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Journal of Molecular Catalysis B: Enzymatic 90 (2013) 98– 106

Contents lists available at SciVerse ScienceDirect

Journal of Molecular Catalysis B: Enzymatic

jou rn al hom epa ge: www.elsev ier .com/ locate /molcatb

nzymatic synthesis of 6- and 6′-O-linoleyl-�-d-maltose: From solvent-free toinary ionic liquid reaction media

abian Fischera,∗, Manuel Happea, Jessica Emerya, Antoine Fornagea, Rolf Schützb

Institute of Life Technologies, University of Applied Sciences Western Switzerland, HES-SO Valais, Route du Rawyl 64, CH-1950 Sion 2, SwitzerlandDSM Nutritional Products Ltd., KAU Building 203/107a, P.O. Box 2676, CH-4002 Basel, Switzerland

r t i c l e i n f o

rticle history:eceived 14 August 2012eceived in revised form 22 January 2013ccepted 23 January 2013vailable online 31 January 2013

eywords:reen solvent

a b s t r a c t

The eco-efficient lipase catalyzed synthesis of sugar fatty acid ester surfactants from renewable com-modities is a stimulating challenge in biotransformation. The biocatalytic investigations went fromsolvent-free and minimal ionic liquid use to bulk organic solvents, and binary solvents thereof. Disaccha-ride maltose was acylated with linoleic acid (C18:2) on the primary O-6,6′ hydroxyl functions. The ionicliquid [emim][MeSO3] and the potentially renewable acetone enabled best conversions. Binary solventslike acetone/DMF and [emim][MeSO3]/[bmpyr][PF6] tended to double conversions. The lipase selectionis also crucial and the enzymes Pseudomonas cepacia and immobilized Candida antarctica allowed high-

co-efficiencyipaseelectivityurfactant

est yields in a screening with 10 different lipases. Also other non-solvent parameters such as reactiontime and molecular sieve content improved maltose transformation further up to 82%. Analysis by HPLC,ESI-MS, and NMR indicated the formation of mono-6 or 6′-O-linoleyl-�-d-maltose as a mixture of tworegioisomers in a 1.4:1 ratio. From an applied point of view, the best solvent is acetone. Unlike ionicliquids and binary mixtures, it is easily removable from reaction mixtures. Moreover, acetone is to some

it ca

degree a green solvent as

. Introduction

Sugar fatty acid ester (SFAE) surfactants [1] are produced fromenewable chemicals. Demands of cosmetics consumers for semi-atural products are ever increasing. At the same time the food

ndustry considers the antimicrobial properties of sugar fatty acidsters conserving convenience foods [2]. In the pharmaceuticalndustry SFAE are thought as auxiliaries in drug delivery and forther purposes [3]. Saccharides and fatty acids provide contrastingolute properties and polyvalent solvents are a requirement for

successful esterification [4]. Monosaccharide acylation is docu-ented in up to complete conversions [5]. In contrast, the lipaseediated acylation of disaccharides and oligosaccharide has been

ess thoroughly examined and higher yielding processes are hardlynown. Other researchers used acetone for the monolauryl maltosester synthesis [6]. t-Butanol also enabled the transesterificationf various disaccharides with ethlyalkanoates [7]. The acylation ofivinyl dicarboxylates on maltose and other disaccharides with arotease from Bacillus subtilis showed high regioselectivity in pyri-

ine [8]. Binary solvent mixtures are an alternative option andnsured maltose acylation with lauric acid in a binary acetone/n-exane solvent [9]. t-Butanol/pyridine mixtures were also used for

∗ Corresponding author.E-mail address: fabian.fischer@hevs.ch (F. Fischer).

381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.molcatb.2013.01.019

n be produced directly from renewable feedstock.© 2013 Elsevier B.V. All rights reserved.

several disaccharide acylations [10]. However, these binary sol-vents are toxic and the E-factor [11] is consequently high, which isnot in line with the green chemistry principles [12,13]. Therefore,green organic solvents, ionic liquids (ILs), supercritical carbon diox-ide (scCO2), perfluorated solvents and liquid substrates can be usedfor this purpose [14,15]. These solvents provide advantages anddisadvantages and their utilization should be optimized. Amongall possible organic solvents, acetone is of interest as it is an apro-tic solvent optionally producible by the acetone–butanol–ethanolfermentation from renewable feedstock [16]. However, due to itsvolatile nature, it is not green like ionic liquids, which in contrast arenon-volatile and, consequently, well retained in open reactor ves-sels. Moreover, many ILs improve or maintain enzymatic activity[17]. A versatile variant are partly fluorinated ionic liquids, whichcombine water repelling and liquid salt properties. Such ILs con-sist mainly of fluorinated anions [PF6]-, [(CF3SO2)2N]-, [RfBF3]-,but also fluorinated cations are described [18]. In some cases, liq-uid substrates also serve as solvents, and product isolation is inquantitative transformations rather simple [19]. The applicabilityof the rare solvent-free approach is extendable through minimalor optimal ionic liquid quantity use to accelerate sluggish solvent-free transformations [20]. The use of ionic liquids (ILs) in enzymatic

sugar fatty acid ester synthesis is a current research field [21]. Ionicliquids, green binary solvents including ionic liquid mixtures arehardly examined in disaccharide acylation [22]. The esterification ofmaltose with linoleic acid in ionic liquids and a green binary solvent

F. Fischer et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 98– 106 99

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1

Scheme 1. Lipase catalyzed regioselective maltos

as not been investigated yet. In this work, a range of ionic liquids,rganic solvents, binary mixtures, minimal and non-solvent param-ters were used to establish an eco-efficient synthesis exemplifiedor 6- and 6′-O-linoleyl-�-d-maltose 3a and 3b (Scheme 1).

