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EKC 271

BIOTECHNOLOGY FOR ENGINEERS

(TERM PAPER)

A Review of Kinetic Studies of Various Esterification via Enzymatic

Route

Name IC Number Matric Number PositionLeong Sim Siong 890119-59-5281 104718 Project Leader

Emily Ooi Shi Yun 910118-07-5394 104702 CheckerMohammad Razif bin Abdul Razak 900509-04-5273 104739 Group Member

Muhammad Hafiz bin Wan 901024-07-5807 104737 Group Member

LECTURER: Prof. Azlina bt Harun @Kamaruddin

SUBMISSION DATE: 15 th OCTOBER 2010


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Content

1.0 Abstract 4

2.0 Introduction 52.1 Enzyme Kinetics 52.2 Esterification 5

3.0 Various Kinetic Studies of Esterification via Enzymatic Route 63.1 Esterification of Oleic Acid for Biodiesel Production Catalyzed by SnCl 2 :

A Kinetic Investigation 63.1.1 Introduction 63.1.2 Results and Discussion 6

3.1.2.1 General aspects 6 3.1.2.2 The SnCl 2 catalyst versus H 2SO4: A comparative study 6 3.1.2.3 Ethanolysis of oleic acid catalyzed by SnCl 22H 2O: kinetic studies 7

3.1.2.3.1 The effect of oleic acid concentration 7 3.1.2.3.2 The effect of SnCl 22H 2O concentration 83.1.2.3.3 The effect of temperature on the ethanolysis of oleic acid

SnCl 2-catalyzed 103.1.3 Conclusions 11

3.2 Kinetics and Yield of Butyl Butyrate Synthesis: Role of Water and

Organic Solvent 123.2.1 Introduction 123.2.2 Results and Discussions 13

3.2.2.1 Standard Condition 133.2.2.2 Effect of enzyme concentration 3.2.2.3 Effect of temperature 3.2.2.4 Effect of continuous removal of water 153.2.2.5 Effect of initial addition of water 16 3.2.2.6 Effect of substrate concentration

3.3 Kinetic study of esterification of rapeseed oil contained in waste activatedbleaching earth using Candida rugosa lipase in organic solvent system 19

3.3.1 Introduction 193.3.2 Results 19

3.3.2.1 Effect of ABE on the esterification of rapeseed oil 193.3.2.2 Effect of the addition order of reactant on esterification of rapeseed oil

contained in waste ABE 193.3.2.3 Effect of initial methanol concentration on FAME formation rate 21


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1.0 Abstract

Various kinetics of esterification has been studied intensively. A better understanding of enzymatic esterification will optimize the reaction for maximum production at lowest cost and shortest

time.

For example, the esterification of oleic acid for biodiesel production catalyzed by SnCl2 wasdeveloped to produce biodiesel from low-cost raw materials which generally contain high amounts of free fatty acids (FFAs) is a valuable alternative that would make their production costs more competitivethan petroleum-derived fuel. [1] Currently, the production of biodiesel from this kind of raw materialscomprises a two-stage process, which requires an initial acid-catalyzed esterification of the FFA,followed by a basecatalyzed transesterification of the triglycerides. [2] Commonly, the acid H 2SO 4 is thecatalyst on the first step of this process. [3] However, the usage of H 2SO 4 has some drawbacks such assubstantial reactor corrosion and the great generation of wastes, including the salts formed due toneutralization of the mineral acid. [4] The SnCl 2 catalyst was shown to be as active as the mineral acidH2SO 4. Its use has relevant advantages in comparison to mineral acids catalysts, such as less corrosionof the reactors and as well as avoiding the unnecessary neutralization of products. [5-6]

Apart from that, organic solvent and water, which are playing an important role in the synthesisof butyl butyrate from n-butanol and butyric acid, have been investigated deeply to get a betterunderstand on the effects of some factors to the esterification rate. [7] The use of enzymes in organicmedia is an actively investigated field of biocatalysts. Mainly three types of media have been advocated:biphasic systems consisting of water and a water-immiscible solvent, reversed micelles or non-aqueoussystems. [8] In the first two systems, the enzyme is solubilized in water, while in non-aqueous media, the

enzyme is in suspension in solvent. Meanwhile, in non aqueous .media, a minimal amount of water isnecessary for the enzyme to be active and, as a consequence, the term micro aqueous was judged moresuitable to define this type of medium. [9]

The effect of activated bleaching earth (ABE) from the oil processing industry on the productionof fatty acid alkyl esters (FAMEs) by the lipase-catalyzed alcoholysis of waste plant oil with methanolin an organic solvent system was investigated. Its obs erved that the esterification rate will become veryhigher if ABE is added to the reaction mixture. Through experiments which have being carried, it wasfound that the optimum ratio of ABE to ABE and rapeseed oil for the esterification of rapeseed oil. Theinhibition kinetics is summarized as a function of methanol on the conditions of the sufficient existenceof rapeseed oil and lipase, which Michaelis Menten Equation is held true. These results indicate that the

presence of ABE relieves the inhibitory effect of methanol on the enzyme because of the adsorption of methanol by ABE. [10]

