Enzymatic synthesis of epoxy fatty acid starch ester in ionic liquid-organic solvent mixture from vernonia oil

RESEARCH ARTICLE

Enzymatic synthesis of epoxy fatty acid starch ester in ionicliquid–organic solvent mixture from vernonia oil

Tegene Desalegn1, Ignacio J. Villar Garcia1,2, Jeremy Titman3, Peter Licence3, Isabel Diaz1,4 and Yonas Chebude1

1Chemistry Department, Addis Ababa University, Addis Ababa, Ethiopia2Department of Chemistry, Faculty of Natural Sciences, Imperial College, London, UK3School of Chemistry, University of Nottingham, Nottingham, UK4 Instituto de Catalisis y Petroleoquimica, CSIC, Madrid, Spain

In this study, epoxy fatty acid esters of cassava starch were synthesized by reacting cassava starchwith vernonia oil methyl ester (epoxy ester) using 1-butyl-3-methylimidazolium hexafluoro-phosphate, [C4C1Im][PF6] ionic liquid (IL) as a reaction medium and DMSO as co-solvent. LipaseCandida antarctica fraction B (Novozyme 435) was used as a catalyst for the esterification reaction.In the optimized reaction conditions, a degree of substitution (DS) of 0.95 was achieved, at areaction temperature of 40°C within 72 h of reaction time. The new cassava starch esters werecharacterized by infrared (FTIR), solid state NMR (CP/MAS 13C NMR), SEM, XRD,thermogravimetric analysis (TGA), and DSC. FTIR and NMR spectroscopy analyses confirmedthe successful esterification of starch and the DS was calculated to be 0.95 by titration methods.SEM and XRD studies showed that the morphology and crystallinity of native cassava starch weresignificantly changed upon esterification producing a continuous and amorphous material.Finally, the thermal behavior of the native and the starch vernolate was investigated using TGAand DSC techniques. The results revealed a change in the characteristic melting point anddecomposition pattern of the new polymer. The onset decomposition temperature of the newstarch vernolate is similar to the native form.

Received: June 13, 2013Revised: August 8, 2013

Accepted: August 15, 2013

Keywords:Cassava starch / Enzymes / Esterification / Ionic liquids / Vernolic acid

1 Introduction

The depletion of geologic hydrocarbon resources and theimpact of petroleum based products on the environmentare the major driving forces in searching for sustainablerenewable resources for both the provision of a vast inventoryof organic molecules and indeed energy generation/storagevectors. The use of renewable raw materials in the chemicalsindustry for non-fuel applications offers a tremendousopportunity to establish a more sustainable developmentprofile, enabling society to meet the needs of current, and

future generations whilst minimizing future environmentaldegradation [1]. Plant oils and naturally occurring triglycer-ides of fatty acids are a key source of renewable feedstocksfor the oleochemical industry [2]. Vernonia galamensis haspotential to become an important industrial oilseed crop.Researchers from Perdue University, USA identified thisplant in 1964 for the first time in Eastern Ethiopia [3]. Theseed of Vernonia galamensis is productive in terms oftriglyceride yield, typical oil yield varies from 35 to 40 wt%. Vernonia oil is uniquely a naturally epoxidized seed oilwith trivernolin contributing for about 60% of the trigly-cerides. The multiple chemical functionality of vernonia oilmakes it a unique candidate for derivatization in order tosynthesize high value-added products and synthetic inter-mediates [4]. Saponification of vernonia oil results in about72–80% of naturally epoxidized fatty acid, vernolic acid(cis-12,13-epoxy-cis-9-octadecenoic acid) [5]. This long-chainunsaturated fatty acid has a reduced viscosity compared toother synthetic epoxide containing oils and offers potential

Correspondence: Yonas Chebude, Addis Ababa University, P. O.Box 1176, Addis Ababa, EthiopiaE-mail: [email protected]: þ251-11239470

Abbreviations: DS, degree of substitution; IL, ionic liquid; VOME,vernonia oil methyl ester

DOI 10.1002/star.201300142Starch/Stärke 2014, 66, 385–392 385

� 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.starch-journal.com

application in the formulation of additives, coatings, andlubricant additives. The combination of its use as rawmaterial and its potential derivatization into value-addedproducts makes vernonia oil a unique renewable candidatefor industrial feedstock [6–9].

