Solvent Free Synthesis Of Bifunctional Esters Biology Essay

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Esters produced from diacids, keto acids and hydroxyl acids are called bifunctional esters. In addition to displaying the characteristics associated with individual functionalities, these esters also show unique properties due to the interaction of their two functional groups (Bakhtiar and Hardy, 1997). Bifunctional esters are synthetically important compounds that mainly used as crosslinking agents, starting materials and building blocks for the synthesis of various bulk chemicals and intermediate products in the synthesis of polymers and resins (Olson, 2001). The excellent properties of bifunctional esters such as good thermal stability, low volatility, low toxicity and high biodegradability make them very useful compounds and significant to many industrial applications especially domestic, food, agricultural, pharmaceutical, textile, plasticizer and lubricant industries.

[0003] Levulinic acid is a platform chemical with numerous potential applications by having a ketone and a carboxylic group (Fang, 2002; Bozell et al., 2000). It is commercially produced from renewable biomass such as starch, cane sugar, and lignocellulosic materials from agricultural wastes. Ethyl levulinate ester is an industrially important derivative of levulinic acid, made by esterifying its carboxylic group with ethanol (Wetzel et al., 2006). The esterification reaction is usually carried out at high temperature in the presence of an acid catalyst such as sulphuric, polyphosphoric or p-toluenesulfonic acids. Ethyl levulinate can be used as an oxygenate additive. A research by Tecaxo and Biofine Inc. showed that a mixture of 20% ethyl levulinate, 1% of co-additive and 79% diesel can be used in regular diesel engines. Ethyl levulinate is also used in the flavoring and fragrance industries. Levulinate esters from high boiling alcohols are used as plasticizer for cellulose plastics. Another potential application of levulinate esters is to replace kerosene as a fuel for the direct firing of gas turbines (Hayes, 2009; Erner, 1982)

[0004] Succinic acid is a dicarboxylic acid which can be used in the synthesis of acyl halides, anhydrides, esters, amides, and nitriles. Succinate esters have potential applications for producing bulk chemicals such as 1,4-butanediol (a precursor to biodegradable plastics), ethylene diamine disuccinate (a biodegradable chelator) and diethyl succinate (a "green" solvent replacement for dichloromethane) with $15 billion market (McKinlay et al., 2007). Synthesis of bio-based succinate by fermentation process using microorganisms has been reported by several researchers (Zeikus et al., 1999; McKinlay et al., 2007). Low productivity due to side reactions, low activity of microorganisms in the presence of high concentration of succinate, and requirement of too expensive media, have caused low bio-based succinate production that is not being able to support the markets (McKinlay et al., 2007). Application of enzymes to produce these high value-added esters may overcome these disadvantages. Immobilized enzyme was used as catalyst for copolymerization of succinic acid and 1,4-butanediol to produce low molecular weight oligomers (Azim et al., 2006). However, so far there is no report on enzymatic synthesis of succinate esters using succinic acid and monohydric alcohols as the substrates.

[0005] Bifunctional esters are traditionally produced by reacting a bifunctional acid and a monohydric alcohol at high temperatures in the presence of metal, acidic or basic catalysts. This method leads to undesirable side reactions and degradation of esters. The use of those chemical catalysts exposed to toxicity and corrosion. Reaction with homogenous chemical catalysts are usually time consuming and also give relatively low yields and high energy consumption. Esterification reaction is a reversible process; hence the long time reaction may result in hydrolysis reaction. Thus, interests have grown on the use of green synthesis of esters in organic medium catalyzed by using enzymes.

[0006] Although organic solvents provide several advantages in enzymatic reactions, their use in industrial processes is not desirable. They are a source of volatile organic compounds (VOCs), causing low level ozone and smog, and their use requires costly post-treatment actions and larger and more expensive reactors and auxiliary equipments (Tufvesson et al., 2007). Performing the reactions under solvent-free conditions can help to overcome these disadvantages. Furthermore, higher volumetric productivity, improved substrate and product concentrations and fewer purification steps are some other advantages of using solvent-free system. It should be added that the avoidance of organic solvents is particularly advantageous to the food and pharmaceutical industries where stringent regulations related to the use of organic solvents are in force (Otero et al., 2001).

[0007] Considering the high benefits and demands of bifunctional esters, process optimization plays a very significant role in their economical synthesis. Furthermore, for any future upscale enzymatic synthesis of esters, finding the optimal reaction conditions is essential. Statistical experimental design approaches can explore the experimental space while studying various variables by requiring a small number of experiments and a short time (Abdel-Fattah et al., 2005). Response surface methodology (RSM) and artificial neural network (ANN) are efficient analytical techniques which can be applied in the study and optimization of biotechnological processes (Jeong et al., 2009; Macedo et al., 2004).