In this paper, the kinetic studies of esterification of oleic acid for biodiesel production catalyzedby SnCl 2, role of water and organic solvent in butyl butyrate synthesis and esterification of rapeseed oilcontained in waste activated bleaching earth using Candida rugosa lipase in organic solvent system willbe discussed.
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2.0 Introduction

2.1 Enzyme KineticsEnzyme kinetics is the study of the chemical reactions that are catalyzed by enzymes. In enzyme

kinetics, the reaction rate is measured and the effects of varying the conditions of the reactioninvestigated. Studying an enzyme's kinetics in this way can reveal the catalytic mechanism of thisenzyme, its role in metabolism, how its activity is controlled, and how a drug ora poison might inhibit the enzyme.

Enzymes are usually protein molecules that manipulate other molecules the enzymes' substrates. These target molecules bind to an enzyme's active site and are transformed into products through a seriesof steps known as the enzymatic mechanism. These mechanisms can be divided into single-substrate andmultiple-substrate mechanisms. Kinetic studies on enzymes that only bind one substrate aim to measurethe affinity with which the enzyme binds this substrate and the turnover rate.

When enzymes bind multiple substrates, enzyme kinetics can also show the sequence in which thesesubstrates bind and the sequence in which products are released. Although these mechanisms are often acomplex series of steps, there is typically one rate-determining step that determines the overall kinetics.This rate-determining step may be a chemical reaction or a conformational change of the enzyme orsubstrates, such as those involved in the release of product(s) from the enzyme. [11]

2.1.1 General PrincipleThe reaction catalyzed by an enzyme uses exactly the same reactants and produces exactly the

same products as the uncatalyzed reaction. Like other catalysts, enzymes do not alter the positionof equilibrium between substrates and products .[12]

Several factors influencing the rate at which an enzyme works are the concentration of substrate molecules, where the more of them available, the quicker the enzyme molecules collideand bind with them. The temperature, as the temperature rises, molecular motion (collisionsbetween enzyme and substrate) speed up. But as enzymes are proteins, there is an upper limitbeyond which the enzyme becomes denatured and ineffective. The presence of inhibitors alsoplays an important role. Competitive inhibitors are molecules that bind to the same site as thesubstrate - preventing the substrate from binding as they do so, but are not changed by the enzyme.Non-competitive inhibitors are molecules that bind to some other site on the enzyme reducing itscatalytic power. Lastly the pH value. The conformation of a protein is influenced by pH and asenzyme activity is crucially dependent on its conformation, its activity is likewise affected. [13]

2.2 EsterificationEsterification is the general name for a chemical reaction in which two reactants (typically an

alcohol and an acid) form an ester as the reaction product. Esters are common in organic chemistry andbiological materials, and often have a characteristic pleasant, fruity odor. Esterification is a reversiblereaction. Hydrolysis, "water splitting", involves adding water and a catalyst to an ester to get the sodiumsalt of the carboxylic acid and alcohol. As a result of this reversibility, many esterification reactionsare equilibrium reactions and therefore need to be driven to completion according to Le Chatelier'sprinciple. Esterifications are among the simplest and most often performed organic transformations. [14]
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3.0 Various Kinetic Studies of Esterification via Enzymatic Route

3.1Esterification of Oleic Acid for Biodiesel Production Catalyzed by SnCl 2 : A KineticInvestigation

3.1.1 IntroductionBiodiesel is a suitable substitute for petroleum-derived diesel. It is biodegradable, almost

sulfurless and a renewable fuel. This alternative fuel consists of methyl or ethyl esters, a result of either transesterification of triacylglycerides (TG) or esterification of free fatty acids (FFAs) [15] .Currently, most of the biodiesel comes up from transesterification of edible resources such asanimal fats, vegetable oils, and even waste cooking oils, under alkaline catalysis conditions [16-18] .The common processes of biodiesel production from low-cost raw materials use mineral acids ascatalysts, owing to the high amounts of FFAs that those resources contain, which make themanufacture of biodiesel from these feedstocks incompatible with alkaline catalysts [19] . Thus, twoalternative approaches are normally used. The first is a two-step process, which requires an initial

acid catalyzed esterification of the FFA, followed by a base-catalyzed transesterification of the TG.Second, a one-step process that only uses an acid catalyst that simultaneously promotes bothesterification and transesterification reactions [20] .

