar to those in pure hydrophobic ILs. Fig. 2 shows the time courses for the enzymatic synthesis of 6-O-lauroyl-d-glucose in pure ILs and ILs mixtures. By using supersaturated glucose solution (222 mM) in pure [Bmim][TfO] and [Bmim][TfO]:[Bmim][Tf2 N] (1:1, v/v) mixture, concentrations of lauroyl glucose of 190 and 153 mM were obtained after 11 h of incubation, respectively. The conversion in the mix-

ture was 300% of the initial concentration of dissolved glucose or 69% of total amount of glucose added to the reaction system. Although, the dissolved glucose concentration (50 mM) in the mixture was much lower than that (222 mM) in pure [Bmim][TfO], conversion in the mixture was not signicantly decreased. In addition, the initial reaction rate of Novozym 435 was increased from 1.3 to 4.4 mol min1 g1 by using supersaturated glucose solution in this mixture, compared with reaction using saturated glucose solution. On the other hand, only the monoester, 6-O-lauroyl-d-glucose, was formed in pure ILs and ILs mixtures, as conrmed by 1 H NMR and LC-MS. As the solubility of monoester in pure ILs and ILs mixtures is lower than 0.1 mM at 40 C, precipitation of monoester as it is formed may increase the conversion of glucose and restrain the further formation of diester. As shown in Fig. 3, the activity of Novozym 435 was well maintained in [Bmim][Tf2 N], while the activity of enzyme in [Bmim][TfO] was drastically decreased. By increasing the content of [Bmim][Tf2 N] in ILs mixtures, stability of Novozym 435 was increased. The stability of Novozym 435 in [Bmim][Tf2 N]:[Bmim][TfO] (1:1, v/v) mixture was similar to that in pure [Bmim][Tf2 N] and the residual activity after 5 times reuse was about 85%. After 11 h reaction with supersaturated glucose solution in [Bmim][TfO], 86% conversion was obtained in the rst reaction. However, conversion in the second reaction with reused enzyme was decreased to 61%. On the other hand, 69% conversion was obtained in the rst reaction with a [Bmim][Tf2 N]:[Bmim][TfO] (1:1, v/v) mixture, while conversion in the reaction with 5 times reused enzyme was still 59%. These results indicate that ILs mixture is more efcient reaction media for the biotransformation of sugars when the economical aspects relating to the yield of product and cost of

Fig. 2. Time courses for the lipase-catalyzed esterication of glucose with vinyl laurate in pure ILs and ILs mixtures. Reaction conditions: 222 mM glucose, 444 mM vinyl laurate, 0.5 mL ILs, 50 mg Novozym 435, 40 C ( , [Bmim][TfO]; , 50% [Bmim][TfO] with 50% [Bmim][Tf2 N]; , [Bmim][Tf2 N]). Filled symbols represent the reaction with supersaturated glucose solution. Empty symbols represent the reaction with saturated solution with undissolved glucose crystals present.

Fig. 3. Residual activity of Novozym 435 after reuse in pure ILs and ILs mixtures. After reaction for 11 h, Novozym 435 was washed with water and tetrahydrofuran and then dried in desiccator to reuse. Reaction conditions: 222 mM glucose, 444 mM vinyl laurate, 0.5 mL ILs, 50 mg Novozym 435, 40 C ( , [Bmim][TfO]; , 75% [Bmim][TfO] with 25% [Bmim][Tf2 N]; , 50% [Bmim][TfO] with 50% [Bmim][Tf2 N]; , [Bmim][Tf2 N]).

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enzyme are considered. For example, 1.3 times higher product could be obtained after 5 times reuse of Novozym 435 in the mixture, compared to pure [Bmim][TfO]. The higher product in the mixture may be obtained with increasing reaction time and the number of reuse of Novozym 435 than those in pure ILs. As the direct esterication may be more interesting from an industrial point of view due to the low price of the substrate and non-toxic by-product (water) (Degn and Zimmermann, 2001), esterication of glucose with lauric acid was also investigated by using supersaturated glucose solution in the [Bmim][Tf2 N]:[Bmim][TfO] (1:1, v/v) mixture. The reaction temperature was increased to 50 C to dissolve lauric acid and molecular sieve (4 A, 15%, w/v) was used to remove water. Although the activity (1.95 mol min1 g1 ) of Novozym 435 was about 70% of transesterication reaction (2.85 mol min1 g1 ), 52% conversion was obtained after 24 h reaction. Although several studies have indicated that the organic solvents mixtures or mixtures of ILs and organic solvents are suitable for biotransformation of underivatized carbohydrates (Degn and Zimmermann, 2001; Ganske and Bornscheuer, 2005a), the enhancement of activity and stability of enzyme is still challenging problem to commercialize the biocatalytic systems. In addition, the use of organic solvents as reaction media causes the environmental problems. As shown in this reports, lipase-catalyzed esterications using supersaturated sugar solution in ILs mixtures can be employed to overcome the problem of restricted sugar solubility and low enzyme stability in various reactions. Acknowledgements This work was nancially supported by the Engineering Research Center for Advanced Bioseparation Technology. The authors also gratefully acknowledge support from the Korea Science and Engineering Foundation (KOSEF).

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