Starch is an abundant, relatively cheap, non-toxic, andrenewable natural resource that can be further modified[10, 11]. Native starches are readily available from diverseorigins includingmaize, potato, cassava, and rice. The commonareas where processed starches find wide application includethe food industry, pharmaceutical excipients and additives,textiles, paper, and packaging [11]. However, the highlyhydrophilic nature, and poor mechanical properties hashindered its wide application [12]. Esterification is one of thecommon routes of modification of starch to attain desiredproperties [13–15]. The main aims are to modify thisbiodegradable but crystalline and brittle material into newpolymeric materials that are not soluble in water and havemore tensile strength and enhanced functionalities. Differentchemical methods of syntheses of starch esters have beenwidely reported [14, 16, 17]. Starch fatty acid esters can also besynthesized by enzymatic methods. The enzymatic synthesisprovides many advantages, which includes chiral (stereo-selectivity), positional (regio-selectivity), and functional groupspecificity (chemo-selectivity). Such high selectivity is verydesirable in chemical synthesis as it offers several benefits suchas reduced or elimination of protecting groups,minimized sidereactions, easier separation, and fewer environmental prob-lems. Other advantages like high catalytic efficiency are alsovery attractive in commercial applications [18].

Ionic liquids (ILs) are materials, composed entirely of ionsthat are liquid below 100°C [19]. They offer a convenientmedia for starch reactions, since a subset of the myriad ofstructures offered in this class of compounds offer goodsolubility for carbohydrates, they are generally inert to addedreagents and exhibit interesting properties such as lowmelting points, wide liquid ranges, and low or negligiblevapor pressures. In particular, ILs bearing the imidazoliumstructure have been found to be non-derivatizing media,capable of dissolving polysaccharides including cellulose andstarch [20, 21]. ILs have also been used in the chemicalsynthesis of sugar esters [10, 11]. Moreover, many of themhave been found to stabilize enzymes [22] and consequently,ILs and mixtures of ILs and organic solvents have beensuccessfully used in the enzymatic synthesis of differentglucose esters [23–26].

The aim of this study was to synthesize a new starch estercontaining the characteristic vernonia oil functionalities. Thesynthetic procedure used for the esterification of starch withvernonia oil methyl ester (VOME), derived from vernoniaoil include the use of an IL, 1-butyl-3-methylimidazoliumhexafluorophosphate, [C4C1Im][PF6], and organic solvent,DMSO, mixture as reaction media and an immobilized lipaseCaLB as catalyst.

2 Materials and methods

2.1 Materials

Native cassava starch was procured from the local market(YASCII, Addis Ababa). The starch was dried for 24 h at 105°Cin vacuo to ensure that the concentration of residual waterwas below 2%, by weight, before use. Vernolic acid methylester (VOME) was prepared following literature procedures [12]using vernonia oil extracted from Vernonia galamensisseed, donated by Adet Agricultural Research Centre,Ethiopia. Immobilized CaLB was donated by Novozymes,Spain. 1-Butyl-3-methylimidazolium hexafluorophosphate,[C4C1Im][PF6] was prepared via literature procedures [27].AnalaR grade DMSO was purchased from Sigma–Aldrich.

2.2 Characterization techniques

FTIR analysis was performed on a Nicolet model Protégé 460magna IR spectrometer. The analysis of the cassava starchand starch vernolate was carried out in KBr pellets using thetransmission mode. SEMmicrographs were collected using aJEOL SEMModel LV 6400microscope operating at 15 kV. Thesamples were coated with platinum in vacuo before analysisin order to make the samples conductive. Powder XRDpatterns were acquired in a PANalytical X’Pert Pro withmonochromatic CuKa1 (l¼ 1.5406A

�, 40 kV, 40mA) radia-

tion, in the angular range of 2°–70° (2u). 13C solid-state CP-MAS NMR analyses were recorded on a Bruker 850MHzinstrument. TGA analyses were performed using a PerkinElmer TGA Q500 in a temperature range between 50 and500°C, with a heating rate of 10°C/min and nitrogen gas flowof 50mL/min. DSC analyses were performed using a DSCQ2000. The samples (about 10mg) were placed in sealedaluminum cups. After a first heating run from roomtemperature to 200°C to erase the thermal history of thematerial, each sample was cooled to 0°C and then heatedagain to 200°C (heating rate 10°C/min).