3.1.2 Results and Discussion

3.1.2.1 General aspects

The ethanolysis of FFA is a typical reversible acid-catalyzed reaction that produces

ester and water as by-product:

First, the esterification of oleic acid with ethanol in the absence of the acidic catalysts(SnCl 22H 2O and H 2SO 4) was conducted. In spite of high molar ratio of ethanol/fatty acidused, there are no significant yields of ethyl oleate even after more than 12 hours of reaction (Figure 1). Conversely, in the presence of SnCl 2, much greater yield (>90%) with ahigh selectivity were obtained after 12 hours of reaction. This proves that the excess of ethanol has no significant effect on the yield or the reaction rate.

Figure 1. Trend of conversion of oleic acid into ethyl oleate.


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In all catalytic runs the ethyl oleate yield increased as the reaction time increased.However, when the reaction time goes beyond 12 hours, the ester concentration remainsalmost invariable.

3.1.2.2 The SnCl 2 catalyst versus H 2SO4: A comparative study

The reaction yields of both acids catalysts increase steadily, reach the maximum values(ca. 90 %) after a reaction time of ca. 120 minutes and thus stay almost invariableafterwards (Figure 2).

The two acidic catalysts have different structures and acid character as well asmechanisms of action, in spite of similar activities displayed (Figure 2). The reaction yieldshave increased steadily to a maximum value of ca. 90 %, in approximately 120 minutesafter setting up the reaction. The monitoring of reaction for periods higher than 120minutes reveals that the yields of both remained invariable after this time (Table 1).

3.1.2.3 Ethanolysis of oleic acid catalyzed by SnCl 22H 2O: kinetic studies

3.1.2.3.1 The effect of oleic acid concentrationLiterature data have attributed a first order dependence on fatty acid

concentration in the majority of esterification reactions [21] . From thisstandpoint, in this present case we can assume that equation 1 could be used todescribe the substrate concentration variation with relation to time:

Figure 2 . Ethanolysis of oleiccatalyzed by Brnsted (H 2SO 4) andLewis acids (SnCl 2).

Table 1. Conversion andselectivity of ethanolysisof oleic acid catalyzed bySnCl 2 and H 2SO 4


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(1) Thus, to obtain the reaction order in relation to the oleic acid concentration,

plots of ln [oleic acid] versus time were thus built for each acid-catalyzedprocess (Figure 3 presents only the data of ethanolysis of the oleic acid tin-catalyzed).

The resulting data fit a first order kinetic behavior. It is important tomention that a high molar excess of ethanol in relation to oleic acid (120:1)was used to assure that the ethanol concentration would remain essentiallyconstant during the reaction course. As displayed in Table 2, the highcorrelation coefficients of the resulting linear equations obtained areindicative of the fact that there is a first order dependenence for bothesterification reaction catalyzed by sulfuric acid and tin chloride. From the

angular coefficients of such equations, the rate constants (k ) and then the half-life times (t1/2 = k /0.693) for each process was thus obtained.

3.1.2.3.2 The effect of SnCl 22H 2O concentrationTo control the accuracy of the kinetic data related to the residual

concentration of oleic acid, it has been essential to maintain the acid catalyzedprocess distant from the equilibrium position, therefore, the kineticmeasurements have been carried out within two hours of reaction. The

Table 2 The effect of substrate concentration on the ethanolysis of oleicacid catalyzed by SnCl 2.

Figure 3 . Arrenhius plotof oleic acidconcentration as functionof time for ethanolysiscatalyzed by SnCl 2.


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concentration of the residual oleic acid was determined directly from thereaction medium via titration with an alcoholic solution of KOH [22] .

To calculate the initial rate, only the first interval of thirty minutes wastaken into account, as the dependence in relation to oleic acid concentration isof pseudo-zero order during this period. This statement is not completely true,

because in this time interval the substrate oleic acid is partially consumed.However, this effect is minimized because the initial concentration of oleic acidused was higher in relation to the other experiments. Thus, with a relativelyacceptable error, we assume that only the tin chloride concentration is rangingin each run. As a general tendency, an increase in the catalyst concentrationcaused an improvement in the ethyl oleate yields at any reaction time.

The results shown in Figure 4 are concomitant with the linear correlationshown in Figure 5.

The increases of the catalyst concentration has a noticeable effect on theconversion rate of the oleic acid into ethyl oleate. This fact can be attributed tohigher number of molecules of substrate activated by polarization of the

carbonyl, in presence of Sn+2

catalyst. Thus, the nucleophilic attack by ethanolbecomes more favorable and consequently, a increases on the formation of esterwas observed. The angular coefficient of the curve obtained is suggestive of afirst-order dependence ( ca. 0.87), in relation to catalyst concentration (seeequation 2 and Figure 5):

(2)

Figure 4 . Effect of theconcentration of SnCl 22H 2O catalyst onthe ethanolysis of


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3.1.2.3.3 The effect of temperature on the ethanolysis of oleic acid SnCl 2-catalyzed

Temperature noticeably affects both reaction rate and conversion of oleic

acid into ethyl oleate. At room temperature, only low conversions was obtainedeven after long time reactions. However, at temperatures above 45 C, asignificance increase of the initial rate of reaction, and consequently, higherconversions were reached.

