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Mesophilic alcohol dehydrogenase behavior in imidazolium based ionic liquids
Journal of Molecular Liquids 161 (2011) 139–143
Contents lists available at ScienceDirect
Journal of Molecular Liquids j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m o l l i q
Mesophilic alcohol dehydrogenase behavior in imidazolium based ionic liquids Bahareh Dabirmanesh a, Khosro Khajeh a,⁎, Jafar Akbari b, Hanieh Falahati c, Somayeh Daneshjoo d, Akbar Heydari b,⁎⁎ a
Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran Department of Chemistry, Faculty of Sciences, Tarbiat Modares University, Tehran, Iran Department of Biotechnology, Faculty of Sciences, Tehran University, Tehran, Iran d Department of Microbiology, Faculty of Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran b c
a r t i c l e
i n f o
Article history: Received 19 February 2011 Received in revised form 10 May 2011 Accepted 12 May 2011 Available online 25 May 2011 Keywords: Activity Alcohol dehydrogenase Fluorescence Ionic liquids Stability
a b s t r a c t The use of ionic liquids to replace organic solvents in biocatalytic processes has recently gained much attention. Despite the wide applications of oxidoreductases, there are few reports of their catalyzed reaction in ionic liquid. We have investigated the influence of four water miscible ionic liquids on the activity, stability and structure of the mesophilic alcohol dehydrogenase from yeast. Upon changes in ionic liquids concentration, both activity and stability of the enzyme were affected. As the concentration of ionic liquids increased, Km increased while kcat decreased. Associated conformational changes caused by ILs (150 mM) were monitored using fluorescence technique. Finally, the effects of ILs cations and anions on the enzyme activity and stability in aqueous IL mixtures were discussed. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Ionic liquids (ILs) are nonvolatile, nonflammable and highly stable organic salts. They hold great potential as environmentally friendly solvents. Their polarity, hydrophobicity and solvent miscibility behaviors can be tuned through the appropriate modification of the cation and anion. Ionic liquids have attracted considerable attention and have emerged as interesting novel reaction media for enzymatic transformation since enzymes show remarkable difference in catalytic features in systems involving different ILs. A number of enzymes have been reported to retain catalytic activity in an ionic liquid medium [1–7]. Lipases have been extensively studied and were more enantioselective in such reaction media than organic solvents. The lipase Novozym 435 was found to be greatly stabilized in the ionic liquid [1]. Some oxidoreductases, such as laccase and peroxidase, chloroperoxidase and D-amino acid oxidase were also active in ionic liquids [8,9]. Walker and Bruce [10] studied the activity
of cofactor dependent enzymes in ILs. Previously, horse liver alcohol dehydrogenase (HLADH) displayed good activity in the system involving [BMIm][Cl] [11]. Eckstein et al. [12] reported the reduction of 2-octanone to (R)-2-octanol catalyzed by alcohol dehydrogenase from Lactobacillus brevis in a biphasic system of [BMIm][NTf2] and phosphate buffer. Several groups reported not just increased activity but increased enzyme stability in ionic liquids as compared with organic solvents [3,13]. Spectroscopic methods have recently been described to associate structural changes in Candida antarctica lipase B and α-chymotrypsin with stability in ILs [14–17]. Despite the importance of alcohol dehydrogenases (ADHs), there are only a few examples concerning the use of them with ionic liquids. So it was of great interest to investigate the influence of various ILs on the catalytic performance and conformational changes of mesophilic alcohol dehydrogenase from yeast (YADH). We herein report the catalytic characteristics, stability and the fluorescence spectra of YADH in the systems involving 1-butyl-3-methylimidazolium ([BMIm][Cl] and [BMIm][BF4]) and 1-methylimidazolium ([MIm][Cl] and [MIm][BF4]).