. Experimental

.1. Material

.1.1. Substratesd(+)-Maltose monohydrate in 99% purity was bought from Fluka

Buchs, Switzerland]. Linoleic acid with a purity of 99% was pur-hased from Acros Organics [Belgium] and an industrial quality ofinoleic acid (60–74%) was provided by DSM-Pentapharm [Aesch,witzerland]. Molecular sieves 3 A [Merck, Darmstadt] were acti-ated before use.

.1.2. EnzymesCandida antarctica lipase B immobilized as Novozym® 435 was

urchased from Novozym [Denmark]. Non immobilized lipases asyophilized powders were bought from Fluka [Buchs, Switzerland]:. antarctica lipase B (10.9 U/mg), Pseudomonas cepacia (40 U/mg),andida cylindracea (3.9 U/mg), Candida rugosa (2.9 U/mg), Rhizo-us niveus (1.7 U/mg), Pseudomonas fluorescens (36 U/mg), Mucoravanicus (9.9 U/mg), Aspergillus oryzae (53.8 U/mg), Rhizopus oryzae58.4 U/mg), Rhizopus arrhizus (9.2 U/mg).

.1.3. Organic solventsAcetone, n-hexane > 99.5%, DMSO > 99.9%, t-butanol > 96%,

oluene puriss, dioxane puriss, and THF analytical grade, wererom Fluka [Buchs, Switzerland]. Acetonitrile and DMF (99%),nhydrous were from Sigma–Aldrich [Buchs, Switzerland].

.1.4. Ionic liquidsThe melting point of non-RTILs was determined by differen-

ial scanning calorimetry (DSC). 1-Butyl-3-methylimidazoliumexafluorophosphate [bmim][PF6] (RTIL, 99%), 1-butyl-4-ethylpyridinium hexafluorophosphate [bmpyr][PF6] (mpDSC =

5.7 ◦C, 99%), 1-butyl-4-dimethylimidazolium hexafluorophos-hate [bdmim][PF6] (mpDSC = 42.3 ◦C, 99%), 1-hexylpyridiniumexafluorophosphate [hpyr][PF6] (mpDSC = 49.4 ◦C, 99%), 1-ethyl-3-propylimidazolium hexafluorophosphate [mpim][PF6]

mpDSC = 44.3 ◦C, 99%), trihexyltetradecylphosphoniumexafluorophosphate [thtdp][PF6] (mpDSC = 43.6 ◦C, 99%), 1-utyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imidebmim][NTf2] (RTIL, 99%), 1-butyl-3-methylimidazolium tetra-uoroborate [bmim][BF4] (RTIL, >99%), tributylmethylammoniumis(trifluoromethylsulfonyl)imide [tbmam][NTf2] (RTIL, 99%),ethyltrioctylammonium bis(trifluoromethylsulfonyl)imide

mtoam][NTf2] (RTIL, 99%). All these ionic liquids were fromolitec [Heilbronn, Germany]. The following ILs: 1-ethyl-3-

ethylimidazolium ethylsulfate [emim][EtSO4] (RTIL, >95%) and-ethyl-3-methylimidazolium methanesulfonate [emim][MeSO3]

3a 3b

cylation with linoleic acid using various solvents.

(RTIL, >95%) were purchased from Sigma–Aldrich (BASF) [Buchs,Switzerland].

2.2. Enzyme screening

The screening mixtures were prepared from 0.06 mmol maltose,0.12 mmol linoleic acid, 40 U lipases as lyophilized powders and100 mg molecular sieves in a 1.5 mL Eppendorf vial and filled upwith ∼1.5 mL acetone. The sealed vials were vertically fixed on arevolving glass cylinder in a hybridization oven, 8 rpm/40 ◦C for1–7 days. Maltose conversion was determined by HPLC.

2.3. Synthesis procedures for 3a and 3b

2.3.1. Method A: transformation in organic solvents10 mmol (2.80 g) linoleic acid, 5 mmol (1.80 g) d(+)-maltose

monohydrate, 0.80 g activated molecular sieve 3 A, 0.90 g (9000TBU) Novozym® 435 (immobilized CALB), and 8.0 mL organic sol-vent were added to a 20 mL glass vial, sealed, and subsequentlyprocessed in an incubator (INFORS AJ110) at 65 ◦C/350 rpm for 3days (basic screening conditions in Methods A–D), best results werereported according to the standard convention for result represen-tation in enzyme catalysis [23]. Temperature choice: the highesttemperature possible for the incubator was 65 ◦C, while the immo-bilized CALB is most active at 70 ◦C but beyond this temperaturedenaturation reduces the activity. At 65 ◦C acetone was alreadysuperheated (bpacetone = 56 ◦C) but the vials remained well sealed.For product isolation the molecular sieve and lipase beads were fil-tered off using a fritted glass funnel (G3). The maltose-containingliquid was transferred to a 200 mL gauged flask and filled up withdistilled water. The maltose content was determined by HPLC.

