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Rute Madeira Lau,a Menno J. Sorgedrager,a Giacomo Carrea,b Fred van Rantwijk,a Francesco Secundob and Roger A. Sheldon*a a Laboratory of Biocatalysis and Organic Chemistry, Delft University of Technology, Julianalaan 136, 2628 BL Delft, The Netherlands. E-mail: firstname.lastname@example.org b Istituto di Chimica del Riconoscimento Moleculare, CNR, Via Mario Bianco 9, Milano, Italy. E-mail: email@example.com
Received 15th April 2004, Accepted 30th July 2004 First published as an Advance Article on the web 25th August 2004
The effects of ionic liquid media on the activity of Candida antarctica lipase B in a simple transesterification reaction were studied. In ionic liquids containing alkylsulfate, nitrate and lactate anions, which dissolved CaLB, the reaction was at least ten times slower than in [BMIm][BF4]. Only [Et3MeN][MeSO4] was an exception, as dissolved CaLB maintained its activity in this solvent. By means of FT-IR spectroscopy, denaturation of CaLB was observed upon dissolution in ionic liquids in which the activity was low, whereas the conformation of enzyme dissolved in [Et3MeN][MeSO4] closely resembled the native one.
The use of ionic liquids as reaction media for enzymatic transformations is a rapidly expanding field.1 Most of these
applications involve dispersions of enzymes in low-coordinating strength ionic liquids, such as the by now archetypical [BMIm][PF6] and [BMIm][BF4]. Any hopes that ionic liquids would provide an easy way to combine enzyme solubility and
Rute Madeira Lau (1974) studied chemical engineering (specialisation in biotechnology) at the Instituto Superior Tecnico (Lisbon, Portugal). She did her diploma work in the Biocatalysis and Organic Chemistry group at the Delft University of Technology (1997). In 2003 she received her PhD under the supervision of Prof. Roger Sheldon and Dr. Fred van Rantwijk. The subject of her thesis was the application of lipases in nonaqueous media, with special attention to the performance of these enzymes in ionic liquids. She is now with DSM. Fred van Rantwijk (1943) studied organic chemistry at the Delft University of Technology where he remained as a staff member. He received his PhD in 1980, for work under the guidance of Professor H. van Bekkum. Since the late 1980s he has been working on the application of enzymes in organic synthesis. His particular research interests are the use of enzymes in nonnatural reactions, enzyme immobilization, and transformations using multi-enzyme systems. Francesco Secundo (1966) received his Laurea in Biological Science from the University of Milan in 1992. This was followed by post-laurea studies with Dr. Robert Phillips (1994)
at the University of Georgia in the US and with Dr. Giacomo Carrea at the Institute of Chemistry of Molecular Recognition (1992–1997) in Milan. Since 1997 he has been research scientist in the same institution. His current research interests are in the area of biocatalysis, devoting particular attention to biocatalyst formulation for catalysis in non-aqueous media. This objective is pursued through studies of enzyme activity, stability and conformation. Roger Sheldon (1942) received a PhD in organic chemistry from the University of Leicester (UK) in 1967. This was followed by post-doctoral studies with Prof. Jay Kochi in the US. From 1969 to 1980 he was with Shell Research in Amsterdam and from 1980 to 1990 he was R&D Director of DSM Andeno. In 1991 he moved to his present position as Professor of Organic Chemistry and Catalysis at the Delft University of Technology (The Netherlands). His primary research interests are in the application of catalytic methodologies—homogeneous, heterogeneous and enzymatic—in organic synthesis, particularly in relation to fine chemicals production. He developed the concepts of E factors and atom utilization for assessing the environmental impact of chemical processes.