2. Materials and methods ⁎ Corresponding author at: Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, P.O. Box 14115-175, Tehran, Iran. Tel./fax: +98 2182884717. ⁎⁎ Corresponding author at: Department of Chemistry, Faculty of Sciences, Tarbiat Modares University, P.O. Box 14115-175, Tehran, Iran. Tel./fax: + 98 2188009730. E-mail addresses: [email protected] (K. Khajeh), [email protected] (A. Heydari). 0167-7322/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.molliq.2011.05.007
2.1. Materials YADH, NaHPO4 and NAD+ were purchased from Sigma (St. Louis, MO, USA). N-methylimidazole, HBF4 (50% aqueous), HCl and 1-chlorobutane were obtained from Aldrich-Fluka (USA). Ionic liquids ([BMIm][Cl], [BMIm][BF4], [MIm][Cl] and [MIm][BF4] ) used in this research were
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prepared in our laboratory with 97% purity. The purity of YADH was confirmed by SDS–PAGE. 2.2. Preparation of imidazolium-based ionic liquids [MIm][Cl] and [MIm][BF4] were obtained by neutralizing N-methylimidazole with aqueous HCl and HBF4. The water was removed by evaporation under reduced pressure. The residue was washed with hexane and then remained under vacuum to produce pure ionic liquid [18,19]. In order to synthesize [BMIm][Cl], N-methylimidazol (5 mmol) and butyl chloride (5 mmol) were mixed in a round bottom flask under nitrogen and then stirred at 85 °C for 72 h. The resulting pale yellow liquid was then washed with ethyl acetate and remained under vacuum overnight to afford white solid, 1-butyl-3-methylimidazolium chloride [20,21]. The resulting white solid, [BMIm][Cl], was dissolved in water and stoichiometric amount of HBF4 was added drop wise overnight. The reaction was stirred at room temperature for an additional 12 h and extracted with CH2Cl2. The organic layer was washed with water until free from acid. Obtained ionic liquid was dried in a vacuum oven overnight at 80 °C to afford a pale yellow liquid and used without further purification [20,22]. All manipulations were performed under nitrogen atmosphere using standard techniques. The structure of ionic liquids 1 was examined by HNMR spectroscopy [22]. Physical and spectroscopic data of ionic liquids are:
2.5. Stability in ionic liquids Enzyme stability was analyzed by incubating enzymes in the absence and presence of 150 mM of each IL at 40 °C for 2 h. Samples were then cooled on ice (for half an hour) and assayed for residual activity as described previously. Sample with no ionic liquid was used as a control. Thermal inactivation of YADH was also determined in 50 mM potassium phosphate (pH 7.8) containing 150 mM [MIm][Cl] at 40 °C. At various time intervals (every 10 min for an hour) samples were removed, cooled on ice and then assayed for residual activity at 25 °C. Plots of the log of residual activity versus time were linear, indicating a first-order decay process under these conditions. Results presented in this paper are the mean from at least 3 repeated experiments in a typical run to confirm reproducibility and the standard deviations were ±5%. 3. Results and discussion The use of room temperature ionic liquids (RTILs), especially those based on 1-alkyl-3-methylimidazolium cations, as media for enzymatic catalysis has aroused the interest of many researchers because of their solvating ability, negligible vapor pressure, easy recyclability and reusability [1–3]. Due to the wide field of application of oxidoreductases, we have studied the influence of room temperature imidazolium based ionic liquids on the activity and stability of YADH.
1
- 1-Methylimidazolium chloride [MIm][Cl]: Mp: 72 °C, H NMR (CDCl3, 500 MHz): δ 10.27 (s, 1H), 8.33 (s, 1H), 7.17 (s, 1H), 7.11 (s, 1H), 3.69 (s, 3H). Yield: 94%. 1 - 1-Methylimidazolium tetrafluoroborate [MIm][BF4]: Mp: 35.8 °C, H NMR (DMSO(d6), 500 MHz): δ 3.85 (s, 3H), 7.64 (s, 1H), 7.67 (s, 1H), 9.01 (s, 1H), 11.09 (s, 1H). Yield: 92%. 1 - 1-Butyl-3-methylimidazolium chloride [BMIm][Cl]: Mp: 65 °C, H 3 NMR (CDCl3, 500 MHz): δ 0.80 (t, 3H, JHH = 7.3), 1.23 (m, 2H), 3 1.75 (m, 2H), 3.98 (s, 3H), 4.19 (t, 2H, JHH = 7.4), 7.46 (s, 1H), 7.63 (s, 1H), 9.55 (s, 1H). Yield: 89%. - 1-Butyl-3-methylimidazolium tetrafluoroborate [BMIm][BF4]: Mp: 1 3 −74 °C, H NMR (CDCl3, 500 MHz): δ 0.95 (t, 3H, JHH=7.3), 1.37 (m, 3 2H), 1.93 (m, 2H), 4.07 (s, 3H), 4.40 (t, 2H, JHH = 7.1), 7.79 (s, 1H), 7.85 (s, 1H), 9.55 (s, 1H). Yield: 91%.
3.1. Effect of ILs on the activity of alcohol dehydrogenases Effects of ionic liquids on the enzyme activity were studied by measuring the initial rate of reaction catalyzed by 0.2 μg/ml of YADH in 50 mM phosphate buffer (pH 7.8) containing 450 mM ethanol and 0.4 mM NAD + in the absence (control) and presence of different amounts of ILs. Variation of YADH activity upon the changes in ILs concentration is shown in Fig. 1, a clear decrease of the initial velocity for the investigated enzyme was observed. The order of YADH activity in solution containing ILs was [BMIm][Cl] N [BMIm][BF4] N [MIm][BF4] ~ [MIm][Cl]. Results show the classical and fast activity decay behavior with ILs concentrations. For these kinds of ILs, this phenomenon has been widely observed (see Ref. [5], for the case of [BMIm][Cl]). In contrast to previous studies that indicated lower activity in more
2.3. Enzyme assay YADH assay was carried out in the 1 ml reaction mixture containing 916 μl of 50 mM sodium phosphate pH 7.8, 17 μl of 24 mM NAD+ and 67 μl of various substrate (ethanol) concentrations ranging from 0.07 M to 6.75 M at 25 °C for 1 min[23,24]. The reduction of NAD + was measured photometrically at 340 nm. One unit of enzymatic activity was defined as the quantity of enzyme necessary to produce 1 μmol NADH per minute. Kinetic parameters (Km and Vmax) were calculated using lineweaver–Burk plots. For the reactions in the presence of different concentrations of ionic liquid the amount of buffer was reduced and replaced by the ionic liquid, controlling pH was also essential to obtain consistent results with ionic liquids. 2.4. Fluorescence spectroscopy Fluorescence spectra of YADH were monitored using a Perkin Elmer LS 55 fluorescence spectrometer. After incubation for 5 min, samples were excited at 295 nm and the emission was recorded between 300 and 400 nm. The excitation and emission slit were both set to 5 nm. In all cases, it was necessary to subtract a blank medium without enzyme to discount the influence of the imidazolium ring fluorescence on the enzyme fluorescence spectrum.