2.3 Experimental procedure

Dried cassava starch 1.0 g (6mmol) was dissolved in 20mL ofa [C4C1Im][PF6]/DMSO mixture (3:1 v/v) in a three neckedround bottom flask at 70°C and under vigorous stirring. Thehomogenous solution was allowed to cool down to 40°C andthen 5.74 g methyl vernolate (18mmol, 3:1 ester/starch unitsratio), 1.0 g Novozyme 435 (catalyst) and 2 g of activatedmolecular sieves were added and the mixture was stirred at40°C for 72 h. After cooling, the enzyme and the molecularsieves were filtered off and the product was precipitatedunder vigorous stirring using absolute ethanol (100mL) andseparated from the liquid phase by centrifugation. Theproduct was further washed three times with ethanol (30, 15,and 10mL, respectively). Finally, the product was dried in a

386 T. Desalegn et al. Starch/Stärke 2014, 66, 385–392


vacuum oven at 60°C for 24 h and obtained as a yellowishpowder.

2.4 The determination of the degree of substitution(DS)

The DS of a starch derivative is defined as the number ofhydroxyl (OH) groups substituted per D-glucopyranosylstructural unit of the starch polymer. Since each glucoseunit possesses three reactive hydroxyl groups, the maximumpossible DS value is 3. The DS varies with the source ofstarch, amylose and amylopectin fractions, stoichiometricamounts, and reaction time [28].

DS was determined using a method based upon thatdescribed by Garg and Jana [29]. Approximately 0.5 g of drystarch vernolate was weighed and added into a 50mL conicalflask. Then water (3mL) and 1.0M NaOH (5mL) were addedand the resulting mixture was stirred at room temperature(19–23°C) for 48 h. Phenolphthalein indicator solution wasadded to the flask and excess of alkali was titrated against0.5M hydrochloric acid. All starch samples, including anative reference sample were analyzed in duplicate by thismethod.

The vernoyl content (A%) was calculated according toEq. (1) where V0 (in mL) is the volume of 0.5M HCl used totitrate the blank, Vn (in mL) the volume of 0.5M HCl used totitrate the derivatized samples, CHCl the concentration of HCl(mol/L) used,Mvernoyl the MWof the vernoyl group (279), andm is the mass of dried starch vernolate sample investigated.

A% ¼ ½ðV0 � VnÞ � CHCl �Mvernoyl � 10�3 � 100�m

ð1Þ

The vernoyl content (A%) was used to calculate the DSaccording to Eq. (2):

DS ¼ Mglucose � A%

ðMvernoyl � 100Þ � ððMvernoyl � 1Þ � A%Þ ð2Þ

where Mglucose is the MW of glucose (162).

2.5 Enzyme activity analysis assay

Two reagents were prepared, Reagent A: 50mM trisbase (2-amino-2-hydroxymethyl-propane-1,3-diol or Tris)(MW¼ 121 g/mol, 0.0655 g) was prepared by dissolving solidtris base in water and the pH was maintained at 8 by addingHCl. Then 0.1 g gum Arabic and 0.4mL triton �-100 wereadded. Reagent B: was prepared by dissolving 0.03775 g ofparanitrophenyl palmitate (p-NPP) in 5mL iso-propanol at37–50°C. Two different 250mL flasks were prepared with19mL reagent A and 1mL reagent B each. To the first flask0.25 g of lipase enzyme (Novozyme 435) were added while thesecond flask was left in the absence of enzyme. The solutionswere stirred at 35°C and the absorbance (410 nm) of the firstsolution containing the enzyme was measured against the

second enzyme-free solution at different time intervals. Theactivity of the enzyme is calculated by measuring the initialslope of the concentration (p-NP extinction coefficient15.1M�1 cm�1) versus time function (0.007M/min). Theactivity of the same enzyme after stirring it in the reactionmedia for 3 h at 40°C (reaction conditions) was assessed thesame way (0.007M/min). Concentration versus time graphsare included in the supporting information.