Table 3 shows the values for the rate constant (k) obtained at eachtemperature (Figure 6).

Figure 5. Plot linear of ln[SnCl 2] (effect of theconcentration of SnCl 22H 2Ocatalyst on the ethanolysis of oleic acid).

Figure 6 . Effectof thetemperature onthe ethanolysis of oleic acidcatalyzed bySnCl 2.

Table 3 . Linear equations for the decrease in oleic acid concentration as afunction of time and the respective values of R 2 and K.


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From of the resulting data shown in Table 3, the curve of the Figure 7 wasconstructed and employing a linear regression method, the angular coefficient (-E/ R) of the curve obtained allow us to calculate the activation energy of thisprocess.

An activation energy of 46.69 kJ.mol -1 was determined which is

approximately similar to the value found by Berrios and co-workers in thekinetic study of the H 2SO 4-catalyzed esterification of free fatty acids insunflower oil [23] .

3.1.3 ConclusionsFrom the evaluation of the catalytic activity of the SnCl 22H 2O in homogeneous phase in the

esterification of FFA for biodiesel production which serves as an alternative to sulfuric acid, tinchloride efficiently promotes the esterification of oleic acid in ethanol solutions. The high yieldsachievable under mild reaction conditions are comparable to those obtained with a common acidcatalysts such as sulfuric acid (H 2SO 4). Therefore, SnCl 2 is a potential catalyst for the productionbiodiesel from low-cost raw materials, which currently have higher amounts of FFAs. Kineticmeasurements revealed that the acid-catalyzed esterification is first-order relative to theconcentration of both oleic acid and the SnCl 2, which is a promising acid-catalyst for theproduction of biodiesel, in lower environmental impact processes.

Figure 7. Linear plot of ln Kversus 1/T resulting fromesterification of oleic acidcatalyzed by tin chloride.


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3.2 Kinetics and Yield of Butyl Butyrate Synthesis: Role of Water and Organic Solvent

3.2.1 Introduction

The ability of lipase to catalyze reactions in organic solvents has been extensively studied. It is

of common knowledge that the rate of enzyme catalyzed reactions in such systems strongly dependboth on the amount of water present in the system and on the nature of the organic solvent used.Each enzyme displays different levels of water activity profile for maximal activity. A smallvariation in water activity can have a significant influence on the catalytic activity and selectivityof lipase. [24]

Organic solvent and water play an important role in the synthesis of butyl butyrate from n-butanol and n-butyric acid in n-hexane by Mucor miehei lipase. [25] The importance of organicsolvent and water in this enzymatic esterification reaction have been investigated by analysis of thekinetics and the reaction balances. Esterificaton was found to take place in both systems as statedbelow:

(a) Low water systems containing solid enzyme in hexane . In the solid enzyme system, theenzyme adsorbed the water produced, thus delaying the appearance of a discrete aqueousphase. As expected, the presence of some water was indispensable for this system, as itsremoval or exclusion by various means (adsorption, distillation) affected enzyme activity.However, water removal had little effect on the final yield of esterification.

(b) Biphasic aqueous enzyme solution/hexane systems . Reaction velocities were quitesimilar to the solid enzyme/hexane system. The butyl butyrate formed was almostexclusively found in the organic phase. Ethyl lbutyrate, a more polar compound, wassynthesized with a lower yield. [26] These results allow the conclusion that the reaction took place in a phase consisting of either solid hydrated enzyme with no discrete aqueous phaseor of an aqueous enzyme solution by basically similar mechanisms according to the amountof water available to the system, the esterification being driven to completion by transfer of the ester into the organic phase because of a favourable partition coefficient. [27]

Low water content thermodynamically favours the reversal of hydrolytic reactions if we usemicroaqueous media in catalysis. [28] Therefore, extensive studies on esterification by lipases havemostly been performed in such media. In fact, esterification reactions constitute a complex easewhen considering production by enzymatic synthesis since water as a product of the reactionaffects both enzyme activity and the thermodynamics of the reaction. [29]

Kinetic studies are necessary to clarify these phenomena. The synthesis of butyl butyrate by alipase from Mucor rniehei (EC 3.1.1.3) had been investigated, emphasis being placed on analyzingthe effects of various experimental conditions on the water content of the medium and on the watercontent in the vicinity of the enzyme. The effect of water was studied from both a kinetic (reactionrate) and a thermodynamic (conversion yield) point of view.