Fig. 1. Variation of enzyme activities upon addition of different concentrations of [BMIm][BF4] (▲), [BMIm][Cl] (■), [MIm][Cl] (□) and [MIm][BF4] (Δ) to 50 mM phosphate buffer (pH 7.8) containing 0.4 mM NAD+, 450 mM ethanol and 0.2 μg/ml of YADH. The relative activity (%) refers to the percentage of the initial reaction rate obtained by the enzyme in the presence of the above ionic liquids compared to the one obtained in the absence of them.
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kosmotropic cations, YADH demonstrated higher activity in the presence of [BMIm][Cl] which is a stronger kosmotrope than [MIm] [Cl] [25]. The observed decay in enzyme activity could be due to either enzyme deactivation or enzyme inhibition. Reversibility of inhibition was determined by measuring the recovery of enzymatic activity after a rapid and large dilution of the enzyme–inhibitor complex. YADH (100 fold over the concentration used above) was incubated with different ionic liquids for 40 min. Then the mixture was diluted about 100 fold with 50 mM phosphate buffer (pH 7.8) containing the enzyme substrates (NAD + and ethanol to the final concentration of 0.4 mM and 450 mM) to initiate reaction. The results showed that the activity was recovered and the enzyme activity remained constant during the incubation time. Therefore, the reduction in the activity might be due to the inhibition of YADH. 3.2. Effect of ILs on kinetic parameters In order to get a deeper insight into how the ionic liquids affect the enzymes kinetics, values of kcat, Km and kcat/Km were determined as the content of each IL increased in the mixture. The results revealed that the presence of ionic liquids affected both the Km and kcat (Fig. 2). The Km and kcat was estimated as 26 mM and 460 s−1 for the uninhibited reaction using ethanol as a substrate. Upon the addition of ionic liquids, kcat was decreased and Km increased. These results infer that the binding affinity of substrates to mesophilic alcohol dehydrogenase significantly weakens owing to the presence of the ionic liquid in the reaction solution. Similar to our result, a simultaneous increase in Km and decrease in Vmax were also observed for laccase [26] when the reaction medium was switched from aqueous solution to ionic liquids such as [BMIm][BF4]. Also, Lee et al. [27] reported an increase in peroxidase Km in the presence of [BMIm] [BF4]. The catalytic characterization of horse liver ADH, using ethanol as substrate, in medium containing [BMIm][BF4] and [BMIm][Cl] was studied by Shi et al. [11]. The results obtained by Shi and colleagues showed that the enzyme was highly affected by [BMIm][BF4] and displayed good activity in the system involving [BMIm][Cl]. The Km value increased and kcat reduced in the order of [BMIm][Cl] N [BMIm][BF4] N [MIm][Cl] ~ [MIm][BF4] (Fig. 3). Ionic liquids normally dissociate into individual ions when dissolved in water, a careful examination of their ionic nature and its impact on enzyme catalysis is very important. Proteins are usually stabilized by kosmotropic anions and destabilized by chaotropic ones; while the opposite is true when regarding cations [25,28,29]. Our results revealed that YADH highest catalytic efficiency was achieved in medium containing [BMIm][Cl] (Fig. 3) because BF4− is a much stronger chaotrope than Cl−. It is likely possible that competition with substrate (NAD+) takes over the kosmotropicity differences between (MIm) and (BMIm) so higher activity is observed in [BMIm] based ionic liquids and in ILs containing similar cation enzymatic performance is affected by the chaotropicity of the anion [28,29]. Moreover, a systematic quantitative characterization of IL cations is worth doing, because only few of them have been known for their physicochemical properties [25]. The complexity of the IL-induced variation in the kinetic parameters indicates the fact that ILs affect enzyme performance depending on a combination of complicated mechanisms rather than on a single simple one such as kosmotropicity/ chaotropicity [25]. 3.3. Fluorescence study An ion may affect the enzyme performance by playing the role as a substrate, a cofactor, or an inhibitor. As the cationic group of [BMIm] [Cl], [BMIm][BF4], [MIm][Cl] and [MIm][BF4] are similar in structure to the adenine moiety of NAD +, it is more likely that these ionic liquids may compete with NAD + for YADH binding site. For this reason we supposed that the decrease in Vmax upon the addition of ionic liquids may be due to the inhibitory effect of ILs. Therefore, in order to follow structural changes, intrinsic fluorescence measurements were made.
Fig. 2. Effects of [MIm][Cl] (Δ), [MIm][BF4] (▲), [BMIm][Cl] (□) and [BMIm][BF4] (■) on the kinetic parameters of YADH.