3 Results and discussion

The esterification of cassava starch with VOME, derived fromnaturally epoxidized seed oil (vernonia oil), was carried outusing Lipase Candida antarctica fraction B (Novozyme 435)as catalyst (Fig. 1). A mixture of [C4C1im][PF6] and DMSO(20 wt%) was found as the perfect compromise betweenstarting materials solubility and enzyme activity. Thesolubility of the starch was found to be much higher inthemixture than for the neat IL (up to 30% by weight) and theenzyme was observed to have similar activity within errorafter being stirred in the reaction mixture for 3 h. The DS wascalculated to be close to unity (0.95) which indicates that theenzymatic reaction in this reaction system might be specificto one of the hydroxyl groups of the starch backbone.

3.1 Solubility studies

The starch vernolate product was subjected to differentsolubility studies. Table 1 shows that the starch vernolate isinsoluble in all tested solvents in which the starting materialsare soluble or partially soluble. The difference in solubility ofthe product of the reaction and the starting materialsindicates that a reaction has occurred and a new product, thestarch vernolate with a DS of 0.95, has been synthesized. Thisinsolubility also means that this new materials is water proofand also resistant to many different organic solvents.

3.2 Spectroscopic studies

The FTIR spectra of both the natural and esterified starchesare shown in Fig. 2. The IR spectrum of the esterified starchshows new bands at 1728, 1373, 1240, and 848 cm�1,characteristic of the vernolate group introduced duringthe esterification process [30]. A new absorption band at1728 cm�1 and characteristic of carbonyl (C––O) groupsappears in the starch ester spectra confirming the successfulesterification of the starch. The presence of unreactedhydroxyl group is indicated by the broad, characteristic bandin the range 3000–3600 cm�1, supports the calculated DS islower than 3. However, the relative intensity of the hydroxylvibration bands in comparison to the observed in the nativecassava starch has substantially decreased as expected due tothe partial esterification of the hydroxyl groups of the original

Starch/Stärke 2014, 66, 385–392 387


Figure 1. Reaction scheme for the synthesis of starch vernolate.

Table 1. Solubility of the new material synthesized (product) and the starting materials, methyl vernolate, and starch

Starch Methyl vernolate Product

Water þ � �Methanol � þ �Ethanol � þ �Chloroform � þ �Benzene � þ �Acetone � � �þ, soluble; �, partially soluble; and �, insoluble.

Figure 2. FTIR spectra of starch and starchvernolate.



starch. It can be seen in Fig. 2 that the relative intensity of theOH peak in comparison to the C–O stretching peak(�1050 cm�1) is higher in the starch spectrum than in thestarch vernolate spectrum, which suggests a higher C–OH/C–OR ratio in the starch than in the starch vernolate due tothe esterification of some of the C–OHgroups.We would alsolike to point out that this difference in ratio cannot be relatedto water content as the native cassava starch was dried toensure that the concentration of residual water was below 2%,by weight, before analysis (see Section 2). A shift in the peak’smaximum from 3381 cm�1 for native cassava starch to3434 cm�1 for esterified starch is also observed. However, thisshift could be caused by the partial saturation of the band inthe case of the native starch spectrum.

Figure 3 shows the solid state CP/MAS 13C NMRspectrum of starch vernolate. In this spectrum, the noticeablesignals in the region between 50 and 110 ppm are mostlyattributed to the different carbons of starch within the starchester product (see spectrum of pure starch in the inside box ofthe spectrum in Fig. 3). In addition, the three representativepeaks at 179.8, 129, and 13.87 ppm in the spectrum arecharacteristic of the carbonyl, alkene, and CH3 carbons in thefatty acid structure and further confirm the successfulesterification of the starch.

3.3 Structural studies

XRD measurements indicate a clear change in crystallinity.The XRD profile of cassava starch (Fig. 4) show a crystallinepattern, with distinguishable diffraction peaks at 15°, 18°, and23° [31]. After esterification, the diffraction peaks disappear

Figure 3. Solid State CP/MAS 13C NMR spectrum of starch and starch vernolate.

Figure 4. XRD patterns of native starch and starch vernolate.