Rute Madeira Lau
Fred van Rantwijk
This journal is © The Royal Society of Chemistry 2004
Green Chem., 2004, 6, 483–487
as a modest transesterification activity was maintained even though dissolution of the biocatalyst was observed. FT-IR has been employed to monitor protein conformation in water.5 mL) at 40 °C for 24 h. the transesterification was carried out in the supernatant. perhaps.6 It would seem that anions that interact with solid enzymes sufficiently strongly to break the intermolecular bonds and effect dissolution also interfere with the intramolecular bonds to such a degree that unfolding takes place. only mildly H-bond accepting. 483–487 . 1-butanol (120 mM). but not with [BMIm][BF4] or [BMIm][PF6]. such as nitrate or lactate. as noted above. For a closer examination. but mainly on the hydrogen bond-accepting properties of the anion.5 The cellulase from Trichoderma reesei likewise was inactive when dissolved in [BMIm][Cl]. 1-butanol (120 mM). as Table 2 Transesterification of ethyl butanoate with CaLB in [Et3MeN][MeSO4]a SP525/mg mL21. a reaction was also observed with 1. which is presumably caused by unfavourable interactions of alkylsulfate anions with CaLB. Conversion in % Solvent [Et3MeN][MeSO4] tert-Butyl alcohol 1. MeSO42 anion is a borderline case and that. although the reaction was always faster in tert-butyl alcohol. [BMIm][BF4] 78 no 0 [BMIm][PF6] 71 no 0 26 see text n. 1) as a suitable test reaction to measure the a Reaction conditions: ethyl butanoate (60 mM). because of low signal intensity and excessive background noise. On the basis of the conversion ( Table 2). the reaction rate was at least 10 times lower (see Table 1).2 14c 18 3 19 46 6 46 63 20 54 68 Supernatantb 25 0 Results and discussion Activity of CaLB in ionic liquids We selected the alcoholysis (transesterification) of ethyl butanoate with 1-butanol (see Fig.7 When the reaction was performed in ionic liquids that contained a nitrate. 24 h) Recovery from the supernatantc (%) Mediuma Dissolution expected. circulans. A similar reconstitution has been observed with alkaline phosphatase8 and lysozyme9 in [EtNH3][NO3]. ethylsulfate or lactate anion. b SP525 (20 mg mL21) was incubated in ionic liquid for 24 h at 40 °C and centrifuged. a reaction was performed with a. indeed dissolved Candida antarctica lipase B (CaLB) but also effected its (partially reversible) deactivation. Infrared spectroscopy is a well-established technique for conformational analysis of proteins. Hence. [BMIm][lactate]. in which CaLB is soluble (see below). 1 Transesterification of ethyl butanoate with 1-butanol. original activity = 100%.4 Kaar et al. Hence. CaLB in [Et2MeN][MeSO4] The behaviour of CaLB in [Et3MeN][MeSO4] did not correspond with the simple pattern set out above. We continued our investigations of the issue of enzyme solubility vs. we performed transesterification experiments with varying amounts of dry solid CaLB (see Table 2). c All enzyme dissolved. Structural changes of CaLB upon dissolution in ionic liquids Conformational changes are an obvious explanation for the activity loss of CaLB upon dissolution in ionic liquids that we wished to confirm experimentally. 3 mg mL21 of CaLB. [EMIm][EtSO4] and [EtNH3][NO3]. presumably due to interactions with the—strongly coordinating— chloride ion. exhibit some tolerance for methylsulfate. after freezedrying (in the absence and in the presence of additives)15–19 and that ionic liquids that interact with the protein sufficiently strongly to effect its dissolution also induce structural changes that lead to loss of activity. While fluorescence spectroscopy and circular dichroism could not be used.2 mg mL21 of CaLB in [Et3MeN][MeSO4].3 We recently have published preliminary results indicating that ionic liquids containing a strongly coordinating anion. c Hydrolytic activity recovered from the supernatant after 24 h incubation at 40 °C. whereas the dispersed solid enzyme maintained its activity. but up to 73% of the original hydrolytic activity was recovered upon rehydration ( Table 1). activity in ionic liquids