[0008] Enzymatic synthesis as ''green'' alternative route to produce high value-added esters has large potential to significantly impact industrial production and contribute to a more sustainable technology. Biotechnological process helps to establish a mild synthesis route which reduces the process steps substantially. In order to achieve a proficient utilization of enzyme for industrial applications, it is necessary to develop highly stable immobilized biocatalyst for ester production. The use of immobilized enzyme has become a valid approach due to its special features which allow the reutilization of the enzyme and separation of products.

[0009] Application of enzymes provides many advantages such as higher quality products, energy efficiency, and reduced environmental hazards. Processing equipment also lasts longer since the milder reaction conditions reduce corrosion. Several bifunctional esters produced in the present invention are useful as starting materials or chemicals additives.

[0010] Yadav and Borkar (2008) reported immobilized Candida antarctica lipase B-catalyzed synthesis of butyl levulinate using levulinic acid and n-butanol in tetrabutyl methyl ether as the solvent.

[0011] It is an object of the present invention to produce bifunctional esters in solvent-free system.

[0012] Optimization of reaction conditions is the most important part in ester production. A statistical based technique commonly used for this purpose is Response Surface Methodology (RSM). RSM has successfully been applied to study and optimize the enzymatic synthesis of various esters (Jeong et al., 2009). However, so far there are few studies on elucidating the effect of several reaction parameters on bifunctional ester synthesis. The main advantage of RSM is the reduced number of experimental runs needed to provide sufficient information for statistically acceptable result. It is a faster and less expensive method than the classical method (Jeong et al., 2009). Knowledge-based approaches such as artificial neural networks (ANNs) and genetic algorithm (GA) also have been successfully applied to the modeling and optimization of various chemical and biochemical processes in recent years. The ability of ANNs to learn the process characteristics with little prior knowledge is desirable and eases their implementation and heightens their modeling potential. These properties make ANNs powerful and flexible tools that are well-suited to modeling enzymatic processes (Bas and Boyaci, 2007).

[0013] It is an object of the present invention to provide a method for producing high quality and purity bifunctional esters.

[0014] Another object of the present invention is to produce a high yield of bifunctional esters by esterification reaction of bifunctional acid preferably levulinic and succinic acids with at least one alcohol in the presence of enzyme.

[0015] An auxiliary objective of the present invention is to carry out the above-specified reaction in a large-scale simple process. Optimization of reaction conditions is conducted by RSM/ANN-GA approaches.

[0016] The present invention related to a process for producing bifunctional esters by enzymatically esterifiying bifunctional acid preferably levulinic and succinic acids with at least one alcohol which contains 1 to 18 carbons per molecule.

[0017] In the preferred embodiment of the present invention, the succinic acid is produced from glucose or lignocellulosic wastes fermentation. In another embodiment of the present invention the succininc acid may be derived from n-butane via petrochemical processes. Levulinic acid is produced via controlled degradation of hexose sugars from waste lignocellulosic materials by mineral acids. The alcohol used in the present invention can be linear or branched alcohol contains 1 to 18 carbons per molecule.

[0018] Synthesis of bifunctional esters including succinate and levulinate esters is accomplished in agreement with the present invention that the mixture of succinic or levulinic acid and alcohol is shaking continuously at temperature in the range from about 25 degree C to 75 degree C. The esterification reaction is carried out in the absence of organic solvent. Preferably immobilized lipase is chosen as the biocatalyst in the reaction mixture to facilitate the esterification reaction. The immobilized lipase is recycled and the yield of the reaction and purity of the product is controlled.

[0019] The effect of parameters on the reaction and their interaction on the production of bifunctional ester is investigated by response surface methodology (RSM). To train an ANN-GA model a set of data containing inputs and outputs are fed. Multilayer feedforward neural network combined with genetic algorithm is used because of its short calculation time and high convergence property in order to characterize the essential behavior of enzymatic production of the esters.


[0020] According to the present invention, solvent-free process for producing bifunctional esters comprises esterifying succinic and levulinic acids compounds with at least one alcohol in the presence of lipase as catalyst in the absence of any organic solvent. In the preferred embodiment of the present invention, stated succinic and levulinic acids are derived from fermentation of lignocellulosic wastes whereas said alcohol is an alcohol with 1 to 18 carbon atoms per molecule.

[0021] Continuous shaking is applied to the reaction mixture and the mixture is allowed to proceed for 300 min for succinate and 30 min for levulinate ester. Then, the biocatalyst is separated from the reaction mixture by filtration. The separated biocatalyst is then recycled and reused in the next batch.