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and the XRD pattern broadens, indicating the amorphouscharacter of the starch vernolate (Fig. 4). During esterifica-tion, some of the hydroxyl groups on starch backbone werereplaced by the vernoyl group, which reduced the formationof intermolecular hydrogen bonding and, thereby, damagedthe orderly crystalline structure of native cassava starch[20, 30, 32]. The SEMmicrographs show a marked differencein the morphology of the starch vernolate in comparison tothe native starch. The cassava starch granulate particles(Fig. 5A) are dramatically changed after esterification, leadingto a continuous and shapeless morphology (Fig. 5B) in thestarch vernolate. The change in morphology might have itsorigin during the dissolution processes under the conditionsdescribed here. It has been seen in the literature that themorphology of starch can change after dissolution, and inparticular in ILs [34]. During the dissolution process, thestarch granules can lose their granular morphology andchange into a more irregular and with an increased surfacearea shape less morphology, facilitating the chemical reagentaccess to the hydroxyl groups enhancing the reaction of starch[10, 11, 33].

3.4 Thermal studies

Native starch and starch vernolate were heated from roomtemperature up to 500°C to examine the changes in thermalstability caused by esterification and to determine the weightloss of the material on heating. The TGA curves areshown in Fig. 6. Native starch shows two main weight lossregions below 500°C. The first region below 120°C is due tothe loss of water with a total weight of about 12%. The secondtransition above 120°C gives a sharp decomposition stepcentered at 320°C due to the degradation of starch with a totalweight loss of about 68%. Examining the decompositionthermogram of starch vernolate in this particular case(dashed line), it can be seen that the shape of the thermogramchanges. The first weight loss is again below 120°C, due tothe loss of water (3%), but the sharp decomposition at highertemperature is now slower in the beginning, with again a

sharp decomposition centered around the same temperature(300°C) with a 55% weight loss, and an extra weight loss athigher temperature (370°C) corresponding to 15%, that isprobably due to the decomposition of the newly introducedester moiety [30].

DSC analysis shows a change in the phase transitionof the starch vernolate in comparison to the original starch.The native cassava starch shows only one endotherm in thetemperature range 25–180°C (Fig. 7A), while a shift in themelting point is clearly observed after the esterification(Fig. 7B). This decrease in transition temperature (Tg¼59.10°C, mp¼ 87.92°C) of starch vernolate compared withthat of native cassava starch (Tg¼ 107.08°C, mp¼ 139.98°C)suggests that the crystallinity of cassava starch is significantlyreduced after the esterification reaction and suggests ahigh degree of esterification. This agrees with the resultsreported in the literature [35, 36]. The introduction of vernoylgroup in the starch granules reduces the strength of theintermolecular bonds and increases the chain mobilityleading to the change of transition temperatures.

Figure 5. SEM micrographs of (A) native starch, and (B) starch vernolate.

Figure 6. Thermogram of starch (solid line) and starch vernolate(dashed line).



4 Conclusions

A vernolate starch ester has been successfully synthesized viathe enzymatic esterification of starch using a mixture oforganic solvent and IL as reaction medium and a Novozyme435 as the catalyst. The IL-based medium provides highsolubility for the starting materials and high activity of theenzyme. The new starch ester presents a DS close to unity(0.95) which suggests a high selectivity of the enzyme usedtoward one of the hydroxyl groups in the native starch. Thenew polymer is insoluble in water (and many other solvents)and retains the functionalities present in the methylvernolate ester such as the epoxyde and the double bond.The reactivity of these functional groups will allow a varietyof future work investigating the further functionalizationof the ester chain in order to tune the properties ofthe material. In particular, the epoxy groups offer a greatpotential for further cross-linking within the material. Inconclusion, the study demonstrates that the enzymatictransesterification of cassava starch with epoxy FAME is avaluable approach for the development of biodegradablematerials that will offer an environmentally benign alterna-tive to petroleum based materials and will widen the scopeof application of vernonia oil and its derivatives in thesynthesis of value added products.

The authors thank the School of Chemistry, University ofNottingham, UK for the provision of all instrumentation for thiswork and for partial funding of TD. We are most grateful to Dr.Jeremy Titman for the 13C solid-state CP-MAS NMR analysesand helpful discussions. IJVG, YC, and ID acknowledge theSpanish Research Council (CSIC) for funds through theprogramme CSIC for development, project number i-COOP014.The Chemistry Department, Addis Ababa University is alsoacknowledged for financial support.