Denaturation process in proteins that contain fluorophore residues can be followed by both changes in the maximal intensity of fluorescence (Imax) and the red shift of the maximal emission wavelength (λmax). Our results revealed that the conformational
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Fig. 4. Thermostability of YADH in 50 mM phosphate buffer (pH 7.8) in the presence of different ionic liquids. Each sample was heated for 3 h at 40 °C followed by activity measurements. Samples with no ionic liquid were used as controls. Fig. 3. Intrinsic fluorescence spectra of YADH in 50 mM phosphate buffer (pH 7.8) in the presence and absence of different ionic liquids (150 mM). For more details refer to Materials and methods.
changes did not differ significantly in the presence of 150 mM imidazolium based ionic liquids (Fig. 3). With respect to our findings, it is suggested that the enzyme may have been inhibited at mentioned concentration without protein denaturation. Zhang et al. [30] reported that imidazole has an inhibitory effect on the activity of YADH in aqueous solution which supports the hypothesis mentioned above. YADH treated with [BMIm] ILs retained higher activity than [MIm] ILs in our experiments. The reason for such result is probably for the length of the side chain, assuming that competition with adenine moiety becomes less favorable as the side chain becomes longer. Shi et al. [11] also reported higher activity for liver alcohol dehydrogenase in [BMIm][Cl] than [EMIm][Cl] (1-ethyl-3-methyl imidazolium chloride). 3.4. Stability in ionic liquids In order to study the effect of ILs on the stability of YADH, 1 ml of enzyme solution (1 mg/ml) was incubated in the absence and presence of [BMIm][Cl], [BMIm][BF4], [MIm][Cl] and [MIm][BF4] at 40 °C for 3 h. According to the results (Fig. 4), the enzyme showed a high tendency for retaining its activity in [MIm][Cl]. The enzyme presented its stability in the order of [MIm][Cl] N [MIm][BF4] N control (no ionic liquids) N [BMIm][BF4] N [BMIm][Cl]. Previously, it was reported that thermostability of horse liver ADH and horseradish peroxidase was improved in the presence of [BMIm][Cl] and [BMIm] [BF4] [11,13]. Because the stability of YADH was 2 fold higher in [MIm] [Cl], irreversible thermoinactivation in the presence of 150 mM [MIm] [Cl] at 40 °C was carried out. Periodically, 100 μl of the enzyme solution was cooled on ice and then 0.2 μl of this solution was used for enzyme activity in phosphate buffer at 25 °C, as mentioned in Materials and methods. Thermostability data seemed to fit the first order enzyme deactivation model. Results show that in aqueous buffer with no [MIm][Cl] the residual activity of the enzyme reduced, indicating a decrease in the stability of the enzyme (Fig. 5). Many research groups have reported that even if the chosen IL had a negative influence on the enzyme activity, some of them are able to stabilize it [31]. Relating the protein stability to protein activity and fluorescence spectroscopic studies we assume that the cationic group of [MIm][Cl] with no side chain is more similar to adenine moiety of NAD + which might place it in the active site and makes YADH more
stable while heating. Utilization of substrate and other ligands occupies a special place among the methods so far devised for enzyme stabilization, as these compounds are inherent in enzymatic reactions [32]. Lactate dehydrogenase and glucose 6-phosphate dehydrogenase were stabilized against thermal denaturation by their substrates NAD + and NADP +, respectively [33,34]. The active center is protected by ligand binding, which results in decreased formation of thermo-unfolded enzyme. 4. Conclusions In summary, our study has demonstrated that both the activity and stability of YADH in aqueous solution can be affected by addition of different ILs. In comparison with aqueous buffer, the enzyme revealed higher stability in the presence of [MIm][Cl] at 40 °C. Stability of an enzyme, in particular thermostability, is an important property for its application in the industry. Alcohol dehydrogenases are of prime interest in the industrial field and they are in general temperature labile. Therefore, [MIm][Cl] would be a suitable medium for storing YADH. Further investigation on the reverse reaction and product enantioselectivity of the enzyme is currently being carried out.
Fig. 5. Thermal inactivation at 40 °C in the absence (▲) and presence (■) of 150 mM [MIm][Cl]. (V/V0) is the relative residual activity (%). A value of 100% refers to the activity obtained by the enzyme prior to incubation.
