Optimized enzymatic synthesis of levulinate ester in solvent-free system
Transcript of Optimized enzymatic synthesis of levulinate ester in solvent-free system
O
Aa
b
a
ARRA
KLEESR
1
fk2awlgucaewAwa(as2
e
(
0d
Industrial Crops and Products 32 (2010) 246–251
Contents lists available at ScienceDirect
Industrial Crops and Products
journa l homepage: www.e lsev ier .com/ locate / indcrop
ptimized enzymatic synthesis of levulinate ester in solvent-free system
lice Leea,1, Naz Chaibakhsha,1, Mohd Basyaruddin Abdul Rahmana,b, Mahiran Basri a, Bimo A. Tejoa,∗
Department of Chemistry, Faculty of Science, 43400 UPM Serdang, Universiti Putra Malaysia, Selangor Darul Ehsan, MalaysiaStructural Biology Research Center, Malaysia Genome Institute, MTDC-UKM, Smart Technology Centre, 43600 Bangi, Selangor, Malaysia
r t i c l e i n f o
rticle history:eceived 28 January 2010eceived in revised form 28 April 2010ccepted 28 April 2010
a b s t r a c t
Ethyl levulinate, produced through esterification of levulinic acid, is a ketoester with various applications.Synthesis of ethyl levulinate was carried out in solvent-free system using immobilized Candida antarcticalipase B (Novozym 435) as the biocatalyst for the reaction. Response surface methodology (RSM) witha four-factor-five-level central composite rotatable design (CCRD) was employed to study and optimize
eywords:evulinic acidnzymatic synthesissterification
the reaction conditions in the synthesis of levulinate ester. The effect of four main reaction parametersincluding time, temperature, ethanol/levulinic acid molar ratio and amount of enzyme on the synthesisof ester were analyzed. A quadratic polynomial model was fitted to the data with an R2 of 0.8993. Modelvalidation experiments show good correspondence between actual and predicted values. A high conver-sion yield (96.2%) was obtained at the optimum conditions of 51.4 ◦C, 41.9 min, 292.3 mg enzyme amount
lar ra
olvent-free systemesponse surface methodologyand 1.1:1 alcohol:acid mo
. Introduction
Levulinic acid, also known as gamma-ketovaleric acid, is a plat-orm chemical with numerous potential applications by having aetone and a carboxylic group (Fang and Hanna, 2002; Bozell et al.,000). It is commercially produced from renewable biomass suchs cane sugar, starch and lignocellulosic materials from agriculturalastes. Ethyl levulinate is an industrially important derivative of
evulinic acid, made by esterifying its carboxylic group with fuel-rade ethanol (Wetzel et al., 2006). The esterification reaction issually carried out at high temperature in the presence of an acidatalyst such as sulphuric, polyphosphoric or p-toluenesulfoniccids. Ethyl levulinate has an oxygen content of 33% and prop-rties similar to the biodiesel fatty acid methyl esters (FAME),hich make it suitable to be used as an oxygenate diesel additive.dding ethyl levulinate to the diesel results in a cleaner burning fuelith high lubricity, flashpoint stability, reduced sulphur content
nd improved viscosity that can be used in regular diesel enginesHayes, 2009). Ethyl levulinate has also been applied in the flavoringnd fragrance industries. It is a substrate for a variety of conden-
ation and addition reactions at the ester and keto groups (Olson,001).Application of enzymes to the synthesis of esters has increasedxtensively in recent years. Although synthesis of esters using
∗ Corresponding author. Tel.: +60 3 89467488; fax: +60 3 89435380.E-mail addresses: [email protected], [email protected]
B.A. Tejo).1 These authors contributed equally to this work.
926-6690/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.indcrop.2010.04.022
tio.© 2010 Elsevier B.V. All rights reserved.
acid catalysts may result in a high yield (Bartoli et al., 2007),enzymatic synthesis offers several advantages over conventionalchemical esterification such as mild reaction conditions, low energyrequirements, minimal waste disposal, ease of product isolation,and biocatalyst reusability (Petersson et al., 2005). Enzymatic syn-thesis of levulinate ester has been reported previously by Yadavand Borkar (2008). Their work focused on the kinetics and mecha-nism study of lipase-catalyzed esterification of levulinic acid withn-butanol using tetrabutyl methyl ether as the solvent. Synthe-sis of levulinylated nucleosides in organic solvent-based systemvia regioselective enzymatic hydrolysis has also been reported byseveral researchers (Garcia et al., 2002; Lavandera et al., 2005).However, so far there is no report on enzymatic esterification oflevulinic acid and monohydric alcohols in a solvent-free system.Furthermore, neither specific study on the interactive effect of reac-tion parameters nor any optimal conditions have been reported forthese esters.