[0022] In the preferred embodiment of the present invention, the immobilized enzyme is used. Immobilization of enzyme to solid carriers improves operational stability of biocatalyst, better operation control, easier product recovery without catalyst contamination and flexibility of reactor design. Furthermore, decreased inhibition by reaction products, selectivity towards non-natural substrates and better functional properties compared to the soluble enzymes, the availability of the product in greater purity, high volumetric productivity with lower residence time, multiple use of a single batch of enzymes and the ability to stop the reaction rapidly by removing the enzyme from the reaction solution, are other advantages of using immobilized enzyme.

[0023] In the present invention, the reaction is carried out in an organic solvent-free system. Organic solvents are a source of volatile organic compounds (VOCs) and their use requires costly post-treatment actions in the form of solvent evaporation and recycling. Using organic solvents results in the need for larger and more expensive reactors and auxiliary equipments. Therefore, a solvent free process would be beneficial both from an environmental as well as from an economical point of view.

[0024] The immobilized enzymes that used are Candida antarctica lipase B (Novozyme 435) or Rhizomucor miehei (Lipozyme IM). Both lipases are measured in the same amount if applied. The protein content of the mixture is controlled to maximize the bifunctional ester production.

[0025] The bifunctional acids used in the present invention are succinic acid or butanedioic acid, and levulinic acid or 4-oxopentanoic acid, which represented by formula (1) and (2), respectively

HOOC(CH2)2COOH Formula 1

CH3C(O)CH2CH2COOH Formula 2

[0026] In the preferred embodiment of the present invention, the bifunctional acids compound used are derived from fermentation of lignocellulosic waste materials.

[0027] The reaction is carried out in temperature ranges from 25 degree C to 75 degree C with preferably 50 degree C. In the present invention, the reaction mixture undergoes continuous shaking in controlled temperatures. Effect of temperature on the reaction yield can be assigned to its effect on substrate solubility, reaction rate, enzyme stability and activity.

[0028] The ratio of bifunctional acid to alcohol in solvent-free system is set to 1:2. In the preferred embodiment of the present invention, to get highest percentage conversion of bifunctional acid and alcohol to bifunctional ester, acid and alcohol will be supplied at a molar ratio in the range of 1:1 to 1:8 with preferably 1:2 for short chain and 1:7 for long chain length of alcohol used. In solvent-free systems, one substrate is generally used in a large excess over another in order to act as a solvent for other reactants.

[0029] The percentage conversion of ester is calculated based on the titration result compared with relative volume of substrate to product. In the present invention, the titration is performed after filtration of biocatalyst with 0.1M NaOH using phenolphthalein as the indicator.

[0030] The product of the esterification reaction is characterized by FT-IR, Thin Layer Chromatography (TLC), Gas Chromatography (GC), and Gas Chromatography-Mass Spectometry (GC-MS).

[0031] Bifunctional esters obtainable from the process according to the present invention are dioleyl succinate and ethyl levulinate which presented by formula (3) and (4), respectively:

H35C18OOC(CH2)2COOC18H35 Formula 3

CH3C(O)CH2CH2COOC2H5 Formula 4

[0032] A response surface methodology approach based on four-factor-five-level central composite rotatable design (CCRD) is employed for optimization. The fractional factorial design consists of 16 factorial points, 8 axial points and 6 center points. The variables selected for the synthesis of bifunctional ester in solvent-free system are temperature, time, amount of enzyme and substrate molar ratio. A software package of Design Expert Version 6.0.6 (State-Ease Inc., Statistics Made Easy, Minneapolis, MN, USA.) is used to fit the model to the independent variables. The optimal condition for the synthesis of ester is generated using the software's numerical optimization function. The ANN in the form of a feed forward-back propagation network integrated with GA is trained and its prediction ability is investigated using MATLAB software.

[0033] Certain detailed routes embodying the present invention will be illustrated in the following examples, it being understood that various changes in the details of the present invention may be made without departing from the scope or sacrificing any of the advantages of the invention.


[0034] Different molar ratios of levulinic acid and ethanol are mixed corresponding to the different substrate molar ratios generated by RSM, in 30mL screw-capped vials. Different amounts of lipase, which are generated by RSM, are subsequently added. The reaction is carried out in a temperature controlled water bath shaker at 150 rpm at different temperatures and for different time periods generated by RSM. The reaction is terminated by dilution with 5 ml of ethanol: acetone (50:50 v/v) and enzyme is removed by filtration. Remaining free acid in the reaction mixture is determined by titration with 0.1M NaOH. The moles of acid reacted are calculated from the values obtained for the control and the test samples. The ester formed is expressed as equivalent to conversion of the acid.