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Acknowledgments We would like to thank the research council of Tarbiat Modares University for the financial support of this investigation. References [1] H. Zhao, J. Mol. Catal. B: Enzym. 37 (2005) 16–25. [2] F. van Rantwijk, R. Madeira Lau, R.A. Sheldon, Trends Biotechnol. 21 (2003) 131–138. [3] S. Park, R.J. Kazlauskas, Curr. Opin. Biotechnol. 14 (2003) 432–437. [4] S. Keskin, D. Kayrak-Talay, U. Akman, O. Hortacsu, J. Supercrit. Fluids 43 (2007) 150–180. [5] F. van Rantwijk, R.A. Sheldon, Chem. Rev. 107 (2007) 2757–2785. [6] Y.H. Moon, S.M. Lee, S.H. Ha, Y.M. Koo, Korean J. Chem. Eng. 23 (2006) 247–263. [7] U. Kragl, M. Eckstein, N. Kafizik, Curr. Opin. Biotechnol. 13 (2002) 565–571. [8] P.C.A.G. Pinto, L.M.F.S. Saraiva, J.L.F.C. Lima, Anal. Sci. 24 (2008) 1231–1238. [9] S. Lutz-Wahl, E.M. Trost, B. Wagner, A. Manns, L. Fischer, J. Biotechnol. 124 (2006) 163–171. [10] A.J. Walker, N.C. Bruce, Chem. Commun. 22 (2004) 2570–2571. [11] X.A. Shi, M.H. Zong, W.Y. Lou, Chin. J. Chem. 24 (2006) 1643–1647. [12] M. Eckstein, M.V. Filho, A. Liese, U. Kragl, Chem. Commun. (2004) 1084–1085. [13] E. Feher, B. Major, K. Belafi-Bako, L. Gubicza, Biochem. Soc. Trans. 35 (2007) 1624–1627. [14] R.M. Lau, M.J. Sorgedrager, G. Carrea, F. van Rantwijk, R. Madeira Lau, R.A. Sheldon, Green Chem. 6 (2004) 483–487. [15] F. van Rantwijk, F. Secundob, R.A. Sheldon, Green Chem. 8 (2006) 282–286.
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Contents lists available at ScienceDirect
Journal of Molecular Liquids j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m o l l i q
Mesophilic alcohol dehydrogenase behavior in imidazolium based ionic liquids Bahareh Dabirmanesh a, Khosro Khajeh a,⁎, Jafar Akbari b, Hanieh Falahati c, Somayeh Daneshjoo d, Akbar Heydari b,⁎⁎ a
Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran Department of Chemistry, Faculty of Sciences, Tarbiat Modares University, Tehran, Iran Department of Biotechnology, Faculty of Sciences, Tehran University, Tehran, Iran d Department of Microbiology, Faculty of Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran b c
a r t i c l e
i n f o
Article history: Received 19 February 2011 Received in revised form 10 May 2011 Accepted 12 May 2011 Available online 25 May 2011 Keywords: Activity Alcohol dehydrogenase Fluorescence Ionic liquids Stability
a b s t r a c t The use of ionic liquids to replace organic solvents in biocatalytic processes has recently gained much attention. Despite the wide applications of oxidoreductases, there are few reports of their catalyzed reaction in ionic liquid. We have investigated the influence of four water miscible ionic liquids on the activity, stability and structure of the mesophilic alcohol dehydrogenase from yeast. Upon changes in ionic liquids concentration, both activity and stability of the enzyme were affected. As the concentration of ionic liquids increased, Km increased while kcat decreased. Associated conformational changes caused by ILs (150 mM) were monitored using fluorescence technique. Finally, the effects of ILs cations and anions on the enzyme activity and stability in aqueous IL mixtures were discussed. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Ionic liquids (ILs) are nonvolatile, nonflammable and highly stable organic salts. They hold great potential as environmentally friendly solvents. Their polarity, hydrophobicity and solvent miscibility behaviors can be tuned through the appropriate modification of the cation and anion. Ionic liquids have attracted considerable attention and have emerged as interesting novel reaction media for enzymatic transformation since enzymes show remarkable difference in catalytic features in systems involving different ILs. A number of enzymes have been reported to retain catalytic activity in an ionic liquid medium [1–7]. Lipases have been extensively studied and were more enantioselective in such reaction media than organic solvents. The lipase Novozym 435 was found to be greatly stabilized in the ionic liquid [1]. Some oxidoreductases, such as laccase and peroxidase, chloroperoxidase and D-amino acid oxidase were also active in ionic liquids [8,9]. Walker and Bruce [10] studied the activity
of cofactor dependent enzymes in ILs. Previously, horse liver alcohol dehydrogenase (HLADH) displayed good activity in the system involving [BMIm][Cl] [11]. Eckstein et al. [12] reported the reduction of 2-octanone to (R)-2-octanol catalyzed by alcohol dehydrogenase from Lactobacillus brevis in a biphasic system of [BMIm][NTf2] and phosphate buffer. Several groups reported not just increased activity but increased enzyme stability in ionic liquids as compared with organic solvents [3,13]. Spectroscopic methods have recently been described to associate structural changes in Candida antarctica lipase B and α-chymotrypsin with stability in ILs [14–17]. Despite the importance of alcohol dehydrogenases (ADHs), there are only a few examples concerning the use of them with ionic liquids. So it was of great interest to investigate the influence of various ILs on the catalytic performance and conformational changes of mesophilic alcohol dehydrogenase from yeast (YADH). We herein report the catalytic characteristics, stability and the fluorescence spectra of YADH in the systems involving 1-butyl-3-methylimidazolium ([BMIm][Cl] and [BMIm][BF4]) and 1-methylimidazolium ([MIm][Cl] and [MIm][BF4]).