and have studied the structural changes of CaLB upon dissolution in ionic liquids using FT-IR spectroscopy. antarctica lipase B in ionic liquids: activity in transesterification. [Et3MeN][MeSO4] [EMIm][EtSO4] 7 yes 53 [BMIm][lactate] 5 yes 42 5 yes 33 [EtNH3][NO3] 3 yes 73 [BMIm][NO3] tert-Butyl alcohol 74 no 0 a For ionic liquid nomenclature see Table 3. rugosa lipase was not active in ionic liquids that contain a nitrate or acetate anion. Novozym 435 (15 mg) in solvent (0. have accordingly found that C. The inescapable conclusion from these results is that dissolution of CaLB in an ionic liquid is not incompatible with catalytic acitivity. in contrast. infrared spectroscopy proved suitable to study the conformation of CaLB in ionic liquids. To our surprise. solution of CaLB in [Et3MeN][MeSO4]. the activity loss is partially reversible. To confirm this latter observation. SP525 in solvent (0.activity in non-aqueous media were extinguished when it became clear that the small fraction of thermolysin that dissolved in [BMIm][PF6] containing 5% water was not active. When the reaction was performed in [BMIm][BF4] or [BMIm][PF6]. which was seen to dissolve. in seeming contradiction with the lack of activity of CaLB in [EMIm][EtSO4] (Table 1). whereas activity was maintained in [BMIm][PF6]. diluted with water and subjected to a triacetin test. The transesterification activity of CaLB in these ionic liquids was low. indicating that the enzyme refolds into the active conformation upon rehydration. solubility and activity recovery upon rehydration Conversionb (%. it would seem Table 1 C. b Reaction conditions: ethyl butanoate (60 mM).. The supernatants were separated. 484 Green Chem.d. Formate dehydrogenase and the b-glycosidase from B. a fluorometric study showed that the enzyme had denaturated. presumably saturated. Fig. 6. the H-bond acidity of the Et3MeN+ cation exerts a stabilising effect. Enzyme dissolution was observed with [BMIm][NO3]. the reaction rate was comparable with that in tert-butyl alcohol.5 mL) at 40 °C for 24 h.2 It has recently been demonstrated that the ability to solvate and dissolve molecules depends on several ionic liquid properties. an immobilised preparation of CaLB. it would seem that [Et3MeN][MeSO4] dissolves approx. 2004. The results will be presented and discussed in the present paper.11–14 Thanks to the possibility of analysing the sample in different physical states. Solubility of free CaLB and recovery of activity We investigated the related issues of enzyme solubility and denaturation more closely by incubating dry solid CaLB (SP525) in various ionic liquids for 24 h at 40 °C.10 It would seem that the. effects of the medium on the activity of Novozym 435. The conversion increased with the amount of enzyme.
[BMIm][lactate] and [BMIm][NO3]. especially in the region 1640–1670 cm21. 1624 cm21 and 1690 cm21 may indicate the presence of aggregates22 that presumably result from incomplete dissolution. a-helix. [BMIm][lactate] and [BMIm][NO3] is caused by changes of the secondary structure of the enzyme which appear much more profound than those caused by the freeze-drying process. in spite of such spectral variations it can be observed that the components at around 1640. 1667.21 In Fig. in this case.. In fact. In contrast. This result is in agreement with that of Vecchio et al. b-turns (both the bands at 1660 and 1667 cm21 could be assigned to the non-hydrogen-bonded carbonyls of b-turns)23–26 and to b-sheets (this latter component was assigned in agreement with Mizutani et al. 1649.14. the similarity of the second-derivative infrared spectrum of CaLB in [Et3NMe][MeSO4] with that in water. The presence of these common components that could be assigned to. the native conformation. and at 1681 cm21 are present with CaLB in water as well as with the dried powder. in contrast. These data suggest that the enzyme preserves its secondary structure in this ionic liquid. The broader components below 1650 cm21 in the spectra of CaLB dissolved in these ionic liquids compared with the enzyme in water. we analyzed