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3.2.2.2 Effect of enzyme concentration

As the enzyme adsorbed water during esterification, the amount of enzyme used couldhave an effect on the partition of water in the medium and on the kinetics. Various lipaseconcentrations were initially introduced into the medium (0.5 g. , 1.0 g. , 2.5 g. and 5.0 g. ). In all cases, good yields were obtained. From the data given, it is shownthat at the beginning of the reaction, the rate of reaction depends on the concentration of theenzyme. In fact, the higher the concentration of the enzyme, the higher the rate of reaction.However, after a long time, the substrate being used up, the enzyme's active sites are nolonger saturated, substrate concentration becomes rate limiting, and the reaction becomesfirst order with respect to concentration of substrate. [31]

The water contents of the different phases were also determined in the assay at 2.5 g.and the results were similar to those obtained using 5 g. lipase. However, at lowerer

enzyme concentrations, lipase was strongly adsorbed to the glass walls of the reactionvessel and determination of the water content of enzyme could not be achieved.

Figure 9 . Effect of lipase concentrationon kinetics of butyl butyrate synthesis.Standard conditions were used except forthe lipase concentration, which was asfollows: [ 0.5 g. ; , 1.0 g. ; ,2.5 g. ; , 5.0 g.


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3.2.2.3 Effect of temperature

The reaction was carried out at 69C, the boiling point of hexane, while enzyme andsubstrate concentrations were as in the standard conditions, to determine if waterdistribution was different at this temperature. As shown on Figure 10, there was no greatdifference between the initial and maximum rates of esterification. This value was lowerthan the maximal rate obtained at 40C. After 5 hours, synthesis slowed down, and theyield was 94% after 100 hours.

At the end of the reaction, the reactor was left at room temperature and the enzyme wasanalysed for water content. The enzyme had adsorbed about 0.75 g. water, for example,the final water content of lipase was 20%, a value possibly somewhat overestimated sincethe equilibrium could have been modified during cooling of the reactor. The water amountin the organic phase was maximal after 5 hours and then decreased. Its concentration wasvery low throughout the reaction and was in the same range as that obtained at 40C. Therest of water probably formed a discrete aqueous phase. [32]

3.2.2.4 Effect of continuous removal of water

Complementary experiments were carried out to try to better understand the role of

Figure 10 . Enzymatic synthesis of butylbutyrate at 69 C in flasks. Enzyme andsubstrate concentrations were as in thestandard conditions. Concentrations of products and substrates as a function of time: , butyl butyrate; , butanol; ,

butyric acid; , water in solvent

Figure 11 . Enzymatic synthesis of butylbutyrate in Dean-Stark apparatus.Enzyme and substrate concentrationswere as in the standard conditions.Concentrations of products as a functionof time: , butyl butyrate; , water insolvent; , water collected


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water on reaction rate and yield by removing water from the medium as it was produced bythe reaction.

First, a 0.3 nm molecular sieve was added into the medium as a dehydrating agent. Nosynthesis was obtained in this ease, probably because complete dehydration of the enzymecaused its inactivation.

Another procedure to remove water was to use a Dean-Stark apparatus at boilingtemperature of the reaction mixture (69C). The results of an esterification carried outunder such conditions are presented in Figure 11. Butyl butyrate was linearly producedduring the first 7 hours at a constant rate. The water content of the organic phase was alittle lower than when performing syntheses without removal of water. The amount of water collected in the trap was approximately equal to the concentration of ester produced.The final water content of the enzyme was determined. Under these conditions, the enzymehas adsorbed only 0.05 g water/g enzyme. The most important difference from Figure 10was that continuous removal of water under conditions of solvent ebullition prevented therapid deactivation of the enzyme observed in the previous case and allowed a highconversion yield to be reached in a short time. [33]

3.2.2.5 Effect of initial addition of water

Figure 12 . Effect of initial water addition onthe kinetics of butyl butyrate synthesis.Standard conditions were used except forinitial water addition, which was as follows: , 0 g. ; , 1 g. ; , 10 g. ; ,50g.

Figure 13 . Effect of initial wateraddition on the initial esterification rate,calculated from the data of Figure 12and similar experiments at other waterconcentrations


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As removal of water had little effect on the yield of esterification, and as previousresults suggested the formation of a discrete aqueous phase during the progress of thereaction, the use of biphasic hexane-water systems could be considered. [34] Variousamounts of water were introduced into the medium, all other conditions being standard.The time course of butyl butyrate production at different water concentrations is shown in

Figure 12. There are two observation that can be deduced from the result of experiment:(a) In biphasic systems, e.g. at 50 g. water, the reaction was complete after about 6hours. Butyl butyrate was exclusively recovered in the hexane phase. The totaltransfer of the ester into the organic phase explains why the reaction could occur tocompletion. [35]