Although organic solvents provide several advantages in enzy-matic reactions, their use in industrial processes is not desirable.They are the source of volatile organic compounds (VOCs) that canaffect the environment and human health, their use requires costlypost-treatment actions, larger and more expensive reactors andauxiliary equipments, and they also have some inhibition effectson the enzyme (Tufvesson et al., 2007). Performing the reactionsunder solvent-free conditions can help to overcome these draw-
backs. Furthermore, higher selectivity and volumetric productivity,improved substrate and product concentrations and fewer purifica-tion steps are some other advantages of using solvent-free system(Otero et al., 2001). In this study, synthesis of levulinate ester hasbeen performed in a solvent-free system.and Products 32 (2010) 246–251 247
f(n(a(
tsba(l
2
2
fCpam
2
bNoao
Table 1Range of variables and their levels for the CCRD.
Variable Levels
−2 −1 0 +1 +2
Temperature, A (◦C) 25.0 37.5 50.00 62.5 75.0Time, B (min) 30.0 82.5 135.00 187.5 240.0
TC
A. Lee et al. / Industrial Crops
In order to optimize the conversion of ester, response sur-ace methodology (RSM) with central composite rotatable designCCRD) was applied. RSM is a fast and economical statistical tech-ique useful for developing, improving and optimizing processesMyers et al., 2009). It has been successfully applied for the studynd optimization of the enzymatic synthesis of various estersJeong et al., 2009; Keng et al., 2005).
The aim of the present work is to investigate the possibility ofhe lipase-catalyzed synthesis of levulinate ester in a solvent-freeystem. This study also helps to better understand relationshipsetween the main reaction parameters (temperature, time, enzymemount, and substrate molar ratio (alcohol:acid)) and the responseconversion yield) and to determine the optimal conditions of ethylevulinate synthesis using CCRD and RSM analyses.
. Materials and methods
.1. Materials
Novozym® 435 (specific activity of 10,000 PLU/g) was purchasedrom NOVO Nordisk A/S (Bagsvaerd, Denmark) and consists ofandida antarctica lipase B (triacylglycerol hydrolase, EC 3.1.1.3)hysically adsorbed within the macroporous acrylic resin. Levuliniccid and ethanol were purchased from Merck Co. (Darmstadt, Ger-any). All other chemicals used were of analytical grade.
.2. Lipase-catalyzed esterification reaction
Different molar ratios of ethanol and levulinic acid, generated
y CCRD, were mixed in 30 mL closed vials. Different amounts ofovozym 435 were subsequently added. The reaction was carriedut in a horizontal water bath at 150 rpm at different temperaturesnd for different time periods, as presented in Table 1. The selectionf Novozym 435 as catalyst for the reaction was based on the pre-able 2omposition of various experiments of the CCRD for the synthesis of ethyl levulinate.
Experiment no.
Temperature (◦C) Time (min) Substra
1 50.0 135.0 3.02 37.5 82.5 2.03 62.5 82.5 2.04 37.5 187.5 2.05 62.5 187.5 2.06 37.5 82.5 4.07 62.5 82.5 4.08 37.5 187.5 4.09 62.5 187.5 4.0
10 50.0 135.0 1.011 50.0 30.0 3.012 25.0 135.0 3.013 50.0 135.0 3.014 50.0 135.0 3.015 50.0 135.0 3.016 50.0 135.0 3.017 50.0 135.0 3.018 50.0 135.0 3.019 75.0 135.0 3.020 55.0 255.0 5.5021 50.0 240.0 3.022 50.0 135.0 5.023 37.5 82.5 2.024 62.5 82.5 2.025 37.5 187.5 2.026 62.5 82.5 2.027 37.5 82.5 4.028 62.5 82.5 4.029 37.5 187.5 4.030 62.5 187.5 4.031 62.5 187.5 2.0
Substrate molar ratio, C 1.0 2.0 3.0 4.0 5.0Enzyme amount, D (mg) 20.0 115.0 210.0 305.0 400.0
vious study (Yadav and Borkar, 2008) in which several commerciallipases including Novozym 435, Lipozyme RM IM and Lipozyme TLIM were screened for activity via lipase-catalyzed synthesis of lev-ulinate ester. Novozym 435 was found to be the best with maximuminitial rate and conversion.