the second-derivative amide I spectra. since CaLB was added to the ionic liquid as a dried solid without any preceding dissolution in water.even after solubilising or suspending them in non-aqueous media.19 We found that the second derivative spectrum of the dried solid sample compared to the sample in water has a new component at around 1673 cm21 (which could be assigned to non hydrogen bonded b-turns) and broader components at around 1619 cm21 and 1690 cm21. this band consists of several overlapping components that can be assigned to different secondary structure elements. even though the broad peaks at approx. in the case of [EtNH3][NO3] hydrogen bonded to the NH3 groups of the solvent). Summarising.13. It can be observed that the second-derivative spectrum of dried solid CaLB shows some differences compared to that of the protein in water. 483–487 485 . as being representative of native CaLB in a fully hydrated and dissolved state. were much less similar to that in water. which might be assigned to intermolecular hydrogen-bonded antiparallel b-sheets. However. at least in part. The spectra of CaLB in [EtNH3][NO3]. 2 Comparison of the second derivative spectra of CalB in aqueous solution (Ã) and in the dried state or in an ionic liquid. Indeed. instead of the two peaks at around 1656 and 1660 cm21 observed with CaLB in water indicate a loss of a-helix and bturns structures due to protein unfolding.22 Nevertheless. 1656. 2 the second-derivative infrared spectra of CaLB in several ionic liquids are compared with the spectrum of the protein in water. The spectrum of [BMIm][lactate] was truncated at 1630 cm21 because of a very low signal to noise ratio at lower wave numbers. A different trend was observed with CaLB in [Et3NMe][MeSO4]. in the order.16.27). might be due to an increased presence of random coil structures and to hydrogen bonded carbonyls (e. Green Chem.14. b-turns (with internally hydrogen-bonded carbonyls). the dried solid sample was also analysed. 2004. which are typically observed with protein aggregates. 1660 (only as shoulder in the dried solid). the complete absence of the bands above 1650 cm21 in the case of CaLB in [EtNH3][NO3] and the presence of only a peak at around 1658 cm21 with CaLB in [BMIm][lactate] and [BMIm][NO3]. random coil conformations.20 The amide I mode of the peptide bond at approximately 1600–1700 cm21 (mainly due to the CNO stretching vibration) is particularly relevant for protein analysis since it is conformationally sensitive. the FT-IR data indicate that the loss of activity in [EtNH3][NO3]. 6. Fig. In order to identify such individual components and to monitor their variations in the different ionic liquids tested. shows that CaLB in the dried state preserves. who have shown that lyophilized CaLB has a different secondary structure pattern compared to the protein dissolved in water. the protein second derivative IR spectrum has an equal number of peaks and at the same wave numbers as those observed in the spectrum of CaLB in water..g. In particular.
K.7 6 3 2 (s. that to maintain the activity of ionic liquid-dissolved enzymes.9 (q. 7.9 (t. which was cooled in an ice-bath under nitrogen. 3H. 7. Ethylamine (26.R. 1.73 g. 3H.043. Queen’s University of Belfast. The formation of the ionic liquid was immediate and caused the initially clear solution to become opaque. NCH3).05 mol) was added dropwise. The ionic liquids [BMIm][lactate] and [EMIm][EtSO4] were received from Prof. All other compounds were purchased from ACROS. 46%) was anhydrous according to 1H NMR. HC = CH). 1H. 2H. The lactate anion. Water and excess ethylamine were evaporated. 3 2 3 CH2CH2CH3).5 mL.3 g. 2H. CH2CH2CH2). CH ). CaLB which dissolved in [BMIm][NO3] with loss of activity. This latter explanation seems unlikely in the case of CaLB. 0. Loss of essential water is sometimes put forward as a possible cause of enzyme deactivation in anhydrous hydrophilic ionic liquids. 9.. These phenomena are not at all reflected in the Jones–Dole constants of the anions (NO32: 20. 