The authors have declared no conflict of interest.

5 References

[1] Eissen,M.,Metzger, J.O., Schmidt, E., Schneidewind, U., 10Years after rio-concepts on the contribution of chemistry to asustainable development. Angew. Chem., Int. Ed. 2002, 41,414–436.

[2] Meier, M. A. R., Metzger, J. O., Schubert, U. S., Plant oilrenewable resources as green alternatives in polymer science.Chem. Soc. Rev. 2007, 36, 1788–1802.

[3] Perdue, R. E., Carlson, K. D., Gilbert, M. G., Vernoniagalamensis, potential new crop source of epoxy fatty acid.Econ. Bot. 1986, 40, 54–68.

[4] Grinberg, S., Kolot, V., Mills, D., New chemical derivativesbased on Vernonia galamensis oil. Indus. Crop. Prod. 1994,3, 113–119.

[5] Carlson, K. D., Schneider, W. J., Chang, S. P., Princen, L. H.,in: Pryde, E. H., Princen, L. H., Mukherjee, F. (Eds.), NewSources of Fats and Oils, AOCS Monograph 9, American OilChemists Society, Champaign, IL, USA 1981, pp. 297–318.

[6] Ayorinde, F. O., Clifton, J., Afolabi, O. A., Shepard, R. L.,Rapid transesterification andmass spectrometric approach toseed oil analysis. J. Am. Oil Chem. Soc. 1988, 65, 942–947.

[7] Trumbo, D. L., Rudelich, J. C.,Mote, B. E., in: Janick, J. (Ed.),Perspectives on New Crops and New Uses, ASHS Press,Alexandria, VA 1999, pp. 266–271.

[8] Thompson, A. E., Dierig, D. A., Kleiman, R., Characterizationof Vernonia galamensis germplasm for seed oil content, fattyacid composition, seed weight, and chromosome number.Indus. Crops Prod. 1994, 2, 299–305.

[9] Ayorinde, F. O., Butler, B. D., Clayton, M. T., Vernoniagalamensis: A rich source of epoxy acid. J. Am. Oil Chem.Soc. 1990, 67, 844–845.

[10] Xie, W., Shao, L., Lui, Y., Synthesis of starch esters in ionicliquids. J. Appl. Polym. Sci. 2010, 116, 218–224.

[11] Xie, W., Wang, Y., Synthesis of high fatty acid starch esterswith 1-butyl-3-methylimidazolium chloride as a reactionmedium. Starch/Stärke 2011, 63, 190–197.

[12] Singh, A. V., Nath, L. K., Guha, M., Synthesis andcharacterization of highly acetylated sago starch. Starch/Stärke 2011, 63, 523–527.

[13] Elhilo, E. B., Melissa, A. A., Ayorinde, F. O., Synthesis of cis-12,13-epoxy-cis-9-octadecenol and 12(13)-hydroxy-cis-9-octadecenol from Vernonia oil using lithium aluminumhydride. J. Am. Oil Chem. Soc. 2000, 77, 873–878.

[14] Junistia, L., Sugih, A. K., Manurung, R., Picchioni, F. et al.,Synthesis of higher fatty acid starch esters using vinyl

Figure 7. DSC curves of (A) cassava starch, and (B) starchvernolate.

Starch/Stärke 2014, 66, 385–392 391


laurate and vinyl stearate. Starch/Stärke 2008, 60, 667–675.

[15] Sagar, A. D., Merrill, E. W., Properties of fatty-acid ester ofstarch. J. Appl. Polym. Sci. 1995, 58, 1647–1656.

[16] Aburto, J., Alric, I., Borredon, E., Preparation of long-chainesters of starch using fatty acid chlorides in the absence of anorganic solvent. Starch/Stärke 1999, 51, 132–135.

[17] Aburto, J., Alric, I., Thiebaud, S., Borredon, E. et al.,Synthesis, characterization, and biodegradability of fatty acidesters of amylose and starch. J. Appl. Polym. Sci. 1999, 74,1440–1451.

[18] Chulalaksananukul, W., Condoret, J. S., Delorme, P.,Willemot, R.M., Kinetic study of esterification by immobilizedlipase in n-hexane. Fed. Eur. Biochem. Soc. Lett. 1990, 276,181–184.