(b) As the initial water concentration increased, the activation phase tended todisappear. Initial rates of esterification as a function of water concentration havebeen represented in Figure 13. Different domains can be observed. First, at lowwater content, the initial rate increased up to a maximum at water concentrationsbetween 1 g. and 5 g. . This result confirms that hydration favoured enzymeactivity. At higher water concentrations, the initial esterification rate decreased to a

minimum at 10 g. and was completely restored at 25 g. and above, where theformation of a biphasic system was visually observed. Between 5 g. and 25 g.concentration of water, the enzyme tended to adsorb on the glass walls of flasks

or to form compact agglomerates. [36]

3.2.2.6 Effect of substrate concentration

Substrate concentration may have an effect on the activity of the enzyme and alsoon the distribution of water within the medium. [37] An important point is that increasingsubstrate concentrations greatly affected the hydrophilicity of the medium and hence thewater solubility (i.e. adding 0.25 M of butanol and butyric acid to hexane increased thewater solubility from 50 ppm to 800 ppm).

Figure 14 . Effect of substrate concentrationon kinetics of butyl butyrate synthesis.Standard conditions were used except thatthe equimolar concentrations of butanol and

butyric acid were as follows: , 0.25 M;0.5 M; , 1 M; , 2 M


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Equimolar concentrations of butanoland butyric acid were used in the range 0.25-2.0 M, and the kinetics obtained are shown in

Figure 14. The maximal rates were dependent on substrate concentration: 0.14 mol.. at 0.25 M; 0.22 mol. . at 0.5 M; 0.121 mol. . at 1M; 0.02 mol. . at 2 M. At high substrate concentration, activation of theenzyme occurred later (8 hours for 1 M, 20 hours for 2 M instead of 3 hours at 0.25 and 0.5M). If water available to the enzyme is defined as water produced minus water in theorganic medium, it can be noticed that activation occurred at equivalent amounts of available water when adding either 0.25 M or 2 M substrate.

Higher substrate concentrations led to higher water solubility in the medium anddelayed the activation of lipase (Figure 15). The final yield at 2 M substrate was only 60%after 200 hours. The dependence of instantaneous esterification rates on the amounts of available water in the synthesis with 2 M substrate is shown in Figure 16. The shape of the

curve obtained is quite similar to that observed with initial rates vs amounts of added water(Figure 13). A maximum was attained at 5 g. of water and a minimum at 10 g. ,suggesting a similar behaviour of the enzyme towards the available water, whether addedto the medium or produced by the reaction. Nevertheless, it appears that at waterconcentrations above 10 g. , synthesis rates were significantly lower than when waterwas introduced exogeneously into the medium.

Figure 15 . Enzymatic synthesis of butylbutyrate at 2 M butanol and butyric acid.Other parameters were as for standardconditions. Concentrations of products as afunction of time: , butyl butyrate; O, waterin solvent

Figure 16 . Enzymatic synthesis of butylbutyrate at 2 M butanol and butyric acid.Changes in the instantaneous esterificationrate as a function of the concentration of available water, calculated from the data of Figure 15


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3.3 Kinetic study of esterification of rapeseed oil contained in waste activated bleachingearth using Candida rugosa lipase in organic solvent system

3.3.1 IntroductionThe synthesis of methyl esters by chemical transesterification has accomplished high yields in

a short reaction time. However, pretreatment of the substrate is required when water is present, andthere are drawbacks to the synthesis, including difficulties in the recovery of catalyst and glycerol,the high energy requirements, and the treatment of wastes, all of which are disadvantages in alkalior acid-catalyzed processes. Therefore, the application of lipase which is environmentally friendlyand requires no pretreatment to hydrolyze or synthesized esters has attracted great interest. Wasteactivated bleaching earth (ABE) is applied to the production of fatty acid methyl ester (FAME)using Candida rugosa lipase in an organic solvent system. [38] Activated bleaching earth is one of the most commonly used absorbents for the removal of carotene, chlorophyll and othercomponents formed during the refining process, due to its high absorption capacity. Waste ABEcontains nearly 40% of its weight as oil, a substrate that should be utilized for the synthesis of awide range of products to be used as bulk chemicals. This waste ABE was applied to produce the

FAME using lipase in either a water or organic solvent system.[38-39]

When the extracted vegetableoil from the waste ABE was used for esterification using lipase, the conversion of vegetable oil inthe organic solvent systems was only 13% (w/w). However, in the presence of ABE in the organicsolvent system, the conversion improved dramatically. [38]

3.3.2 Results

3.3.2.1 Effect of ABE on the esterification of rapeseed oilWhen the ABE ratio was lower than 0.3, no FAME was detected. However, the initial

FAME formation rate increased rapidly with the increase in the ABE ratio from 0.5 to 0.7(Figure 17). Higher than 0.7, the initial FAME formation rate decreased rapidly, which

might be due to incomplete mixing of the viscous reactant. The optimum ABE ratio was0.7 where the initial FAME formation rate was the highest.