2.3. Analysis and characterization
The reaction was terminated by adding ethanol:acetone (50:50,v/v) and the immobilized enzyme was filtered. The remainingunreacted acid was measured by titration with 0.1 M NaOH to anend point pH 8.25 using phenolphthalein indicator. The moles ofreacted acid were calculated from the values obtained for the con-trol (without enzyme) and the test samples. The ester producedwas expressed as equivalent to the acid conversion (Abdul Rahmanet al., 2008; Radzi et al., 2005). Product was also identified by thin-layer chromatography (TLC) using chloroform as the solvent andgas chromatography/mass spectroscopy (GC/MS) using a Shimadzu
(model GC 14B; model MS QP5050A; Shimadzu Corp., Tokyo, Japan)instrument equipped with a FID and a semi polar column BP-10(0.33 mm × 50 mm, 0.25 �m). The carrier gas was nitrogen and thetotal gas flow rate was 50 mL min−1. The injector and the detectortemperatures were set at 280 and 310 ◦C, respectively. The ovenVariables
te molar ratio Enzyme amount (mg) Actual conversion (%)
20.0 29.1115.0 52.4115.0 45.2115.0 52.1115.0 47.8115.0 49.5115.0 36.1115.0 56.1115.0 43.2210.0 82.9210.0 60.1210.0 65.4210.0 50.5210.0 52.5210.0 56.3210.0 59.7210.0 57.4210.0 59.7210.0 66.9300 94.1210.0 53.2210.0 50.5305.0 93.0305.0 74.7305.0 86.6305.0 74.7305.0 61.8305.0 57.1305.0 84.7305.0 57.5305.0 74.4
248 A. Lee et al. / Industrial Crops and Products 32 (2010) 246–251
Table 3Analysis of variance (ANOVA).
Source Sum of squares Degree of freedom Mean square F-Value P-Value
Model 6631.71 9 736.86 19.84 <0.0001Temperature, A 393.17 1 393.17 10.59 0.0040Reaction time, B 14.88 1 14.88 0.40 0.5339Substrate molar ratio, C 876.28 1 876.28 23.59 <0.0001Enzyme amount, D 4778.77 1 4778.77 128.66 <0.0001A2 122.67 1 122.67 3.30 0.0842C2 138.67 1 138.67 3.73 0.0676AD 37.70 1 37.70 1.01 0.3258BC 106.71 1 106.71 2.87 0.1056CD 188.93 1 188.93 5.09 0.0355
tr
2
(vf2b
N
wnfpgte(revdi
Itm
y
watasUaivtef
The base peak of the fragmentation of the ester is related to CH3CO(m/z = 43). The other two important ion peaks are due to the for-mation of ion acylium, RCO+ that gives the fragment ions at m/z 99(because of the loss of alkoxy group from the ester, R-O) and m/z129 because of the loss of methyl group from the ester. Other bonds
Residual 742.88 20Lack of fit 670.63 15Pure error 72.25 5Corrected total 7374.59 29
emperature was kept at 100 ◦C for 3 min, increased to 300 ◦C at aate of 10 ◦C min−1 and held for 7 min.