2. Materials and methods ⁎ Corresponding author at: Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, P.O. Box 14115-175, Tehran, Iran. Tel./fax: +98 2182884717. ⁎⁎ Corresponding author at: Department of Chemistry, Faculty of Sciences, Tarbiat Modares University, P.O. Box 14115-175, Tehran, Iran. Tel./fax: + 98 2188009730. E-mail addresses: [email protected] (K. Khajeh), [email protected] (A. Heydari). 0167-7322/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.molliq.2011.05.007
2.1. Materials YADH, NaHPO4 and NAD+ were purchased from Sigma (St. Louis, MO, USA). N-methylimidazole, HBF4 (50% aqueous), HCl and 1-chlorobutane were obtained from Aldrich-Fluka (USA). Ionic liquids ([BMIm][Cl], [BMIm][BF4], [MIm][Cl] and [MIm][BF4] ) used in this research were
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prepared in our laboratory with 97% purity. The purity of YADH was confirmed by SDS–PAGE. 2.2. Preparation of imidazolium-based ionic liquids [MIm][Cl] and [MIm][BF4] were obtained by neutralizing N-methylimidazole with aqueous HCl and HBF4. The water was removed by evaporation under reduced pressure. The residue was washed with hexane and then remained under vacuum to produce pure ionic liquid [18,19]. In order to synthesize [BMIm][Cl], N-methylimidazol (5 mmol) and butyl chloride (5 mmol) were mixed in a round bottom flask under nitrogen and then stirred at 85 °C for 72 h. The resulting pale yellow liquid was then washed with ethyl acetate and remained under vacuum overnight to afford white solid, 1-butyl-3-methylimidazolium chloride [20,21]. The resulting white solid, [BMIm][Cl], was dissolved in water and stoichiometric amount of HBF4 was added drop wise overnight. The reaction was stirred at room temperature for an additional 12 h and extracted with CH2Cl2. The organic layer was washed with water until free from acid. Obtained ionic liquid was dried in a vacuum oven overnight at 80 °C to afford a pale yellow liquid and used without further purification [20,22]. All manipulations were performed under nitrogen atmosphere using standard techniques. The structure of ionic liquids 1 was examined by HNMR spectroscopy [22]. Physical and spectroscopic data of ionic liquids are:
2.5. Stability in ionic liquids Enzyme stability was analyzed by incubating enzymes in the absence and presence of 150 mM of each IL at 40 °C for 2 h. Samples were then cooled on ice (for half an hour) and assayed for residual activity as described previously. Sample with no ionic liquid was used as a control. Thermal inactivation of YADH was also determined in 50 mM potassium phosphate (pH 7.8) containing 150 mM [MIm][Cl] at 40 °C. At various time intervals (every 10 min for an hour) samples were removed, cooled on ice and then assayed for residual activity at 25 °C. Plots of the log of residual activity versus time were linear, indicating a first-order decay process under these conditions. Results presented in this paper are the mean from at least 3 repeated experiments in a typical run to confirm reproducibility and the standard deviations were ±5%. 3. Results and discussion The use of room temperature ionic liquids (RTILs), especially those based on 1-alkyl-3-methylimidazolium cations, as media for enzymatic catalysis has aroused the interest of many researchers because of their solvating ability, negligible vapor pressure, easy recyclability and reusability [1–3]. Due to the wide field of application of oxidoreductases, we have studied the influence of room temperature imidazolium based ionic liquids on the activity and stability of YADH.
1
- 1-Methylimidazolium chloride [MIm][Cl]: Mp: 72 °C, H NMR (CDCl3, 500 MHz): δ 10.27 (s, 1H), 8.33 (s, 1H), 7.17 (s, 1H), 7.11 (s, 1H), 3.69 (s, 3H). Yield: 94%. 1 - 1-Methylimidazolium tetrafluoroborate [MIm][BF4]: Mp: 35.8 °C, H NMR (DMSO(d6), 500 MHz): δ 3.85 (s, 3H), 7.64 (s, 1H), 7.67 (s, 1H), 9.01 (s, 1H), 11.09 (s, 1H). Yield: 92%. 1 - 1-Butyl-3-methylimidazolium chloride [BMIm][Cl]: Mp: 65 °C, H 3 NMR (CDCl3, 500 MHz): δ 0.80 (t, 3H, JHH = 7.3), 1.23 (m, 2H), 3 1.75 (m, 2H), 3.98 (s, 3H), 4.19 (t, 2H, JHH = 7.4), 7.46 (s, 1H), 7.63 (s, 1H), 9.55 (s, 1H). Yield: 89%. - 1-Butyl-3-methylimidazolium tetrafluoroborate [BMIm][BF4]: Mp: 1 3 −74 °C, H NMR (CDCl3, 500 MHz): δ 0.95 (t, 3H, JHH=7.3), 1.37 (m, 3 2H), 1.93 (m, 2H), 4.07 (s, 3H), 4.40 (t, 2H, JHH = 7.1), 7.79 (s, 1H), 7.85 (s, 1H), 9.55 (s, 1H). Yield: 91%.