To find out why the FAME formation rate was affected by the ABE ratio, the lipaseactivity was measured after the reaction. The residual lipase activity increased with theincrease in the ABE ratio (Figure 17). The ABE maintained the enzyme activity at a highlevel without the inactivation of lipase. However, when methanol was not added in thereactant, the enzyme activity remained without inactivation regardless of the presence of ABE in the reaction which indicates that methanol is the main inhibitor deactivating thelipase in the esterification of rapeseed oil.

3.3.2.2 Effect of the addition order of reactant on esterification of rapeseed oil contained in waste

ABE

The waste ABE, methanol, enzyme and solvent were added in six different orders, asshown in Table 2. In the case of the first addition of methanol, the initial FAME formationrate was 2.5 3 times as high as that of the third addition of methanol (Table 2) . Theaddition of lipase and solvent did not show a significant influence on the FAME formationrate. This result surmises that the decrease in the FAME formation rate in the third addition
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of methanol may be caused by the inactivation of lipase due to the presence of methanol inthe reaction mixture.

Run no. Addition order Initial FAME formation rate (mM/min )a Correlation coefficient

1 Waste ABE, MEOH, enzyme, solvent 1.2 0.98

2 Waste ABE, MEOH, solvent, enzyme 1.2 0.99

3 Waste ABE, enzyme, MEOH, solvent 0.7 0.97

4 Waste ABE, solvent, MEOH, enzyme 1.1 0.99

5 Waste ABE, solvent, enzyme, MEOH 0.4 0.96

6 Waste ABE, enzyme, solvent, MEOH 0.5 0.97

Table 2

Figure 17 . Effect of ABE ratio on the initial FAME formation rate (open circle) and residual lipaseactivity (closed circle). Rapeseed oil 70 g, n-hexane 120 ml, lipase 6 g and the ABE ratio to ABE andthe rapeseed oil of 0 0.75 were mixed on a 500 ml Erlenmeyer flask capped with rubber stopper andincubated at 37 C for 30 min with shaking at 120 strokes per minute (spm). The reaction was startedby the addition of methanol 10.2 g at the same condition and carried out for 8 h.
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3.3.2.3 Effect of initial methanol concentration on FAME formation rateThe methanol concentration in the reaction mixture was varied. The effect of this

variation on the esterification of rapeseed oil in the presence of ABE and the FAMEformation rate were investigated, as shown in Figure 18. In the absence of ABE in thereaction, the maximum initial FAME formation rate was low. With the increase in the ABE

concentration in the reaction mixture the FAME formation increased rapidly. However,when the ABE concentration was higher than 260 g/l, the peak of FAME formation ratesshifted from left to right and broadened widely. This indicates that the presence of ABErelieves the inhibitory effect of methanol on the enzyme, because of the adsorption of methanol by ABE.

3.3.3 DiscussionABE with its activated, porous, three-layer structure of silica-alumina-silica has been used in

adsorbing the dark color of crude oil, which is caused by chromophoric chloroplast-relatedmaterials that undergo different degrees of polymerization.

The esterification reaction in n-hexane was carried out with and without ABE. When ABE wasadded the FAME formation rate improved remarkably as its presence in the reactants absorbed therapeseed oil and methanol. The rest of the rapeseed oil was dissolved in the solvent, but there wasvery little free methanol in the solvent due to the low solubility of methanol in an organic solvent.However, the lipase acts a catalyst in the solid phase of an organic solvent, and converts thedissolved and adsorbed rapeseed oil to the FAME. The esterification reaction is assumed to becarried out on the surface of the ABE after a co-immobilization of rapeseed oil and methanol. Theorganic solvent may be effective in extracting the triglycerides embedded in waste ABE and

Figure 18 . The effect of the initial methanol concentration in the presence of variousABE concentrations. A reaction mixture consisting of 114 mM of rapeseed oil, 1.8 g/l of lipase (added as powder), ABE ranging from 0 g/l to 450 g/l, n-hexane and methanol.The methanol concentration in the reaction mixture was varied from 25.6 mM to800 mM. The working volume was 28 ml. The ABE concentrations were: 0 g/l (openrhombus), 60 g/l (open triangle), 130 g/l (open square), 190 g/l (open circle), 260 g/l(closed rhombus), 320 g/l (closed triangle), 380 g/l (closed square) and 450 g/l (closedcircle), respectively. An arrow indicates the increase of the ABE concentration in thereaction mixture.
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facilitating the generation of an active conformation of lipase, which contributed to the markedimprovement in FAME production. The ABE may also play an important role in the activation of lipase, probably caused by the interaction of its functional groups with those involved in theconformational change of the enzyme. The interfacial activation of the enzyme may be enhancedby its adhesion to the surface of ABE, leading to a more active conformation of the enzyme and to

a better dispersion of the enzyme molecules.However, when ABE is absent, the lipase exists as a solid phase, but rapeseed oil and methanol

exist in a liquid phase. The affinity of methanol to lipase in the reaction mixture is higher than thatof organic solvent, which causes an easy inactivation of lipase. This phenomenon is shown in theresults (Figure 18) .