.4. Design of experiments, statistical analysis and optimization
A four-factor-five-level central composite rotatable designCCRD) was applied in this study. Rotatability indicates that theariation in the predicted response is constant at a given distancerom the center point of the design (Anderson and Whitcomb,005). The total number of required experiments was 30 obtainedy the following equation:
= 2k + 2k + n0 (1)
here k is the number of independent variables and n0 is theumber of replicated center points (Mohammad et al., 2006). The
ractional factorial design comprised of 16 factorial points, 8 axialoints and 6 center points. Center point is repeated six times toive a good estimate of the experimental error. The parameters andheir corresponding ranges selected for the synthesis of levulinatester in solvent-free system were: temperature (25–75 ◦C); time30–240 min); enzyme amount (20–400 mg) and substrate molaratio of ethanol to levulinic acid (1:1–5:1). High and low levels ofach variable were coded as 2 and −2, respectively, and the meanalue was coded as zero (Table 1). The employed experimentalesign is presented in Table 2. All the experiments were performed
n triplicate.A software package of Design Expert Version 6.0.6 (State-Ease
nc., Statistics Made Easy, Minneapolis, MN, USA) was used to fithe data obtained for the response to a second-order polynomial
odel using the following equation:
= b0 +4∑
i=1
bixi +4∑
i=1
biix2i +
3∑
i=j
4∑
j=i+1
bijxij + e (2)
here y is the dependent variable (percentage of conversion), xind xj are the independent variables (factors), b0, bi, bii and bij arehe regression coefficients of model and e is the error of model. Annalysis of variance (ANOVA) and R2 (coefficient of determination)tatistic were used to check the adequacy of the developed model.sing an F-test, it was possible to test the variation of the dataround the fitted model (lack of fit). The significance of each factor
n the model was estimated by testing the null hypothesis. Small P-alue results in rejection of the null hypothesis, which means thathe factor is significant. The optimal conditions for the synthesis ofster were obtained using the software’s numerical optimizationunction.37.1444.71 3.09 0.108814.45
3. Results and discussion
3.1. Identification of ester product
GC–MS analysis of the reaction mixture shows the presenceof ethyl levulinate at a retention time of 10.098 min. The massspectrum of the product exhibits molecular ion at m/z 144 thatcorresponds to molecular formula of ethyl levulinate (C7H12O3).
Fig. 1. Plots showing correlation of actual conversions and values predicted by themodel (a) and normal probability of residuals (b).
A. Lee et al. / Industrial Crops and Products 32 (2010) 246–251 249
F vulinO
ca
3
qsiphecoe
C
wr
podtatEoiaansb
ig. 2. Effect of various individual parameters, temperature (a), time (b), ethanol:lene parameter is varied whilst the others are kept constant at their center points.
leavages occur through some pathways and gave fragments ionst m/z 41, 56, 74, 84 and 116.
.2. Model fitting and analysis of variance (ANOVA)
Fitting of the data to various models (linear, two factorial,uadratic and cubic) and their subsequent analysis of variancehows that the esterification reaction of levulinic acid and ethanoln solvent-free system is most properly described with a quadraticolynomial model. The R2 of the quadratic model (0.8993) wasigher than that of linear (0.8222) and two factorial (0.8710) mod-ls. The cubic model was found to be aliased, since the centralomposite matrix provided too few design points to determine allf the terms in the cubic model (Decaestecker et al., 2004). The finalquation of the model (based on the coded values) is as follows:
onversion (%) = +56.94 − 4.05A + 0.79B − 6.04C + 14.11D
+2.08A2 + 2.21C2 − 1.54AD + 2.58BC − 3.44CD
(3)
here A is the temperature, B is the time, C is the substrate molaratio and D is the enzyme amount.
The ANOVA for the model is presented in Table 3. The com-uted F-value of the model (19.84) is higher than the tabular valuef F9,20 (=2.39), implying the model is significant at the 95% confi-ence level. A very small P-value (<0.0001) and a suitable R2 showshat the model can satisfactorily represent the real relationshipmong the reaction parameters (Gunawan et al., 2005). Fig. 1 showshe experimental versus predicted conversions obtained from thequation (3). A linear distribution is observed which is indicativef a well-fitting model. Normal probability plot is also presentedn Fig. 1. The plot indicates that the residuals (difference between
ctual and predicted values) follow a normal distribution and formn approximately straight line. Adequate precision shows signal tooise ratio. Ratios greater than 4 are suitable. The adequate preci-ion of the developed model is 23.231 indicating that the model cane used to navigate the design space. The lack of fit F-value of 3.09ic acid molar ratio (c) and enzyme amount (d), on the synthesis of ethyl levulinate.
is lower than the tabular F15,5-value (4.62), implying that there isno lack of fit in the model at 95% level of significance. The coeffi-cients of the response surface model are also presented in Table 3.A P-value less than 0.05 indicates that the model term is signifi-cant. In this case A, C, D, and CD are significant terms. Equation (3)was used then to study the effect of various parameters and theirinteractions on the conversion of ester.