[19] De Diego, T., Lozano, P., Abad, M. A., Steffensky, K. et al.,On the nature of ionic liquids and their effects on lipases thatcatalyze ester synthesis. J. Biotechnol. 2009, 140, 234–241.

[20] Biswas, A., Shogren, R. L., Stevenson, D. G., Willett, J. L.,Bhowmik, P. K., Ionic liquids as solvents for biopolymers:Acylation of starch and zein protein. Carbohydr. Polym.2006, 66, 546–550.

[21] Seoud, E. O. A., Koschella, A., Fidale, L. C., Dorn, S., Heinze,T., Applications of ionic liquids in carbohydrate chemistry: Awindow of opportunities. Biomacromolecules 2007, 8,2629–2647.

[22] Zhao, H., Methods for stabilizing and activating enzymes inionic liquids-a review. J. Chem. Technol. Biotechnol. 2010,85, 891–907.

[23] Ganske, F., Bornscheuer, U. T., Optimization of lipase-catalyzed glucose fatty acid ester synthesis in a two-phasesystem containing ionic liquids and t-BuOH. J. Mol. Catal. B:Enzymatic 2005, 36, 40–42.

[24] Ganske, F., Bornscheuer, U. T., Lipase-catalyzed glucosefatty acid ester synthesis in ionic liquids. Org. Lett. 2005, 7,3097–3098.

[25] Ha, S., Hiep, N., Lee, S., Koo, Y.-M., Optimization of lipase-catalyzed glucose ester synthesis in ionic liquids. BioprocessBiosyst. Eng. 2010, 33, 63–70.

[26] Lee, S. H., Ha, S. H., Hiep, N. M., Chang, W.-J., Koo, Y.-M.,Lipase-catalyzed synthesis of glucose fatty acid ester usingionic liquids mixtures. J. Biotechnol. 2008, 133, 486–489.

[27] Huddleston, J. G., Visser, A. E., Reichert, W. M.,Willauer, H.D. et al., Characterization and comparison of hydrophilic andhydrophobic room temperature ionic liquids incorporating theimidazolium cation. Green Chem. 2001, 3, 156–164.

[28] Mathew, S., Abraham, T. E., Physico-chemical characteriza-tion of starch ferulates of different degrees of substitution.Food Chem. 2007, 105, 579–589.

[29] Garg, S., Jana, A. K., Characterization and evaluation ofacylated starch with different acyl groups and degreesof substitution. Carbohydr. Polym. 2011, 83, 1623–1630.

[30] Zhang, L., Xie, W., Zhao, X., Liu, Y., Gao, W., Study onmorphology crystalline structure and thermal properties ofyellow ginger starch acetates with different degrees ofsubstitution. Thermochim. Acta 2009, 495, 57–62.

[31] Defloor, I., Dehing, I., Delcour, J. A., Physico-chemicalproperties of cassava starch.Starch/Stärke1998,50, 58–64.

[32] Garcia, V., Colonna, P., Bouchet, B., Gallant, D. J., Structuralchanges of cassava starch granules after heating atintermediate water contents. Starch/Stärke 1997, 49,171–179.

[33] Paulos, G., Endale, A., Bultosa, G., Gebre-Mariam, T.,Isolation and physicochemical characterization of cassavastarches obtained from different regions of Ethiopia. Ethiop.Pharm. J. 2009, 27, 42–54.

[34] Stevenson, D. G., Biswas, A., Jane, J-l., Inglett, G. E.,Changes in structure and properties of starch of fourbotanical sources dispersed in the ionic liquid, 1-butyl-3-methylimidazolium chloride. Carbohydr. Polym. 2007, 67,21–31.

[35] Shogren, R. L., Preparation, thermal properties, and extrusionof high-amylose starch acetates. Carbohydr. Polym. 1996,29, 57–62.

[36] Horchani, H., Chaabouni, M., Gargouri, Y., Sayari, A.,Solvent free lipase-catalyzed synthesis of long-chainesters using microwave heating: Optimization by responsesurface methodology. Carbohydr. Polym. 2010, 79, 466–474.



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Enzymatic synthesis of epoxy fatty acid starch ester in ionic liquid-organic solvent mixture from vernonia oil