3 (a) Schematic representation of hydrogen bonding in a b-sheet. The filtrate was concentrated in vacuo.3 The interaction should not be too strong. PF62: 20.5 at 1. Water is a powerful hydrogen bonding medium and an ionic liquid must mimic water in this respect to dissolve proteins.3 (m. 1. 1H NMR (CDCl ): d = 0.9 (m.2 (t. CH ). CH CH ). Concluding remarks It has become clear that ionic liquids with very similar structures differ greatly in their behaviour towards enzymes.30 such as drying over phosphorus pentoxide.211 mol) was added dropwise to a solution of triethylamine (27. as a gift.4 g.046 mol) an aqueous solution (30 mL) of silver nitrate (8. 2H. indicates that the enzyme preserves its native structure in this ionic liquid. 1H NMR (DMSO-d ): d = 1.211 mol) in toluene (100 mL). which stays active under strictly anhydrous conditions. 2. at such a rate that a temperature below 0° C could be maintained (the reaction is highly exothermic). Experimental Materials Novozym 435 (immobilised Candida antarctica lipase B). Dimethyl sulfate (20 mL. followed by the separation of the denser Fig. H2C-NH3). chaotropicity28 and the Jones–Dole B constant. 0. The transesterification of ethyl butyrate was monitored by HPLC on a Waters custom-packed 8 3 100 mm 5 mm Symmetry C18 column.05 M acetate buffer pH 4.33 Structural details of the ionic liquids used are listed in Table 3.31 Hydrogen bonding could be the key to understanding the interactions of proteins and ionic liquids. 2H. 0. 0. 483–487 .48 g. Analytical methods NMR spectra were measured on a Varian Unity Inova-300 instrument. 4.0 mL min21 with refractive index detection (Shodex SE-51 RI). Consider.7 (s. 70% aqueous solution) was slowly added to nitric acid (18. a balance of mild hydrogen bond-accepting and donating properties is required. because otherwise dissociation of the hydrogen bonds that maintain the structural integrity of the ahelices and b-sheets will cause the protein to unfold. such as cosmotropicity vs. Table 3 Structures of ionic liquids Abbreviation [BMIm][BF4] [BMIm][PF6] [BMIm][NO3] [BMIm][lactate] [EMIm][EtSO4] [Et3MeN][MeSO4] [EtNH3][NO3] Type 1 1 1 1 1 2 2 R n-C4H9 n-C4H9 n-C4H9 n-C4H9 C2H5 R1 CH3 C2H5 X2 BF42 PF62 NO32 CH3CH(OH)COO2 C2H5OSO32 R2 C2H5 H R3 C2H5 H R4 C2H5 H CH3OSO32 NO32 486 Green Chem.21). Sterically demanding ions. The parameters that quantify interactions of proteins and aqueous electrolytes.29 which classify these ions as chaotropic and protein-destabilising. [Et3NMe][MeSO4]36. (b) putative hydrogen bonds between a peptide chain and a lacate ion. 4. but maintained its activity while suspended in [BMIm][BF4] or [BMIm][PF6]. which do not easily penetrate the protein matrix as this would require the dissociation of many intermolecular hydrogen bonds to create a few new ones. could easily form stable hydrogen bonds with the polypeptide backbone (see Fig. BF42: 20. in particular. the residue was further dried in vacuo over P2O5.1 (t. [EtNH3][NO3]35. 3H. we suggest.0 (s. CH). In conclusion. for example. Synthesis of ionic liquids [BMIm][NO3]34. The resulting ethylammonium nitrate (20. 6.1 g. in particular as regards the hydrogen bond-accepting properties of the anion. yield 9. do not seem particularly relevant for understanding enzymes in anhydrous ionic liquids. could also contribute to maintaining activity.093. The solution was slowly brought to room temperature and the white precipitate was removed by filtration. To a stirred and ice-cooled aqueous solution (50 mL) of 1-butyl-3-methylimidazolium chloride (8.especially in the region 1640–1670 cm21. 3H. Seddon. 2004. 65% aqueous solution) while stirring and cooling in ice-ethanol to maintain a temperature of less than 25 °C. eluent 65 : 35 (v/v) MeOH–aqueous 0. SP525 (dry solid Candida antarctica lipase B) and purified CaLB lyophilisate were kindly donated by Novozymes. 2H. however.5 (d. on the basis of our results. in agreement with the fact that it also preserves its catalytic activity.3 g (93%). [BMIm][BF4] and [BMIm][PF6] were synthesised according to standard procedures32 and checked for the absence of chloride and acid. NCH2). 3).