3.3. Effect of reaction parameters
The effect of the four independent variables on the synthesis oflevulinate ester is shown in Fig. 2. The percentage of conversiongradually decreases from 63.0 to 55.0% by increasing temperatureat the center point of other variables (Fig. 2(a)). Increasing tem-perature causes increase in the acid solubility and dissociation anddecrease in the binding equilibrium, leading to unfavorable ester-ification conditions (Hari Krishna et al., 2001). According to theanalysis of variance, time is not a significant parameter that caninfluence the conversion. This also can be observed in Fig. 2(b) inwhich increasing time from 30 to 240 min causes a small increase inthe conversion from 56.1 to 57.7% due to equilibrium of the esteri-fication reaction. In solvent-free systems, one substrate is generallyused in a large excess over another in order to act as a solvent forother reactants (Yamane, 2001). Yadav and Borkar (2008) foundthat by increasing the mole ratio of n-butanol from 1 to 3, the con-version and initial rates were increased in solvent-based synthesisof levulinate ester. In this study, the mole ratio of ethanol wasincreased from 1 to 5. However, the highest conversion of esterwas obtained at alcohol:acid molar ratio of 1:1 (Fig. 2(c)). Accord-ing to Carta et al. (1992), ethanol inhibits the catalytic function oflipase at even low concentrations. An irreversible denaturation ofthe enzyme also occurs at high concentrations of ethanol.
As the amount of enzyme is increased, the ester production isalso increased (Fig. 2(d)). The presence of higher amount of enzymeprovides more active sites for the acyl–enzyme complex formationand also increases the probability of enzyme–substrate collisionand subsequent reaction (Soo et al., 2004).
250 A. Lee et al. / Industrial Crops and Products 32 (2010) 246–251
FtOp
lsimia
tTemteta8st
2
TO
�
ig. 3. Response surface (a) and contour (b) plots showing the interaction betweenwo parameters, enzyme amount and temperature, in synthesis of ethyl levulinate.ther variables are constant at their center points. The numbers inside the contourlots indicate conversion yield (%) of the ester.
The interaction effects of two parameters on the synthesis ofevulinate ester were examined by three-dimensional responseurface plots (Figs. 3–5). Contour plots are also very helpful fornterpreting the main effects of reaction parameters and their
utual interactions (Myers et al., 2009). Contour plots represent-ng the effect of varying parameters on the synthesis of the esterre shown in Figs. 3–5.
Fig. 3 depicts the effect of temperature and enzyme amount onhe ester synthesis at 135 min and alcohol:acid molar ratio of 3:1.he effect of temperature is more significant at higher amounts ofnzyme. Maximum conversion of the ester can be obtained usingore enzyme quantity at lower temperatures. Fig. 4 represents
he effect of time and substrate molar ratio on the synthesis ofthyl levulinate. Temperature and enzyme amount were fixed at
heir center points. The reaction with low incubation time (30 min)nd low substrate molar ratio (1:1) gives the highest conversion of6.1%. In alcohol:acid molar ratio of 2.7:1, the percentage conver-ion is constant at 58.9% during 4 h of the reaction, indicating thathe equilibrium condition is achieved. The effect of varying alco-able 4ptimum conditions for lipase-catalyzed synthesis of ethyl levulinate in solvent-free syst
Experiment Temperature (◦C) Time (min) Enzyme amount (mg) S
1 50.0 65.6 386.4 22 50.0 71.8 250.0 13 52.0 125.0 392.8 24 45.0 100.0 370.0 25 51.4 41.9 292.3 1
2 =∑
(ya−yp)2
yp= 0.56.
Fig. 4. Response surface (a) and contour (b) plots showing the interaction betweentwo parameters, time and substrate molar ratio, in synthesis of ethyl levulinate.Other variables are constant at their center points. The numbers inside the contourplots indicate conversion yield (%) of the ester.
hol:acid molar ratio and enzyme amount and at 50 ◦C and 135 min isshown in Fig. 5. As the amount of enzyme increased, the conversionalso increased at each molar ratio. According to the analysis of vari-ance, the interaction of enzyme amount and substrate molar ratiois a significant term (P-value = 0.0355). Using substrate molar ratio1:1 to 2:1 and enzyme amount 311–400 mg results in a predictedconversion of 100%.
