2088
E. Durand et al. / Process Biochemistry 47 (2012) 2081–2089
100
90
80
70
60
50
40
30
20
10
0
to different behaviors of compounds. However, this study provides
T=0
valuable information about the most widely used enzyme in indus-
trial biocatalysis (lipase B from C. antarctica) in these new “green”
solvents.
1 day
2 days
3 days
5 days
Acknowledgements
The authors thank Dr. Robert Lortie, Pr. Elisée Ferré and Dr Loic
Remy for helpful discussions and Emilie PEREZ for the revisions in
English.
ChCl:Gly
ChCl:Urea
Toluene
References
Solvents
[1] Bornscheuer UT. Lipase-catalyzed syntheses of monoacylglycerols. Enzyme
Microb Technol 1995;17:578–86.
[2] Soumanou MM, Bornscheuer UT. Lipase-catalyzed alcoholysis of vegetable oils.
Eur J Lipid Sci Technol 2003;105:656–60.
Fig. 7. Relative initial specific activity for transesterification of vinyl laurate with
octanol at 50 ◦C using crushed iCALB after different pre-incubation times of the
catalyst in DESs or toluene.
[3] Gog A, Roman M, Tosa M, Paizs C, Irimie FD. Biodiesel production using
enzymatic transesterification—current state and perspectives. Renew Energ
2012;39:10–6.
[4] Tarahomjoo S, Alemzadeh I. Surfactant production by an enzymatic method.
Enzyme Microb Technol 2003;33:33–7.
[5] Willis WM, Marangoni AG. Enzymatic interesterification. In: Akoh CC, Min DB,
editors. Food lipids: chemistry, nutrition, and biotechnology. 2002. p. 839–75.
[6] Winska K, Grudniewska A, Chojnacka A, Bialonska A, Wawrzenczyk C.
Enzymatic resolution of racemic secondary cyclic allylic alcohols. Tetrahedron-
Asymmetr 2010;21:670–8.
[7] Li D, Xuebing X, Haraldsson GG, Tianwei T, Fang W. Enzymatic production of
alkyl esters through alcoholysis: a critical evaluation of lipases and alcohols. J
Am Oil Chem Soc 2005;82:341–7.
structure in glycerol-containing DES, which leads to a decrease of
activity during this period. Indeed, glycerol is likely to have a neg-
ative effect on the lipase activity and stability by being adsorbed
over the catalyst which reduces the diffusion of the hydrophobic
substrate toward the active site of the lipase [45,46]. In this case,
comparing the activity after 5 h and after 5 days incubation time
shows that iCALB is maintaining the same rate and thus preserves
its activity without any apparent denaturation. In ChCl:U, iCALB
loses only 5% after one day incubation and less than 38% after 5 days
in comparison with the rate before incubation. Consequently, the
stability of iCALB in these two solvents should allow to carry out
long and more complex reactions with a minimal modification of
the protein structure.
[8] Yang Z, Pan WB. Ionic liquids: green solvents for nonaqueous biocatalysis.
Enzyme Microb Technol 2005;37:19–28.
[9] Van Rantwijk F, Sheldon RA. Biocatalysis in ionic liquids. Chem Rev
2007;107:2757–85.
[10] Shi YG, Cai Y, Li JR, Chu YH. Enzyme-catalyzed regioselective synthesis of car-
bohydrate fatty acid esters in ionic liquids. Prog Chem 2011;23:2247–57.
[11] Park S, Viklund F, Hult K, Kazlauskas RJ. Ionic liquids create new opportunities
for nonaqueous biocatalysis with polar substrates: acylation of glucose and
ascorbic acid. Ion Liq Greensolv Progress Prospects 2003;856:225–38.
[12] Madeira Lau R, van Rantwijk F, Seddon KR, Sheldon RA. Lipase-catalyzed reac-
tions in ionic liquids. Org Lett 2000;2:4189–91.
[13] Hernandez-Fernandez FJ, Rios A, Lozano-Blanco LJ, Godinez C. Biocatalytic ester
synthesis in ionic liquid media. J Chem Technol Biotechnol 2010;85:1423–35.
[14] Canongia Lopes JN, Costa Gomes MF, Padua AAH. Nonpolar, polar, and associ-
ating solutes in ionic liquids. J Phys Chem B 2006;110:16816–8.
[15] Pan C, Pelzer K, Philippot K, Chaudret B, Dassenoy F, Lecante P, et al. Ligand-
stabilized ruthenium nanoparticles: synthesis, organization, and dynamics. J
Am Chem Soc 2001;123:7584–93.
4. Conclusion
It was already reported that iCALB was active in DESs com-
posed of choline chloride or ethylammonium chloride paired with
hydrogen-bond donors such as amide, hydroxyl or acid [24,26].