2.3.2. Method B: catalysis in ionic liquidsThe preparation of the reaction mixtures followed Method

A and the organic solvent was replaced by 3.0 g ionic liq-uid. After processing 65 ◦C/350 rpm/3 days the reaction mixturewas dissolved with 10 mL CH3CN/H2O/THF (70/20/10, v/v/v) andhomogenized using an ultrasound bath, 2 min/20 ◦C. The work-upwas similar to Method A using instead a paper filter (Schleicher undSchüll) and the aforementioned ternary solvent.

2.3.3. Method C: catalysis with ionic liquid coated lipase beads

mixed for 20 min with a rotavapor at 22 ◦C/1 bar. Then the aux-iliary solvent was removed under reduced pressure at 22 ◦C andthe remaining coated beads were stored in a dessiccator overnight(15 h) at room temperature.

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.3.4. Method D: transformation in binary solvent mixturesThe reaction mixtures were prepared according to Methods A

nd B. Binary solvents were 1:1 (v/v) mixtures of 4.0 mL organic sol-ent each, or 3.0 g of ionic liquid. In the case of binary ionic liquids,r an organic solvent/ionic liquid mixture either g or mL quantitiesere employed. For the best combination fractions were varied andlotted against maltose conversions.

.4. Determination of maltose conversion by HPLC analysis

Maltose concentrations were directly determined (the sameay) from reaction mixtures after dilution in a 200 mL gauged flaskt room temperature by HPLC (Agilent 1100 Series with RID) usingn Aminex-H87 column (300 mm × 7.8 mm i.d.) at 35 ◦C with 5 mM2SO4 as eluent (0.5 mL/min).

.5. Product isolation

The filtered raw product was separated based on an improvedPLC protocol from Method A. The preparative HPLC consisted of

Knauer (Germany) setup with Luna 5u C18(2) column (100 A,50 mm × 15 mm, 5 �m) [Phenomenex], and UV200 nm detector.2O/CH3CN eluted the isomeric mixture after 15.9 min, several

ractions were collected and the eluent removed at 40 ◦C/60 mbar.he initial eluent was a mixture of 40% H2O and 60% CH3CN and

gradient was applied to reach 100% CH3CN in 10 min time andubsequently kept isocratic for 20 min.

.6. Polarity determination of ionic liquids

Polarity values of ILs not found in the literature [24,25]ere determined by the Reichardt method [26] using betaine

2,6-diphenyl-4-(2,4,6-triphenylpyridinium-1-yl)phenolate) a dyehose UV–vis absorption maxima (�max) shifts in function of the ILolarity. These measurements were realized at room temperaturend at 50 ◦C for ILs with melting points above room temperature.

.7. Product characterization

.7.1. NMR, MS, and IR analysisThe maltose linoleic acid ester 3a and 3b was dissolved in

.75 mL DMSO-d6 for NMR analyses: 1H and 13C NMR, COSY1H–1H correlation), HSQC (1H–13C heteronuclear single quantumoherence), and HMBC (1H–13C heteronuclear multiple bond (3J)orrelation). All these experiments were conducted on a 400 MHzMR spectrometer. A supplementary 800 MHz 1H NMR spectrum

mproved hydroxyl proton resolution (both NMR-spectrometersere of the Bruker Avance type). A Varian 320-MS TQ mass spec-

rometer with electrospray (ESI) unit identified molecular masseaks. A Nicolet FT-IR Thermo 5700 spectrometer (KBr tablet) wassed for carbonyl function analysis.

.8. NMR-data

The two NMR-data sets below were read from spectra con-aining a 1:1.4 regioisomer mixture of 3a and 3b. Overlappingignals are indicated as multiplets (m). Readable chemical couplingonstants with overlapping signals are reported as identified for theespective isomer.

.8.1. 6-O-linoleyl-˛-d-maltose 3aıH (800 MHz, DMSO-d6) 6.42, (1H, d, JOH,1 = 4.54 Hz, CHOH,

H-1), 5.57 (1H, d, JOH,2′ = 6.32 Hz, CHOH, OH-2′), 5.41 (1H, d,

OH,3′ = 2.96 Hz, CHOH, OH-3′), 5.26 (4H, m, HC CH, H-9′′, H-0′′, H-12′′, H-13′′), 5.11 (2H, m, CHOH, OH-4′, OH-3), 4.95 (1H,, J1′ ,2′ = 3.95 Hz, CH, H-1′), 4.90 (1H, t, J = 3.97 Hz, CHOH, H-1),.76 (1H, d, J = 6.85 Hz, CHOH, OH-2), 4.52 (1H, t, JOH,6′ = 5.80 Hz,

lysis B: Enzymatic 90 (2013) 98– 106

CHOH, OH-6′), 4.23 (1H, bd, JABX = 11 Hz, Ha-6), 3.98 (1H, ddd,JABX = 11.2 Hz, 6.73, 2.26 Hz, CH2,Hb-6), 3.67 (1H, m, CH, H-5), 3.60(1H, m, CH2OH, Ha-6′), 3.47 (1H, m, CH2OH, Hb-6′), 3.35 (2H, m,CHOH, H-3,H-3′), 3.24 (2H, m, CHOH, H-2′,H-4), 3.18 (1H, m, CHOH,H-5′), 3.03 (1H, m, CHOH, H-4′), 2.95 (1H, m, CHOH, H-2). 2.67 (2H,t, J = 6.94, CHCH2CH , H-11′′), 2.25 (2H, t, J = 7.38, C(O)CH2, H-2′′),1.95 (4H, m, CH2, H-8′′, H-14′′), 1.46 (2H, m, CH2, H-3′′), 1.27–1.20(16H, m, CH2, H-4′′. . .7′′,14′′. . .17′′), 0.80 (3H, t, J = 7.05, CH3, H-18′′).