3.4. Model validation and optimum conditions
The �2 goodness-of-fit test was used to examine the validityof the model (Table 4) (Mooney and Swift, 1999). The test showsthat there is not a significant difference between the predicted andactual values since the �2 value (0.56) is much smaller than the
cut-off value of � .05 for 4 degrees of freedom (9.49). This indicatesthat the generated model is valid at 95% confidence level.Table 4 presents the optimal combination of parameters that canbe used to obtain high percentage of conversions. The optimumconditions can be used for future upscale synthesis of the ester
em.
ubstrate molar ratio Predicted conversion (%) (yp) Actual conversion(%) (ya)
.0 100.0 95.2
.1 90.0 89.5
.5 90.0 91.5
.5 90.5 94.3
.1 100.0 96.2
A. Lee et al. / Industrial Crops and P
Ftlt
(tpaea8
4
epfrmsiwm
R
A
New Partnerships in the Bioeconomy, 1st edn. Springer, the Netherlands.Yadav, G.D., Borkar, I.V., 2008. Kinetic modeling of immobilized lipase catalysis in
ig. 5. Response surface (a) and contour (b) plots showing the interaction betweenwo parameters, enzyme amount and substrate molar ratio, in synthesis of ethylevulinate. Other variables are constant at their center points. The numbers insidehe contour plots indicate conversion yield (%) of the ester.
Ismail et al., 1999). A short time (41.9 min) is required to attainhe maximum conversion of ester (96.2%). From economic stand-oint, it would be favorable to use the minimum time and enzymemount to attain maximum conversion. Maximum conversion ofster (90.0%) is predicted using 50 mg enzyme at 50.0 ◦C, 71.8 minnd substrate molar ratio 1.1:1. The actual conversion obtained is9.5 with 0.5% deviation.
. Conclusion
Immobilized Candida antarctica lipase B-catalyzed synthesis ofthyl levulinate in an organic solvent-free system is successfullyerformed. Central composite rotatable design and response sur-ace methodology are effectively applied to the optimization of theeaction parameters. Temperature, enzyme amount and substrateolar ratio are the significant process variables that affected the
ynthesis of levulinate ester. A high percentage conversion (96.0%)s achieved in a short reaction time (41.9 min) which matches well
ith the predicted value of 96.1%. The developed model and opti-um conditions can be used for future process scale-up.
eferences
bdul Rahman, M.B., Chaibakhsh, N., Basri, M., Rahman, R.N.Z.R.A., Salleh, A.B., Radzi,S.M., 2008. Modelling and optimization of lipase-catalyzed synthesis of dilauryladipate ester by response surface methodology. J. Chem. Technol. Biotechnol.83, 1534–1540.
roducts 32 (2010) 246–251 251
Anderson, M.J., Whitcomb, P.J., 2005. RSM Simplified: Optimizing Processes UsingResponse Surface Methods for Design of Experiments, 1st edn. ProductivityPress, New York.
Bartoli, G., Bosco, M., Carlone, A., Dalpozzo, R., Marcantoni, E., Melchiorre, P., Sambri,L., 2007. Reaction of dicarbonates with carboxylic acids catalyzed by weak Lewisacids: general method for the synthesis of anhydrides and esters. Synthesis 22,3489–3496.
Bozell, J.J., Moens, L., Elliott, D.C., Wang, Y., Neuenscwander, G.G., Fitzpatrick, S.W.,Bilski, R.J., Jarnefeld, J.L., 2000. Production of levulinic acid and use as a platformchemical for derived products. Resour. Conserv. Recycl. 28, 227–239.
Carta, G., Gainer, J.L., Gibson, M.E., 1992. Synthesis of esters using a nylon-immobilized lipase in batch and continuous reactors. Enzyme Microb. Technol.14, 904–910.
Decaestecker, T.N., Lambert, W.E., Van Peteghem, C.H., Deforce, D., Van Bocxlaer,J.F., 2004. Optimization of solid-phase extraction for a liquid chromatographic-tandem mass spectrometric general unknown screening procedure by means ofcomputational techniques. J. Chromatogr. A 1056, 57–65.
Fang, Q., Hanna, M.A., 2002. Experimental studies for levulinic acid production fromwhole kernel grain sorghum. Bioresour. Technol. 81, 187–192.
Garcia, J., Fernandez, S., Ferrero, M., Sanghvi, Y.S., Gotor, V., 2002. Building blocksfor the solution phase synthesis of oligonucleotides: regioselective hydrolysisof 3′ ,5′-di-O-levulinylnucleosides using an enzymatic approach. J. Org. Chem.67, 4513–4519.
Gunawan, E.R., Basri, M., Abdul Rahman, M.B., Salleh, A.B., Rahman, R.N.Z.A., 2005.Study on response surface methodology of lipase-catalyzed synthesis of palm-based wax ester. Enzyme Microb. Technol. 37, 739–744.