3.1. Effect of ILs on the activity of alcohol dehydrogenases Effects of ionic liquids on the enzyme activity were studied by measuring the initial rate of reaction catalyzed by 0.2 μg/ml of YADH in 50 mM phosphate buffer (pH 7.8) containing 450 mM ethanol and 0.4 mM NAD + in the absence (control) and presence of different amounts of ILs. Variation of YADH activity upon the changes in ILs concentration is shown in Fig. 1, a clear decrease of the initial velocity for the investigated enzyme was observed. The order of YADH activity in solution containing ILs was [BMIm][Cl] N [BMIm][BF4] N [MIm][BF4] ~ [MIm][Cl]. Results show the classical and fast activity decay behavior with ILs concentrations. For these kinds of ILs, this phenomenon has been widely observed (see Ref. [5], for the case of [BMIm][Cl]). In contrast to previous studies that indicated lower activity in more
2.3. Enzyme assay YADH assay was carried out in the 1 ml reaction mixture containing 916 μl of 50 mM sodium phosphate pH 7.8, 17 μl of 24 mM NAD+ and 67 μl of various substrate (ethanol) concentrations ranging from 0.07 M to 6.75 M at 25 °C for 1 min[23,24]. The reduction of NAD + was measured photometrically at 340 nm. One unit of enzymatic activity was defined as the quantity of enzyme necessary to produce 1 μmol NADH per minute. Kinetic parameters (Km and Vmax) were calculated using lineweaver–Burk plots. For the reactions in the presence of different concentrations of ionic liquid the amount of buffer was reduced and replaced by the ionic liquid, controlling pH was also essential to obtain consistent results with ionic liquids. 2.4. Fluorescence spectroscopy Fluorescence spectra of YADH were monitored using a Perkin Elmer LS 55 fluorescence spectrometer. After incubation for 5 min, samples were excited at 295 nm and the emission was recorded between 300 and 400 nm. The excitation and emission slit were both set to 5 nm. In all cases, it was necessary to subtract a blank medium without enzyme to discount the influence of the imidazolium ring fluorescence on the enzyme fluorescence spectrum.
Fig. 1. Variation of enzyme activities upon addition of different concentrations of [BMIm][BF4] (▲), [BMIm][Cl] (■), [MIm][Cl] (□) and [MIm][BF4] (Δ) to 50 mM phosphate buffer (pH 7.8) containing 0.4 mM NAD+, 450 mM ethanol and 0.2 μg/ml of YADH. The relative activity (%) refers to the percentage of the initial reaction rate obtained by the enzyme in the presence of the above ionic liquids compared to the one obtained in the absence of them.
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kosmotropic cations, YADH demonstrated higher activity in the presence of [BMIm][Cl] which is a stronger kosmotrope than [MIm] [Cl] [25]. The observed decay in enzyme activity could be due to either enzyme deactivation or enzyme inhibition. Reversibility of inhibition was determined by measuring the recovery of enzymatic activity after a rapid and large dilution of the enzyme–inhibitor complex. YADH (100 fold over the concentration used above) was incubated with different ionic liquids for 40 min. Then the mixture was diluted about 100 fold with 50 mM phosphate buffer (pH 7.8) containing the enzyme substrates (NAD + and ethanol to the final concentration of 0.4 mM and 450 mM) to initiate reaction. The results showed that the activity was recovered and the enzyme activity remained constant during the incubation time. Therefore, the reduction in the activity might be due to the inhibition of YADH. 3.2. Effect of ILs on kinetic parameters In order to get a deeper insight into how the ionic liquids affect the enzymes kinetics, values of kcat, Km and kcat/Km were determined as the content of each IL increased in the mixture. The results revealed that the presence of ionic liquids affected both the Km and kcat (Fig. 2). The Km and kcat was estimated as 26 mM and 460 s−1 for the uninhibited reaction using ethanol as a substrate. Upon the addition of ionic liquids, kcat was decreased and Km increased. These results infer that the binding affinity of substrates to mesophilic alcohol dehydrogenase significantly weakens owing to the presence of the ionic liquid in the reaction solution. Similar to our result, a simultaneous increase in Km and decrease in Vmax were also observed for laccase [26] when the reaction medium was switched from aqueous solution to ionic liquids such as [BMIm][BF4]. Also, Lee et al. [27] reported an increase in peroxidase Km in the presence of [BMIm] [BF4]. The catalytic characterization of horse liver ADH, using ethanol as substrate, in medium containing [BMIm][BF4] and [BMIm][Cl] was studied by Shi et al. [11]. The results obtained by Shi and colleagues showed that the enzyme was highly affected by [BMIm][BF4] and displayed good activity in the system involving [BMIm][Cl]. The Km value increased and kcat reduced in the order of [BMIm][Cl] N [BMIm][BF4] N [MIm][Cl] ~ [MIm][BF4] (Fig. 3). Ionic liquids normally dissociate into individual ions when dissolved in water, a careful examination of their ionic nature and its impact on enzyme catalysis is very important. Proteins are usually stabilized by kosmotropic anions and destabilized by chaotropic ones; while the opposite is true when regarding cations [25,28,29]. Our results revealed that YADH highest catalytic efficiency was achieved in medium containing [BMIm][Cl] (Fig. 3) because BF4− is a much stronger chaotrope than Cl−. It is likely possible that competition with substrate (NAD+) takes over the kosmotropicity differences between (MIm) and (BMIm) so higher activity is observed in [BMIm] based ionic liquids and in ILs containing similar cation enzymatic performance is affected by the chaotropicity of the anion [28,29]. Moreover, a systematic quantitative characterization of IL cations is worth doing, because only few of them have been known for their physicochemical properties [25]. The complexity of the IL-induced variation in the kinetic parameters indicates the fact that ILs affect enzyme performance depending on a combination of complicated mechanisms rather than on a single simple one such as kosmotropicity/ chaotropicity [25]. 3.3. Fluorescence study An ion may affect the enzyme performance by playing the role as a substrate, a cofactor, or an inhibitor. As the cationic group of [BMIm] [Cl], [BMIm][BF4], [MIm][Cl] and [MIm][BF4] are similar in structure to the adenine moiety of NAD +, it is more likely that these ionic liquids may compete with NAD + for YADH binding site. For this reason we supposed that the decrease in Vmax upon the addition of ionic liquids may be due to the inhibitory effect of ILs. Therefore, in order to follow structural changes, intrinsic fluorescence measurements were made.