ıC (100 MHz, DMSO-d6): 172.9(C O, C-1′′), 129.7(C C, C-9′′, C-10′′), 127.7(C C, C-12′′, C-13′′), 101.0(OCHO, C-1′), 92.1(OCHOH, C-1), 81.1(CHO, C-4), 76.5(CHOH, C-3′), 74.3(CHOH, C-2), 73.2(CHOH,C-3), 72.9(CHOH, C-5′), 71.8(CH, C-5), 70.4(CHOH, C-2′), 70.0(CH,C-4′), 63.5(CH2OH, C-6′), 60.8(CH2OCO, C-6), 33.3(C(O)CH2, C-2′′), 30.9, 29.0, 28.7, 28.6, 28.53, 28.47 and 22.0 (CH2, C-4′′. . .8′′,14′′. . .17′′), 26.62 and 26.59 (CH2CH , C-8′′, C-14′′),25.2(CH2, C-11′′), 24.4(CH2, C-3′′), 13.9(CH3, C-18′′).

2.8.2. 6′-O-linoleyl-˛-d-maltose 3bıH (NMR 800 MHz, DMSO-d6) 6.81 (1H, d, JOH,1 = 6.72, CHOH, OH-

1), 5.62 (1H, d, JOH,2′ = 6.32, CHOH, OH-2′), 5.53 (1H, d, JOH,3′ = 2.96,CHOH, OH-3′), 5.26 (4H, m, HC CH, H-9′′, H-10′′, H-12′′, H-13′′), 5.11(3H, m, CHOH, OH-4′, OH-3, OH-2), 4.96 (1H, d, J1′ ,2′ = 3.95, CH, H-1′), 4.63 (1H, JOH,6 = 5.80, CHOH, OH-6), 4.30 (1H, t, J1,2 = 6.95, CHOH,H-1), 4.23 (1H, bd, JABX = 11 Hz, Ha-6′), 3.98 (1H, ddd, JABX = 11.2 Hz,6.73, 2.26 Hz, CH2,Hb-6′), 3.68 (1H, m, CH2OH, Ha-6), 3.67 (1H, m,CH, H-5′), 3.45 (1H, m, CH2OH, Hb-6), 3.35 (1H, m, CHOH, H-3, H-3′), 3.24 (1H, m, CHOH, H-2′, H-4), 3.18 (1H, m, CHOH, H-5), 3.03(1H, m, CHOH, H-4′), 2.95 (1H, m, CHOH, H-2), 2.67 (2H, t, J = 6.94,

CHCH2CH , H-11′′), 2.25 (2H, t, J = 7.38, C(O)CH2, H-2′′), 1.95 (4H,m, CH2, H-8′′, H-14′′), 1.46 (2H, m, CH2, H-3′′), 1.27–1.20 (16H, m,CH2, H-4′′. . .7′′,14′′. . .17′′), 0.80 (3H, t, J = 7.05, CH3, H-18′′).

�C (NMR 100 MHz, DMSO-d6) 172.9(C O, C-1′′), 129.7(C C,C-9′′, C-10′′), 127.7(C C, C-12′′, C-13′′), 101.0 (OCHOC, C-1′),96.8(OCHOH, C-1), 80.6(CH, C-4), 76.5(CHOH, C-3′), 75.2(CHCO, C-5), 74.2(CHOH, C-2), 73.2(CHOH, C-3), 72.9(CHC, C-5′), 72,4(CHOH,C-2′), 70.0(OCH, C-4′), 63.5(CH2OCO, C-6), 60.8(CH2OH, C-6′),33.3(C(O)CH2, C-2′′), 30.9, 29.0, 28.7, 28.6, 28.53, 28.47 and 22.0(CH2, C-4′′. . .8′′,14′′. . .17′′), 26.62 and 26.59 (CH2CH , C-8′′, C-14′′),25.2(CH2, C-11′′), 24.4(CH2, C-3′′), 13.9(CH3, C-18′′).

2.9. MS and IR data of the regioisomeric mixture 3aand 3b

ESI-MS: 603.23 (72%) + H+ = 604.24 m/z; (expected (C30H52O12):604.7417 m/z).

FT-IR (KBr) cm−1: 3353 (s), 2926–2855 (s), 1723 (s), 1617 (m),1475 (s).

3. Results

3.1. Enzyme screening and other non-solvent parameters

The investigations started with the screening of 10 lipases(Fig. 1A) in acetone to identify the best lipase for the biotransforma-tion of maltose and linoleic acid into the corresponding SFAE 3a and3b. These mostly non-immobilized lipases were processed in ace-tone at 40 ◦C. Native lipases show their best activity in pure wateraround 40 ◦C and denaturize above it. Acetone was chosen for thescreenings because it is known to work well in inverse hydrolysiswith lipases. The obtained results showed that C. antarctica lipaseB (immobilized) and native P. cepacia worked best with 57.1% and58.0% after 3 days of processing. In the following the immobilized

CALB (Novozyme® 435) was used as it performs well, allows simplework up and remains stable up to 70 ◦C without activity loss.