Dong. ionic liquid. Arakawa and J. Denmark) for gifts of Novozym 435 and SP525. A. Dong. Fennell Evans.. 282–287. Broker. K. 2000. antartica lipase B. 303. Zambianchi. 1989. Acta. Vargas-Mora. Novozym 435 (15 mg) and 1. 3.. Acad. The original protein spectra were smoothed with a 9-point Savitsky–Golay method to remove white noise. J. 201–207. Triacetin (0.18 (m. A.. M. 7 R. Moulton and A. 2000. T. Manning and J. 1998. Mol. 1996. W. 11695–11700. 10969–10976. Collins. Biochemistry. W. L. Magnuson. 6 M. 17 H. Welton and D. Res.. 1996. T. M. 65–76. 1997. Soc. M. Org. Carrea. 24 H. S. 33. D. Biophys. G. 118. 1993. Green Chem. F. A. The spectra of CaLB obtained as dried powder or in a given ionic liquid were normalized for area and overlaid for comparison. T.. Solution Chem. Klibanov. A. Susi.. Cull. 95–120. Bioeng. F. 469–487. Agric. Biophys J. Recl. J.. Swatloski. R. M. 125. 2000. J. Surewicz. 2003. Torres. M. 95. Sheldon and K. Vecchio. K. The reactions were monitored for 24 hours by taking 50 mL samples that were diluted with 100 mL of HPLC eluent. Eckstein and N. J. CH CH ). Reichert. J. 5 J.2 mL sample of ionic liquid supernatant was diluted with 10 mL of 0. M. 1129–1131.16 The spectra were corrected by subtraction of the background spectrum (water. 13 W. 1995. Crit. F. Nakanishi and T. Bioeng. Am. Susi. K. 35 P... CH2CH3). 2275–2287. 1991. 1995. Chim. 583–587. Am. Fasman.87 (s. I. D. 2056–2063. Kragl. H. J. 32. 1995. Protein Sci. Rogers. 2002.-J. Ding. Transesterification of ethyl butyrate with butanol The transesterification of ethyl butyrate (0. Seddon and G. phase was decanted and the lower. Biotechnol. Seddon and R. Biochim. 1996. 4. Proc. 9H. Kaftzik.06 M) with n-butanol (0. 1992. Rogers. 12 F. Rev. Byler and H. Griebenow and A. W. Russell. Matzumura.. Sci. J. A. J. 3. 33 K. H. Griebenow and A. R. van Rantwijk. Anderson. 65. 6 2 3 OCH3). Byler and H. Biochem. Ashburn and P. Wilkes and M. 34 J. 553–557. Queen’s University of Belfast. Kaftzik. J. G. USA. 21 A. 565–571.12 M) was carried out in 0. 4 R. 71. H. 6. L.. 4125–4131.37 (s. 23 W. Kirk. A. J. 1986. 16. 51. 2000. Prestrelski.8 g (79%). 3303–3308. D. 1993. Chem. Wasserscheid and U. Solubility of CaLB SP525 (15 mg) was added to 500 mL of ionic liquid and was incubated for 24 hours at 40 °C. When the addition of dimethylsulfate was complete. P. Sheldon. S. 2002.. S. 2695–2724. 131–138. C. Jesionowski.. Stark and M. 661–671.. 72. R. F. Kragl. 181–204. are gratefully acknowledged.. 3H. A spectrum of purified dried solid CaLB was measured by mixing a 1 mg sample with 200 mg of KBr in a mortar. 4189–4191. Biochemistry. Arch. Larsson and O. Pèzolet. Klibanov. Flowers II. Madeira Lau.. 22 K. 2002. 147–151.. J. N. F. T. K. Berberich. The lipase activity in the supernatant was assayed in triacetin hydrolysis. Biochemistry. 2 M.. Secundo and G. van Bekkum.14 Acknowledgements Generous gifts of ionic liquids by Prof. Madeira Lau and R. Natl. Russell.. 334. Arakawa and J. 26 N. 27 Y. S. Carpenter and J. D. 25 M. M. Mantsch. J. Am. R. Griebenow. Walden. R.5. S. 1999. V. Biotechnol. van Rantwijk. 389–394. Green Chem. D. Green Chem. M. 28 R. R. 1995. N-CH3). R. Holbrey. Commun. Second-derivative spectra were obtained with the Savitsky–Golay algorithm for a 9 data point window with Spectra Analysis software (Jasco). H. Biopolymers. D. 5. Erbeldinger. Pitner. Lett. Halverson. 29. 2001–2008. Hollosi and G. Imamura.. 