Fig. 2. Effects of [MIm][Cl] (Δ), [MIm][BF4] (▲), [BMIm][Cl] (□) and [BMIm][BF4] (■) on the kinetic parameters of YADH.
Denaturation process in proteins that contain fluorophore residues can be followed by both changes in the maximal intensity of fluorescence (Imax) and the red shift of the maximal emission wavelength (λmax). Our results revealed that the conformational
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Fig. 4. Thermostability of YADH in 50 mM phosphate buffer (pH 7.8) in the presence of different ionic liquids. Each sample was heated for 3 h at 40 °C followed by activity measurements. Samples with no ionic liquid were used as controls. Fig. 3. Intrinsic fluorescence spectra of YADH in 50 mM phosphate buffer (pH 7.8) in the presence and absence of different ionic liquids (150 mM). For more details refer to Materials and methods.
changes did not differ significantly in the presence of 150 mM imidazolium based ionic liquids (Fig. 3). With respect to our findings, it is suggested that the enzyme may have been inhibited at mentioned concentration without protein denaturation. Zhang et al. [30] reported that imidazole has an inhibitory effect on the activity of YADH in aqueous solution which supports the hypothesis mentioned above. YADH treated with [BMIm] ILs retained higher activity than [MIm] ILs in our experiments. The reason for such result is probably for the length of the side chain, assuming that competition with adenine moiety becomes less favorable as the side chain becomes longer. Shi et al. [11] also reported higher activity for liver alcohol dehydrogenase in [BMIm][Cl] than [EMIm][Cl] (1-ethyl-3-methyl imidazolium chloride). 3.4. Stability in ionic liquids In order to study the effect of ILs on the stability of YADH, 1 ml of enzyme solution (1 mg/ml) was incubated in the absence and presence of [BMIm][Cl], [BMIm][BF4], [MIm][Cl] and [MIm][BF4] at 40 °C for 3 h. According to the results (Fig. 4), the enzyme showed a high tendency for retaining its activity in [MIm][Cl]. The enzyme presented its stability in the order of [MIm][Cl] N [MIm][BF4] N control (no ionic liquids) N [BMIm][BF4] N [BMIm][Cl]. Previously, it was reported that thermostability of horse liver ADH and horseradish peroxidase was improved in the presence of [BMIm][Cl] and [BMIm] [BF4] [11,13]. Because the stability of YADH was 2 fold higher in [MIm] [Cl], irreversible thermoinactivation in the presence of 150 mM [MIm] [Cl] at 40 °C was carried out. Periodically, 100 μl of the enzyme solution was cooled on ice and then 0.2 μl of this solution was used for enzyme activity in phosphate buffer at 25 °C, as mentioned in Materials and methods. Thermostability data seemed to fit the first order enzyme deactivation model. Results show that in aqueous buffer with no [MIm][Cl] the residual activity of the enzyme reduced, indicating a decrease in the stability of the enzyme (Fig. 5). Many research groups have reported that even if the chosen IL had a negative influence on the enzyme activity, some of them are able to stabilize it [31]. Relating the protein stability to protein activity and fluorescence spectroscopic studies we assume that the cationic group of [MIm][Cl] with no side chain is more similar to adenine moiety of NAD + which might place it in the active site and makes YADH more
stable while heating. Utilization of substrate and other ligands occupies a special place among the methods so far devised for enzyme stabilization, as these compounds are inherent in enzymatic reactions [32]. Lactate dehydrogenase and glucose 6-phosphate dehydrogenase were stabilized against thermal denaturation by their substrates NAD + and NADP +, respectively [33,34]. The active center is protected by ligand binding, which results in decreased formation of thermo-unfolded enzyme. 4. Conclusions In summary, our study has demonstrated that both the activity and stability of YADH in aqueous solution can be affected by addition of different ILs. In comparison with aqueous buffer, the enzyme revealed higher stability in the presence of [MIm][Cl] at 40 °C. Stability of an enzyme, in particular thermostability, is an important property for its application in the industry. Alcohol dehydrogenases are of prime interest in the industrial field and they are in general temperature labile. Therefore, [MIm][Cl] would be a suitable medium for storing YADH. Further investigation on the reverse reaction and product enantioselectivity of the enzyme is currently being carried out.
Fig. 5. Thermal inactivation at 40 °C in the absence (▲) and presence (■) of 150 mM [MIm][Cl]. (V/V0) is the relative residual activity (%). A value of 100% refers to the activity obtained by the enzyme prior to incubation.
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