Beside enzyme activity and solvent choice also molecular sievecontent and reaction time were assessed. The mixing of the more

F. Fischer et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 98– 106 101

Fig. 1. Maltose conversions in acetone/40 ◦C: (A) with various 9 native lipases andC[r

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Fig. 2. Maltose conversions in relation to solvent polarity in lipase catalyzed mono-6 and 6′-O-linoleyl maltose 3a and 3b synthesis. The reaction mixtures consisted of:10 mmol linoleic acid, 5 mmol maltose, 0.8 g molecular sieve, 9000 U of immobilized

◦

ALB immobilized as Novozyme® 435; (B) dependence on Candida antarctica activityU] under irreversible conditions; (C) increasing molecular sieve 3 A content andeaction time: 3 days (�) and 7 days (�).

iscous solutions, as this was the case for ILs and binary mixtures,equired vigorous shaking at 350 rpm, what was possible with anNFORS incubator, whose highest operation temperature was 65 ◦C.he screening vials remained well sealed under these conditionslthough the boiling point of acetone is 56 ◦C. The optimal activityor Novozym® 435 is around 70 ◦C [27] with higher temperaturehe activity drops therefore 65 ◦C were close to optimal processonditions. The examinations showed also that the molecular sieveontent and reaction time influenced conversions in a proportionalanner and after one week 82% of maltose was converted using a

:2 (n:n) maltose/linoleic acid ratio (Fig. 1B and C).

.2. Maltose conversion in pure medium

The solvent free biotransformation with liquid linoleic acid asolvent yielded after 3 days 20.3% of 3a and 3b. This result served

CALB, and were vigorously shaken for 3 days at 65 C/350 rpm. (A) Conversions inorganic solvents with log P values in brackets. (B) Ionic liquid use in solvent quanti-ties. (C) Minimal solvent conditions with ionic liquid coated immobilized CALB.

as a benchmark against various pure solvents. Because solvent freeconversion, if efficient, is preferred over any solvent use. The slowreaction was visibly caused by insufficient maltose solubility andtherefore an aprotic solvent was searched that would acceleratethe inverse hydrolysis. A range of pure organic and ionic liquidswas selected to screen pure solvent conditions.

3.3. Pure organic solvents

Nonpolar to polar pure organic solvents were chosen (Fig. 2A).N

Apolar hexane and toluene, ET = 0.01–0.1, yielded lower (9–14%)

3a and 3b than the more polar DMF, THF, acetone and ace-tonitrile, EN

T = 0.21 − 0.46, which enabled maltose conversionsbetween 30 and 40%. Some polar organic solvents were clearly not

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ppropriate, like DMSO or t-BuOH. An experiment in an NMR tubeas attempted to verify the reason for DMSO and it was confirmed

hat DMSO-d6 dissolves maltose very well, but in combination withinoleic acid the well resolved hydroxyl protons were no longer sep-rated because of an interaction between the linoleic acid and thealtose.

.4. Pure ionic liquids

Room temperature ionic liquids (RTILs) and ILs with a meltingoint below 65 ◦C were chosen. The melting point did not influencehe conversion but fluorinated ILs, with [PF6]- and [NTf2]- anionshowed in most cases clearly low conversions, and they were evenelow solvent-free conditions (Fig. 2B). The water miscible RTILemim][MeSO3] converted the maltose at 65 ◦C up to 36.8%, whatas equally good as with well suited organic solvents. The polarity

f most ILs was higher than for the organic solvents but the conver-ion did not increase or decline systematically with solvent polarity.he polarity of the used ILs was determined, if data were not avail-ble from literature, by the Reichardt method at room temperaturer above the melting point of the respective IL (at 50 ◦C).

.5. Use of IL coated lipase beads for maltose conversion

The use of minimal solvent quantities is a means to accelerateeactions due to concentration effects or because the ILs stabilizenzymes and in some cases even co-catalyze enzyme reactions.onic liquid coated immobilized CALB beads were therefore pre-ared as earlier described [24]. The Novozym® 435 beads became

mpregnated by the respective IL what is described as coatingFig. 2C). The IL coated Novozym® 435 converted maltose and

inoleic acid somewhat more efficient than pure IL solvent quan-ities. Conversely, the best IL [emim] [MeSO3] found for solventonditions was less efficient as coating.

ig. 3. (A) Maltose conversion in various 1:1 binary solvents, processed in an incu-ator, 65 ◦C/350 rpm/3 days. (B) Binary solvent partition versus maltose conversionor the best combination DMF/acetone.

lysis B: Enzymatic 90 (2013) 98– 106

3.6. Maltose conversion in binary solvents

In all pure solvents the biotransformation was slow and a betterprocessing mode was searched. It was therefore examined if theconversion rates increase by combining two of the better suitedpure solvents found before experimentally. These binary mixturesconverted the substrates more efficient than pure solvents (Fig. 3A).In fact the maltose conversions tended to double, while the repeat-ability decreased. A means to find the best working binary solventratio is to variate solvent fractions. Therefore the best combina-tion found, DMF/acetone, was examined for various solvent ratiosand the obtained curve indicated that the best ratio was a 1:1 (v/v)mixture (Fig. 3B). The work up from binary solvent reaction mix-tures was more laborious than from pure organic solvent. After all,binary solvent mixtures accelerated reactions due to the combi-nation of favorable solvent properties of two already more or lessappropriate solvents. They optimized the solubility of linoleic acidand maltose including the interaction with the immobilized CALB.

4. Discussion

4.1. Biotransformation in organic solvents

Several organic solvents enabled the maltose acylation withlinoleic acid using identical screening parameters, 65 ◦C/350 rpm/3days (Method A). The reasons for fair to good conversions, how-ever, are complex. The most suited organic solvent was bysurprise DMF, which allowed 39.7% maltose conversion. Ace-tone with 38.1% worked equally well, while THF (33.9%) andacetonitrile (29.7%) performed somewhat lower. The rest ofthe tested organic solvents showed a descending suitability: t-butanol > hexane > dioxane > DMSO > toluene (Fig. 2A).