However, in the present study we showed that some conditions
are necessary to promote biocatalysis in DESs media and that not all
eutectic mixtures can be used as efficient media for lipase-catalyzed
reactions. First, in the specific case of immobilized C. antarctica B
lipase, we noticed that a preliminary grinding was crucial in order
to get an efficient reaction kinetic.
[16] Pertici P, Simonelli G, Vitulli G, Deganello G, Sandrini P, Mantovani A.
(Eta-4-cyclo-octa-1,4-diene)(eta-6-cyclo-octa-1,3,5-triene)ruthenium(0)
chemistry—role of molecular-hydrogen in a new synthetic route to cyclo-olefin
ruthenium complexes. J Chem Soc Chem Comm 1977:132–3.
Secondly, some DESs such as ChCl:Mo, ChCl:Ox and ChCl:EG can
react and compete with the substrates in alcoholysis reactions lead-
ing to byproduct formation and DES destruction. Moreover, in the
specific case of the DESs based on dicarboxylic acids, the viscosity
that is already high from the beginning, increased significantly with
side reactions, making stirring and recycling difficult. However, in
most cases, the strong hydrogen bonds between DES components
can dramatically lower their reactivity as it was observed in DESs
based on glycerol or urea. In addition, results revealed that the
reactivity for alcoholysis in deep eutectic mixtures may depend
on the polarity of the nucleophilic substrate. The DESs composed
of glycerol or urea combined with choline chloride as quaternary
ammonium salt allowed the best initial specific activity of the
lipase, comparable to those in conventional organic media. More-
over, in these DESs, the activity of iCALB seems to be less influenced
by the alcohol chain length in alcoholysis reaction with vinyl lau-
rate compared to its activity in toluene. Although we know that
immobilized C. antarctica lipase B denaturates in solutions of urea,
it did not denaturate quickly in DESs containing urea or glycerol
and the stability in these DESs is sufficient to allow the reaction
for several days. It is difficult to extrapolate these results to other
lipases and DES combinations since the reactivity and stability of
the enzymes in DESs are influenced by the components of the DES
itself. The network formed by hydrogen-bonds can vary and lead
[17] Pery T, Pelzer K, Buntkowsky G, Philippot K, Limbach HH, Chaudret B, et al. Evi-
dence for the presence of mobile surface hydrides on ruthenium nanoparticles.
Chem Phys Chem 2005;6:605–7.
[18] Triolo A, Russina O, Bleif HJ, Di Cola E. Nanoscale segregation in room temper-
ature ionic liquids. J Phys Chem B 2007;111:4641–4.
[19] Seddon KR, Stark A, Torres MJ. Influence of chloride, water, and organic solvents
on the physical properties of ionic liquids. Pure Appl Chem 2000;72:2275–87.
[20] Jastorff B, Stormann R, Ranke J, Molter K, Stock F, Oberheitmann B, et al.
How hazardous are ionic liquids? Structure–activity relationships and biolog-
ical testing as important elements for sustainability evaluation. Green Chem
2003;5:136–42.
[21] Ranke J, Molter K, Stock F, Bottin-Weber U, Poczobutt J, Hoffmann J, et al.
Biological effects of imidazolium ionic liquids with varying chain lengths in
acute Vibrio fischeri and WST-1 cell viability assays. Ecotoxicol Environ Saf
2004;58:396–404.
[22] Ventura SPM, Marques CS, Rosatella AA, Afonso CAM, Goncalves F, Coutinho
JAP. Toxicity assessment of various ionic liquid families towards Vibrio fischeri
marine bacteria. Ecotoxicol Environ Saf 2012;76:162–8.
[23] Abbott AP, Capper G, Davies DL, Rasheed RK, Tambyrajah V. Novel solvent
properties of choline chloride/urea mixtures. Chem Commun 2003;7:0–7, 1.
[24] Gorke JT, Srienc F, Kazlauskas RJ. Hydrolase-catalyzed biotransformations in
deep eutectic solvents. Chem Commun 2008;123:5–123, 7.
[25] Zhao H, Baker GA, Holmes S. Protease activation in glycerol-based deep eutectic
solvents. J Mol Catal B-Enzym 2011;72:163–7.
[26] Zhao H, Baker GA, Holmes S. New eutectic ionic liquids for lipase activation and
enzymatic preparation of biodiesel. Org Biomol Chem 2011;9:1908–16.
[27] Lindberg D, Revenga MD, Widersten M. Deep eutectic solvents (DESs) are
viable cosolvents for enzyme-catalyzed epoxide hydrolysis.
2010;147:169–71.
J Biotechnol
[28] Abbott AP, Cullis PM, Gibson MJ, Harris RC, Raven E. Extraction of glycerol from
biodiesel into a eutectic based ionic liquid. Green Chem 2007;9:868–72.