405–422. 1993. 113. Caughey. 1984. Yang. 1253. 2003. Dev. Nauk. 72. J. It has been previously shown that the use of KBr pellets does not affect the protein integrity. Holbrey. Chem. Food Chem. D. Jackson and H. 1914. R. K. A. K.118 M KOH. Tedeschi.. Klibanov. F. F. Biocatal. 6H. P. 2002. Sucholeiki. F. A. K. J. 2. 14 A. 30. The supernatant was separated from the solid enzyme by filtration followed by centrifugation. 14247–14254. 15 J. Bodley and D. A. P. phase was washed with toluene (50 mL) and dried by heating in vacuo to remove residual organic solvents. 2000. 2. P. 483–487 487 . Wellner. 6. Chem. 351–362. 124.5 mL of ionic liquid as the solvent. 227–233. Griffiths. 16. 64. Holbrey and R. Baldwin. M. 465–473. P.ionic liquid phase from the toluene solution. T. Mantsch. de Goede. Biochem. K. 20 K. Solutions of pure CaLB in water (30 mg mL21) or in ionic liquids (10 mg mL21) were loaded in a demountable CaF2 cell (Hellma) with optical path length of 6-m or 12-m (in the case of water) for FTIR measurements. Proc. 2003. 1993. 2004. R. Biophys. M. Prog. J. 39. R. J. A... Imp. 2003. 1996. the resulting mixture was pressed into a pellet using a Perkin-Elmer kit and a 10 ton hydraulic press. J. Biotechnol. Griebenow and A. T. 19 G. K. Summers and R. L. Dousseau and M.1 M sodium phosphate buffer pH 7. Crowe. yield: 37.1 M) was added and the reaction was monitored by titration with 0. Seddon. 1985.. 1993. van Rantwijk and K. Anderson.. Madeira Lau. J. 92. 32 S. M. Pays-Bas. Perczel. Belton and A. Mori.. 53. Chapman. The upper. 741–747. 8 D. S. Zacchetti. Biotechnol.. Chem. Biol. Biotechnol. Seddon. Biochem. Klibanov.06 M) were added and the reaction was carried out at 40 °C. W. Kaar. Izv. 4. Spectrosc. Rev. Curr. 8771–8779. B. 9. Sorgedrager. Interference of water vapour and CO2 was avoided by purging the sample and detector compartments with N2. R. Carpenter. 13.. organic. 9 C.-J. Soc. Chem. Acad. Carpenter. Arch. Biophys. Trends Biotechnol. J. Mantsch and D. Kendrick. Maat and H. 3H. 29. Biopolymers. S. Bioeng. D. Turner. Seddon. van Rantwijk. 229–235. 112. 3 J. 567–572. 3916–3922. K. the reaction mixture was stirred at room temperature for 1 h. R. Zaworotko. 319. Soc. Prestrelski. Biophys. 28. 69–74. J. R. [Et3MeN][MeSO4] was obtained as a colorless ionic liquid. 407–413. F. Sheldon. Mishra. F. 11 D. Meyer. 6701–6703. Huang and W. R. P. Biophys. Huddleston. References 1 For reviews see: U.. Soc. T. Y. Mizutani. Seddon. G. The authors wish to thank Novozymes (Bagsvaerd. 545–551. 0. L.. 1990.3-dimethoxybenzene (internal standard. J. Trav. K. 2002. Tatham. 29 H. Am. 30 E. M. 1990. as well as a purified sample of C. J. J. Chem. Biochem. 13. M. Activity test of CaLB A 0. K. Opin. D. at a flow of 6 L min21. Soc. 406–414. 25. ionic liquid. R. The system was managed by Jasco software. H. 1H-NMR (DMSO-d ): d = 1. 69. 36 J. Carpenter.. Green Chem. Lye.25 (m. Costantino. Langer and A. KBr blank as appropriate). W. J. Appl.. Fourier Transform Infrared (FT-IR) measurements FT-IR spectra were recorded using a Jasco 610 instrument equipped with a DTGS detector as the average of 32 scans at 2 cm21 resolution. 965–967. Jenkins and B. 10 N. Spear. M. Pure Appl. 31 A. Biochemistry. Benckhuijsen. Donald. Messiano and A. 18 K. 21. J. Armstrong. J. B. Chem. Biotransform. M. 16 S. Chem. 1997. 443–447. Org. Lansbury. W.
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