The solubility properties of maltose and linoleic acid in organicsolvents differ and a versatile solvent is vital for a productiveesterification. Highly polar organic solvents dissolve disaccharidesbetter, but lipase activity decreases with rising solvent polarity [28].Recorded maltose conversions were therefore related to organicsolvent polarity (Fig. 2A) indicating that polarity values betweenEN

T = 0.2 − 0.5 on Reichardt’s scale are preferred. The non-polar n-hexane EN

T = 0.01 and toluene [25] ENT = 0.10 yielded low, which

is due to the poor solubility of maltose in these apolar solvents.In comparison DMSO dissolves maltose very well but conversionswere low with 12%. The polarity of DMSO EN

T = 0.45 is close tothe polarity of acetone EN

T = 0.36 or DMF ENT = 0.40, which yielded

higher 3a and 3b (Fig. 2A). Also dioxane ENT = 0.44 and t-butanol

ENT = 0.32 resulted in sluggish esterifications. This is in line with

earlier results using �-methyl-d-glucose and myristic acid [24].Also there no direct correlation between saccharide conversion andsolvent polarity was observed. An alternative parameter to evalu-ate enzymatic activity with organic solvents is the log P, a constantfor hydrophobic behavior respectively organic solvent solubilityin pure water [29]. The results with clearly hydrophobic solvents,such as hexane (log P = 3.5) and toluene (log P = 2.5), correlated withthe polarity. That maltose conversions were low with these twosolvents was therefore an effect of the poor solubility of maltoseand not of reduced lipase activity. The hydrophobic properties ofthe best performing organic solvents THF (log P = 0.49), acetone(log P = −0.23), acetonitrile (log P = −0.33) and DMF (log P = −1.0)are close together. In contrast, the polarity and log P = 0.37 oft-BuOH [30] would predict similar suitability but the maltoseconversion was less productive with 14.9%. In this case nei-

ther the solvent polarity nor the log P predict this behavior andanother explanation has to be found. The two solvents withthe least hydrophobic qualities, DMSO log P = −1.3 and dioxanelog P = −1.1 were both leading to unproductive transformations

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s predicted due to enzyme deactivation. The excellent wateroluble organic solvents, with a low log P ≤ 2, strip off residualater from enzymes what causes deactivation. In this respect the

ow conversions with DMSO and dioxane had to be expected.he log P = −1 for DMF would indicate no activity either by thisheory, but it worked well and this is in line with documentedesults were DMF equally performed well under similar condi-ions [8]. It is noteworthy that log P values in the literature differo some extend as their values were determined by various assay

ethods.The investigations in organic solvents showed that many sol-

ents increase conversions as the synthesis without an additionalolvent enabled only 20.3% maltose conversion in three days. Theields with suitable organic solvents doubled conversions and ace-one was found as the preferred solvent, due to highest yields, easyork-up and because it can be sourced from renewable feedstock

y the acetone–butanol–ethanol fermentation [16].

.2. Ionic liquids as solvent and coating of immobilized lipase

Several ionic liquids (ILs) in bulk solvent quantities were sub-ected to screening conditions with immobilized CALB (Method B)nd [emim][MeSO3] clearly excelled (Fig. 2B). There are over a mil-ion ILs imaginable but the choice is limited by their availability31]. The solvent properties and other physical characteristics of allhese ILs are certainly diverse but unknown; and even for obtain-ble ILs, such data is often unavailable.

What kind of ILs have to be chosen for the here examinediotransformation keeping in mind that there is insufficient knowl-dge about all parameters? Ionic liquids stabilize or destabilizeative lipases [32] and the immobilization on any support improveshe compatibility with less suited ILs. Nevertheless, the possi-le parameter variations are diverse; in a systematic IL screeningome physical solvent parameters can and need to be fixed. Theere used ILs were either room temperature ionic liquids (RTIL)r melted between 42.3 and 49.4 ◦C. Secondly also water mis-ible and immiscible ILs were distinguished. The water miscibleemim][MeSO3] [33] enabled maltose conversion of 36.8%, whichs equally efficient as well suited organic solvents, like acetone orMF. Similar results were reported with Novozym® 435 mediated

ransesterification using a methanesulfone ionic liquid [34]. It isoteworthy that the solvent polarity of [emim][MeSO3] EN

T = 0.65s much higher than for well suited organic solvents, EN

T = 0.2 − 0.5.onic liquids are not only greener than organic solvents, but in manyolar ILs enzymes remain active, which contrasts with highly polarrganic solvents where enzyme activity declines the higher theolarity [28]. The water immiscible fluorinated ILs with [PF6]- asnion [35] yielded low and only [bmpyr][PF6] converted maltoseomewhat better (25.1%). Anion variation using [EtSO4]-, [BF4]- andNTf2]- hardly improved maltose conversions (Fig. 2B). Even thoughll ionic liquids provide higher polarity, it is obvious that IL polarityoes not necessarily lead to higher conversion. The hydrophobicityxpressed as log P values would also be of interest here as seen withrganic solvents. But for ionic liquids log P values are not estab-ished to the same extend and experimental determinations areare [36]. log P calculations are possible but obtained values dependhen also on the calculation method used. The hydrophobicity in ILss a function of the anions and cations and often discussed for thenions. From the used anions [BF4]− is the least hydrophobic andncreases then in the series: [BF4]− < [PF6]− < [Tf2N]− [37]. The more

ater miscible ILs are the more likely to serve as enabling solvents

ecause maltose becomes potentially better dissolved and a moreroductive biotransformation should result. The good conversionsing the [MeSO3]− anion in combination with the not to hydropho-ic [emim]+ cation is in line with this idea that a certain water

lysis B: Enzymatic 90 (2013) 98– 106 103

miscibility is needed for a fair maltose and linoleic acid conversioninto 3a and 3b.