The methanol inhibition is generally observed during lipase esterification andtransesterification reactions [40-41] . Methanol which is not only substrate, but also inhibitor of lipase,binds to lipase and makes the lipase methanol complex, catalytically inactive. To avoid theinhibitory effect of alcohol on the ethanolysis of sunflower oil, silica gel is added to the reaction toobtain a higher conversion yield of sunflower oil from ethanolysis than that obtained in thestandard condition as the inhibition of methanol due to the interference of the interaction of the

lipase molecule with methanol and that three-step esterification successfully converted 94%soybean oil to methyl esters. [42-43]

ABE was a good enhancer of the FAME production in lipase-catalyzed esterification. Theglycerol produced during alcoholysis is adsorbed in the waste ABE, which may favor the reactiontowards product formation.

When using waste ABE, a FAME is obtained directly following filtration after esterification.FAME could be recovered from the oil-free waste ABE by extraction using n-hexane. The organicsolvent may be recovered and recycled into the process. [43]

4.0 Conclusion

Enzymatic esterification plays a vital role in the current food and cosmetic industries. In the foodindustry, esters produced which has a pleasant scent and taste are widely used for flavouring and also inmanufacturing of butter and margarine. Emulsions containing sucrose esters have been recognized in thecosmetic industry to provide measurable improvements in skin tolerance and sensorial properties. Besidesthat, esters such as ethyl oleate, can be used as industrial solvents for pharmaceutical manipulations, aslubricant or plasticizer. Hence, a better understanding towards esterification is vital as this leads to theadvancement of technology where esterification can be optimized for different functions.

Corresponding to this, various experiments have been conducted to find out the most desirable conditionto carry out the esterification process. In order to find a way to minimize the cost of production as well asthe time of reaction, but at the same time increase the yield of useful product, researchers all over the world

have done researches and carried out investigations intensively.Various factors which lead to significant effect on the rate of esterification have been investigated.First of all, the effect of concentration on the reaction rate for both enzymes and substrates. We shall

first look into the concentration of enzymes. Enzymes are very specific proteins that catalyze a chemicalreaction. Both the enzyme and the substrate possess specific complementary geometric shapes that fitexactly into one another. This is often referred to as "the lock and key" model. In a reaction which iscatalyzed by an enzyme, the substrate is bound onto the enzyme which lowers the activation energy of thereaction. This results in the proportionality of the concentration of the enzymes to the rate of reaction,
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where the higher the concentration of the enzymes, the higher the rate of reaction. However, as time goesby, the substrates are gradually being used up. During this time, the concentration of the substratesbecomes the limiting factor where enzymes exist in excess. The reaction will then become first order withrespect to concentration of substrate.

Next we come upon the concentration of substrates. According to the Michaelis-Menten kinetics theory,

the rate of the reaction increases together with the increase in the substrates concentration. The reaction ratereaches maximum as the mixture becomes saturated with substrates for reaction to occur. However, the rateof reaction doesnt increase linearly with the substrate concentration. In the esterification reaction, theconcentration of substrate plays a role on distribution of water in the medium as well as the hydrophilicityof the medium. Higher substrate concentrations led to higher water solubility in the medium and delayedthe activation of lipase.

Secondly, the effect of temperature is studied. Commonly, a specified enzyme can only catalyze specificreaction(s) in a certain temperature. An optimum temperature is whereby a specified enzyme has thehighest efficiency before denatured. The activity of enzyme increases with temperature until it reachesmaximum at optimum temperature. An optimum temperature is whereby a specified enzyme has thehighest efficiency before denatured. The geometric shape of the enzyme will be changed resulting in the

substrate can no longer fit into the enzyme. Therefore, the enzyme lost its function to catalyze the reactionand hence, the rate of reaction drops.The next factor is the continuous removal of water. Esterification produces water and since this is a

reversible reaction, the increment of water concentration as the reaction proceed will slow down theforward reaction, the production of ester. Therefore, the removal of water which decreases the waterconcentration in the esterification medium will speed up the forward reaction rate.

Finally, the initial addition of water into the medium of esterification will also affect the rate of esterification. When initially introduced into the medium, water allows maximum rates to be obtained atthe beginning of synthesis. Activation of the enzyme by the water produced was observed in dehydratedmedia. In biphasic system, the yield of the ester will increased as the ester will be transferred into theorganic phase. Due to equilibrium, the ester concentration in aqueous phase will be reduced in order toproduce more ester.

As a compendious ending, various factors, such as enzymes and substrates concentration, temperature,initial addition of water and removal of water can affect kinetic and yield of esterification via enzymaticroute. Further studies on esterification must be carried out in order to produce

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