[0035] A high conversion yield (96.2%) is obtained at the optimum conditions. The mass spectrum of the product exhibits molecular ion at m/z 144 that corresponds to molecular formula of ethyl levulinate. 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 formation of ion acylium, RCO+ (m/z = 99) and m/z 129 because of the loss of methyl group from the ester.


[0036] Example 1 was repeated with succinic acid and oleyl alcohol. The esterification percentage was 85.0% at the optimum condition.


[0038] Fitting of the data to the various models and their subsequent analysis of variance showed that the synthesis of levulinate ester was suitably described with quadratic polynomial model. A very small P-value (< 0.0001) and a suitable coefficient of determination (R2 = 0.8993) shows that the model can satisfactorily represent the real relationship among the reaction parameters.


[0039] Through examining different ANN configurations, the best network is characterized. The network is trained by Levenberg-Marquardt (LM) algorithm. The values of coefficient of determination (R2) between the actual and predicted responses were determined as 0.99 for training dataset.

[0040] It is to be understood that the present invention may be embodied in other specific forms and is not limited to the sole embodiment described above. However modification and equivalents of the disclosed concepts such as those which readily occur to one skilled in the art are intended to be included within the scope of the claims which are appended thereto.


1. A process for synthesis of bifunctional esters, said process includes the steps of:

(a) mixing different substrates with certain molar ratio in the absence of any organic solvent;

(b) adding a certain amount of immobilized enzyme to the reaction mixture;

(c) incubating the mixture in a controlled temperature for certain time;

(d) terminating the reaction and filtering the immobilized enzyme;

(e) titration of the unreacted acid and calculating the conversion of said ester;

(f) purification and characterization of said ester;

(g) optimization of reaction conditions;

2. The process according to claim 1, wherein the bifunctional esters include mainly levulinate and succinate esters.

3. The process according to claim 2, wherein said bifunctional ester is produced from esterification of a bifunctional acid and a monohydric alcohol.

4. The process according to claim 3, wherein said bifunctional acid can be a dicarboxylic acid or a β-keto acid.

5. The process according to claim 3, wherein said alcohol can be linear or branched alcohol contains 1 to 18 carbons per molecule as well as fatty alcohols.

6. The process according to claim 3, wherein the said esterification reaction is carried out in an organic solvent-free system.

7. The process according to claim 1, wherein the enzyme is an immobilized lipase including Candida antartica lipase B (Novozyme 435) or Rhizomucor miehei (Lipozyme IM).

8. The process according to claim 3, wherein the molar ratio of said bifunctional acid to said alcohol is from 1:1 to 1:8.

9. The process according to claim 1, wherein the synthesis is affected at a temperature from 25 to 75 degree C.

10. The process according to claim 1, wherein the immobilized enzyme is used in a quantity of about 17 to 250% by weight of the bifunctional acid.

11. The process according to claim 1, wherein the required time ranges from 30 to 300 min.

12. The process according to claim 1, wherein after the esterification of the bifuctional acid, the solution comprising the ester is filtered and the immobilized enzyme is isolated and reutilized in another batch.

13. The process according to claim 1, wherein the optimization of reaction conditions is performed by statistical techniques including response surface methodology approach or artificial neural network integrated with genetic algorithm.

14. The process according to claim 13, wherein the optimization is performed to maximize the esterification yield and minimize required amount of enzyme, time, energy consumption and raw materials.


Figure 1 represents contour plots showing the interaction between two parameters, substrate molar ratio and temperature, in synthesis of dioleyl succinate.

Figure 2 represents the relative yield of the dioleyl succinate obtained in various incubation time.

Figure 3 represents mass spectrum of ethyl levulinate ester.

Figure 4 represents response surface plot showing the interaction between two parameters, enzyme amount and substrate molar ratio, in synthesis of ethyl levulinate.

Figure 5 represents correlation of actual yields and values predicted by the response surface model in the synthesis of succinate ester

Figure 6 represents effect of the amount of enzyme on the percentage yield of levulinate ester.

Figure 7 represents schematic diagram of artificial neural network (ANN).

Figure 8 represents conversion of ester versus experimental number for training the network.



The present invention relates to a process for synthesis of bifunctional esters. The process includes the steps of (a) mixing different amount of substrates in solvent-free system based on design of experiments study, (b) mixing said substrates with immobilized enzyme, (c) incubating the reaction mixture in a temperature controlled system for a certain time, (d) terminating the reaction and removing the immobilized enzyme by filtration, (e) titration of unreacted acid to calculate the percentage of conversion, (f) purification and characterization of the product, and (g) optimization of the reaction conditions and maximizing the yield.