To improve reaction rates with ionic liquids, a minimal sol-vent approach was attempted too [20]. For this purpose the sameionic liquids were used as coatings on immobilized C. antarcticalipase B [24,38] and subjected to non-solvent conditions, Method C(Fig. 2C). Ionic liquid coated immobilized lipases outperformed ILsolvent conditions. This is consistent with results from monosac-charide use [24] but overall conversions into 3a and 3b remainedbelow the use of solvent quantities of [emim][MeSO3] and alsoacetone. The results show that a task enabling ionic liquid [39]was found for the synthesis of mono-6- or 6′-O-linoleyl-�-d-maltose 3a and 3b. Another approach to improve reaction ratesis binary solvent mixtures, whose results are discussed in thefollowing.

4.3. Biotransformation in binary solvents

Binary solvents improved maltose conversions and tended todouble the efficiency in comparison to mono solvent use (MethodD). This is expected with regard to the theory about solvent mix-tures [40]. The binary solvent approach is particularly appropriatefor hardly miscible substrates such as maltose and linoleic acid,whose physical properties differ considerably. To examine thebinary solvent effect more closely, well performing DMF, ace-tone, THF, acetonitrile, [emim][MeSO3] including [bmpyr][PF6]were combined as 1:1 mixtures (Method D). DMF/acetone enabledmaltose conversion of up to 72.1% (Fig. 1A). The best greensolvent variant acetone/[emim][MeSO3] yielded 63.6% and theacetone/[bmpyr][PF6] 60.9%. For the 1:1 ionic liquid mixture,[emim][MeSO3]/[bmpyr][PF6], improved maltose conversion up to60%. The good performance of ionic liquid mixtures is in line withthe enzymatic catalysis of 6-O-lauroyl-d-glucose in a binary IL sys-tem [41]. It has to be taken into account that the combination oftwo ionic liquids results in a quarternary solvent respectively asalt in salt mixture, [bmpyr][emim][MeSO3][PF6]. “Binary ionic liq-uids” are in this respect rather different from binary organic solventmixtures. In accordance with the standards for reporting biocat-alytic reactions the results shown for the binary mixtures werethe best obtained. In particular binary solvent mixtures yieldedless often consistently than single solvent use. The reasons are asoften unclear as multiple factors (solvent purity, mixing behav-ior, temperature and others) influence enzymatic reactions, whatis a widely observed and accepted fact in the field [23]. The bestyielding mixture, DMF/acetone, was therefore further examinedby gradually changing the solvent fractions what resulted in a bellshaped conversion curve with a maximum for the 1:1 (v/v) mixture(Fig. 3B). The work up from binary solutions demands more prepar-ative working skills than mono solvent use and consequently theeco-efficiency is comparable to IL use.

4.4. Non-solvent parameters

Non-solvent parameters are also decisive to ensure productivegreen biotransformations. This was shown for lipase choice, sub-strate ratio, molecular sieve content, and reaction time. A series of10 lipases were subjected, at the beginning of the investigationswith pure solvents, to acetone based reaction conditions (MethodA) (Fig. 1A). As most native lipases exercise their optimal activ-ity between 30 and 50 ◦C, the enzyme screening was conductedat 40 ◦C. The lowest conversion was provided by R. arrhizus with44.6% and the best activity was shown by native P. cepacia at 58%

and immobilized C. antarctica with 57.1%. Enzyme quantities alsoinfluenced substrate conversions (Fig. 1B). An excess of linoleic acidof four or six equivalents improved maltose conversions only up to10%. However, water removal with molecular sieves 3 A correlated

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ell with maltose conversions (Fig. 1C) and the more molecularieve used the faster the water extraction and consequently lessntermittent hydrolysis occurred. Finally, prolonging the processo 7 days improved maltose conversion up to 82% in acetone. Inonclusion solvent choice is a crucial parameter in mono-6- and′-O-linoleyl-�-d-maltose 3a and 3b synthesis. In addition, processime, enzyme choice, and water removal are influential non-solventarameters that potentially enhance the eco-efficiency of a lipaseediated acylation.