Hari Krishna, S., Sattur, A.P., Karanth, N.G., 2001. Lipase-catalyzed synthesis ofisoamyl isobutyrate-optimization using a central composite rotatable design.Process Biochem. 37, 9–16.
Hayes, D.J., 2009. An examination of biorefining processes, catalysts and challenges.Catal. Today 145, 138–151.
Ismail, A., Linder, M., Ghoul, M., 1999. Optimization of butylgalactoside synthesisby �-galactosidase from Aspergillus oryzae. Enzyme Microb. Technol. 25, 208–213.
Jeong, G.T., Yang, H.S., Park, D.H., 2009. Optimization of transesterification of ani-mal fat ester using response surface methodology. Bioresour. Technol. 100, 25–30.
Keng, P.S., Basri, M., Abdul Rahman, M.B., Salleh, A.B., Rahman, R.N.Z.A., Ariff, A., 2005.Optimization of palm based wax esters production using statistical experimentaldesigns. J. Oleo Sci. 54, 519–528.
Lavandera, I., Garcia, J., Fernandez, S., Ferrero, M., Gotor, V., Sanghvi, Y.S., 2005.Enzymatic regioselective levulinylation of 2′-deoxyribonucleosides and 2′-o-methylribonucleosides. In: Beaucage, S.L., Bergstrom, D.E., Glick, G.D., Jones, R.A.(Eds.), Current Protocols in Nucleic Acid Chemistry. Wiley, New York, pp. Unit2.11.
Mohammad, P., Azarmidokht, H., Fatollah, M., Mahboubeh, B., 2006. Applicationof response surface methodology for optimization of important parameters indecolorizing treated distillery wastewater using Aspergillus fumigatus UB2 60.Int. Biodeter. Biodegr. 57, 195–199.
Mooney, D.D., Swift, R.J., 1999. A Course in Mathematical Modeling, 1st edn. TheMathematical Association of America.
Myers, R.H., Montgomery, D.C., Anderson-Cook, C.M., 2009. Response SurfaceMethodology: Process and Product Optimization Using Designed Experiments,3rd edn. John Wiley & sons, New Jersey.
Olson, E.S., 2001. Conversion of Lignocellulosic Material to Chemicals and Fuels.Energy & Environmental Research Center. University of North Dakota.
Otero, C., Arcos, J.A., Garcia, H.S., Hill, C.G., 2001. Enzymatic synthesis and hydrolysisreactions of acylglycerols in solvent-free systems. In: Vulfson, E.N., Halling, P.J.,Holland, H.L. (Eds.), Enzymes in Nonaqueous Solvents: Methods and Protocols.Humana Press Inc., Totowa, NJ, pp. 479–496.
Petersson, A.E.V., Gustafsson, L.M., Nordblad, M., Borjesson, P., Mattiasson, B., Adler-creutz, P., 2005. Wax esters produced by solvent-free energy-efficient enzymaticsynthesis and their applicability as wood coatings. Green Chem. 7, 837–843.
Radzi, S.M., Basri, M., Salleh, A.B., Ariff, A., Mohammad, R., Abdul Rahman, M.B., Rah-man, R.N.Z.R., 2005. High performance enzymatic synthesis of oleyl oleate usingimmobilised lipase from Candida antartica. Electron. J. Biotechnol. 8, 291–298.
Soo, E.L., Salleh, A.B., Basri, M., Rahman, R.N.Z.A., Kamaruddin, K., 2004. Responsesurface methodological study on lipase-catalyzed synthesis of amino acid sur-factants. Process Biochem. 39, 1511–1518.
Tufvesson, P., Annerling, A., Hatti-Kaul, R., Adlercreutz, D., 2007. Solvent-free enzy-matic synthesis of fatty alkanolamides. Biotechnol. Bioeng. 97, 447–453.
Wetzel, S., Duchesne, L.C., Laporte, M.F., 2006. Bioproducts from Canada’s Forests:
synthesis of n-butyl levulinate. Ind. Eng. Chem. Res. 47, 3358–3363.Yamane, T., 2001. Solvent-free biotransformations of lipids. In: Vulfson, E.N., Halling,
P.J., Holland, H.L. (Eds.), Enzymes in Nonaqueous Solvents: Methods and Proto-cols. Humana Press Inc., Totowa, NJ, pp. 509–516.