.5. Regioisomer analysis

The disaccharide fatty acid ester analysis is challenging as aixture of inseparable regioisomers is potentially obtained. NMR

pectroscopy is the method of choice in the structure elucidationf regioselectively acylated maltose 3a and 3b. Initially, analyti-al HPLC separated the isolated product mixture into two isomers,hich eluted at 7.77 and 8.14 min in a 54.8% to 45.2% ratio. An

lectro spray mass spectrum showed a single molecular mass peakt 604 m/z confirming the presence of two structural isomers. A3C–1H NMR correlation (HSQC) permitted to distinguish the malt-se hydroxyl protons from carbon linked protons as no correlationith carbon atoms is possible. The main difficulty in the disac-

haride fatty acid 3a and 3b analysis by 1H NMR is overlappingroton signals as recorded on a 400 MHz NMR spectrometer. Aigh field 800 MHz spectrometer substantially reduced this signalverlapping of the hydroxyl protons (Fig. 4). This spectrum showslso impressive ıH-shift differences for the two isomers 3a and 3b,hich originate from intra molecular hydrogen bonds [42]. The 1HMR spectrum confirmed the presence of two regioisomers visi-le as sets of two doublets beginning with OH-(C1) at ıH = 6.81 and.42 ppm. From the well resolved hydroxyl protons HO-C(1,2′,3) the

somer ratio was calculated as 58.2% for 6′-O-linoleyl-�-d-maltoseb and 41.8% for 6-O-linoleyl-�-d-maltose 3a (Fig. 4). This cor-esponds to the isomer ratio found with HPLC. The non-acylated

riplet hydroxyls HO-C(6,6′) at ıOH = 4.63 and 4.52 ppm also show

1.4:1 ratio, which is again in line with the isomer ratio found forhe secondary hydroxyl groups, HO-C(1,2′,3). Most importantly, ithows that no double acylation occurred.

ig. 4. 1H NMR (800 MHz, DMSO-d6) of hydroxyl protons at lower field of linoleyl-�-d-m3a) ratio.

lysis B: Enzymatic 90 (2013) 98– 106

To verify the regio-selective nature of the acylation on the O-6 and 6′ positions a long range 13C–1H NMR correlation (HMBC)experiment indicated that the carbonyl C(1′′) at 172.9 ppm cou-pled over three bonds (3J) to the methylene proton Ha-C(6,6′) atıH = 4.22 ppm (Fig. 5). This broad doublet is part of the ABX systemand the peaks at 3.98 ppm are the Hb-C(6,6′). A detailed assign-ment of all chemical shifts of the isomeric mixture with manyover lapping signals was in almost all cases realized. A prepara-tive separation or improved regioselectivity remains to be achievedto obtain pure isomers for a spectroscopic verification. All in all,two regioisomers were obtained where the primary O-6 hydroxylfunction close to the reducible anomeric carbon C(1) is less fre-quently esterified than the O-6′ position on the second glucosemonomer.

4.6. Which is the best solvent?

Solvent use should be simple even though solvent interactionwith substrates and enzyme are complex. Moreover, the solventshould fit to an economic and environmental context, when used atan industrial scale. The solvent choice is particularly decisive as twonon-miscible substrates and a biocatalyst had to be combined. Butalso residual water should not be stripped off from the lipase by thechosen solvent. Among the pure organic solvents acetone and DMFwere most efficient, but the latter is unstable and its high boilingpoint turns product isolation energy intensive. The application ofgreen pure ILs showed that water miscible [emim][MeSO3] wasbest suited, while non-miscible ILs exercised no solvent function.Water miscible ILs are at least as difficult to remove from reactionmixtures as high boiling organic solvents. The third option, binarysolvent mixtures, leaded to higher conversions but are equally notsuited for efficient workup as this is the case for ILs or high boilingorganic solvents.

In final conclusion, acetone turned out to be the best solvent

as it more or less complies with the requirements for a versatilegreen solvent. More precisely, it enabled the investigated biotrans-formation, is distilled from reaction mixtures with ease and can beproduced from biomass by fermentation.

altose 3a and 3b indicate selective O-6 and O-6′ acylation in a 58.2% (3b) to 41.8%

F. Fischer et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 98– 106 105

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. Conclusions

Solvent choice is an important parameter in lipase mediatedynthesis of mono-6- and 6′-O-linoleyl-�-d-maltose 3a and 3b.ono solvent reaction mixtures, based on acetone, DMF, THF, ace-

onitrile or the ionic liquid [emim][MeSO3], enabled fair maltoseonversions. Acetone and [emim][MeSO3] excelled as green sol-ents and are equally well suited as mineral oil based organicolvents. Binary green solvents, [emim][MeSO3]/[bmpyr][PF6] andhe acetone/[emim][MeSO3] mixture tended to double maltoseonversions in comparison to single solvent use. IL combinationsre of broad interest and considering the virtual ionic liquid library,imitless options are possible.

An evaluation of non-solvent reaction parameters indicates thatheir influence is potentially as decisive as the solvent choice. Aipase screening with 10 enzymes identified native P. cepacia andmmobilized C. antarctica as the best acylation catalysts. Furtheron-solvent factors such as molecular sieve content and reactionime (7 days) also improved maltose conversion up to 82% in ace-one using CALB (Novozym® 435).

The product mixture, mono-6- and 6′-O-linoleyl-�-d-maltosea and 3b, contained two regioisomers. In particular 1H,3C NMR, and 13C–1H correlation experiments showed thathe mono-acylation occurred mainly on 6′-O-linoleyl-�-d-

altose 3b (58.2%) and secondly on 6-O-linoleyl-�-d-maltose 3a41.8%).

From an industrial point of view, the best solvent is acetone.nlike ionic liquids and binary mixtures, it is easily removable

rom reaction mixtures. Moreover, acetone is to some extentreen as it can be produced from renewable feedstock. All inll, this study hopefully contributes also to the critical discussionn green solvent use for eco-efficient chemical and biochemicalngineering.

cknowledgements

This project was financially supported by DSM Nutritionalroducts, Basel, Switzerland. We also thank the DSM-Branch Pen-apharm in Aesch Switzerland for chemical donations.

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a regioisomer mixture with O-6 and O-6′ acylated 3a and 3b.

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