Kinetics of lipase-catalyzed hydrolysis of palm oil in lecithin/isooctane reversed micelles

Article Review Presented to Dr. Melaine McNeil San Jos State University

ChE - 218

by Priyanka Tiwari February 13, 2012

Contents
1 2 Purpose of Study...................................................................................................................... 3 Methods (Mathematical and Experimental Approach) ........................................................... 3 2.1 2.2 2.3 2.4 2.5 3 4 Summary .......................................................................................................................... 3 Assumptions and Variables .............................................................................................. 3 Mathematical Method employed ...................................................................................... 4 Experimental Methods employed..................................................................................... 5 Experimental and Theoretical Results.............................................................................. 6

Conclusions ............................................................................................................................. 6 Evaluation of the study ............................................................................................................ 7 4.1 4.2 4.3 4.4 Was the study worth doing? ............................................................................................. 7 Analysis of the experimental data .................................................................................... 8 Credibility of findings ...................................................................................................... 8 Are conclusions reasonable? ............................................................................................ 9

Reference ............................................................................................................................... 10

Purpose of Study This report analyses reaction kinetics explained for enzyme catalyzed hydrolysis process of

palm oil.

2 2.1

Methods (Mathematical and Experimental Approach) Summary

The article selected discusses the kinetics involved in the process of hydrolysis of palm oil by using an enzyme called Candida rugosa lipase. Hydrolysis of oil leads to the production of fatty acids and glycerol. Fatty acids act as a feedstock for several chemicals such as lubricants, soaps, etc. These are also used as raw materials for food and cosmetic products. Authors have used lecithin/isooctane reversed micellar scheme for extraction. This set up results in easy separation of products from the enzyme. Because of its application in food industry, the hydrolysis utilizes natural micellar system called lecithin which is non-toxic in nature [1]. 2.2 Assumptions and Variables

The researchers have made the following assumptions for the process: The reaction kinetics of the process follows Michaelis-Menten mechanism. E+S v vm ES E + P. Therefore we get the following relationship
m

where,

v is the reaction rate of product formation , vm is the maximum reaction rate molmin-1mg-1, [S] is substrate concentration mol/dm3, Km is called as MichaelisMenten constant (mol/dm3). The values of Km and vm are estimated using the Lineweaver-Burk Plot (obtained from linear transformation of Michaelis-Menten equation). 3

It is also assumed that the reaction is first order, single substrate reversible kinetic model. Therefore the reaction mechanism can be represented as: S+W P + Q where,

[S] is concentration of palm oil (ester bonds), [W] is the concentration of water, [P] and [Q] are concentrations of products. 2.3 Mathematical Method employed From the reaction mechanism, the degree of hydrolysis is calculated as: ------------------------------------------------------------------[ ] is the initial substrate concentration. The rate equation for the process S + W v Where, [ ] [ ] P + Q is proposed as: (2) is the rate of (1)

[ ][ ] -------------------------------------------

is the rate of forward reaction (decomposition of ester bonds) and

backward reaction (formation of ester bonds). Also [P] = [Q] = [ ] Using (1) and (2), one gets [ ][ At equilibrium value of

[ ] --------- (3)

]. ----------------------------------------------- (4)

and X = Xe (degree of hydrolysis at equilibrium). So on calculating

and substituting its value in equation (3), the following equation is obtained: [ ] ------------------------------------ (5)

Integrating equation (5), researchers obtained the following equation:

[

------------------------------------------------------------ (6)

At t = tm , [ Solving for [ Where 2.4 ] [

from equation (6),

is calculated as

] ------------------------------------------------------- (7)

, the researchers arrived at following equation ] ------------------------------------------------------------------- (8)

Experimental Methods employed Researchers have derived a mathematical model for hydrolysis of palm oil (equation 8). The

authors have performed palm oil hydrolysis in laboratory to obtain experimental data. The reaction is formed as a batch process. 100cm3 of bottles are filled with 12 cm3 of lecithin/organic solution containing varying amount of substrate concentration (palm oil). Two organic solvents are tested e.g., iso-octane and n-hexane. The concentration of substrate is varied from 0.059mol/dm3 to 0.480mol/dm3. To start the reaction, buffer solution of lipase with pH 7.0 is added to the reaction mixture. The process is carried out at a fixed temperature of 37C. The degree of hydrolysis is calculated by finding the acid value and saponification value of the oil in the reaction mixture.

Following parameters are obtained with respect to time: Degree of hydrolysis Vs. reaction time Formation of products ( Free fatty acids) Vs. reaction time

2.5

Experimental and Theoretical Results

The experimental results show that the kinetics of the hydrolysis follows Michaelis-Menten kinetics for low initial substrate concentrations[ ]. It is found that for high [ ] (for e.g., 0.746mol/dm3), lower reaction rates are obtained. Theoretical results (obtained from mathematical model, equation (8) predict a higher rate for high[ ]. Therefore the data doesnt fit the model equation well at large values of substrate concentrations (Figure 1).

Figure 1. Shows the deviation of experimental and theoretical data at high substrate concentration (0.480 dm3).

Conclusions Parameters such as

m

vm are obtained from Lineweaver-Burk plot. The values of vm are

m

1.29 mol min-1mg-1 and 1.02 mol min-1mg-1 and the values of

are 0.243mol/dm3 and

0.6mol/dm3 for isooctane and n-hexane respectively. This shows a significant difference in the two values of
m.

As the authors obtained a low value of

m for

isooctane system, they inferred

that use of lecithin isooctane solvent increases the enzyme affinity for the substrate during reaction. Thus it was considered as more efficient solvent for the process.

The authors concluded that the experimental results of the process fitted the model equation well for low values of[ ]. This discrepancy between experimental and theoretical results for high value of initial substrate concentration [ ] were explained as an outcome of following possibilities: Substrate inhibition resulting in inhibited enzyme kinetics. In this case, the basic Michaelis-Menten equation doesnt hold true. Due to the reduction in size of micellar radius. Due to deactivation of enzyme during the course of the reaction.

4 4.1

Evaluation of the study Was the study worth doing? In order to carry out the hydrolysis process the researchers have adopted a high energy

efficient method. The use of enzymatic splitting of palm oil by lipase requires less energy consumption. This is because the process occurs at low temperature. The industrial process of hydrolysis works at high operating temperatures and pressures e.g., 250 C and 50 bars. Under these conditions polymerization of fats occur that results in the formation of extremely dark colored fatty acids. Therefore distillation is further required for products purification. This makes the whole process a high energy intensive mechanism [2]. Enzyme catalyzed reactions thus reduce thermal degradation of the products formed. The process is also economical as the enzyme, lipase, is available at low cost. This is because the microorganisms producing the enzyme can be genetically manipulated. Its also considered as an environment friendly process since it involves mild operating temperature, i.e., 37C 3 . Low operating temperatures prevent enzyme denaturation and thus help in reducing process cost. In addition to this, the researchers 7

have utilized a reverse micellar system for the process. This set up requires less energy for increasing interfacial area to perform hydrolysis. Thus it can be inferred that the study is energy efficient, low cost and environment friendly process that makes it a worth studying process. 4.2 Analysis of the experimental data The authors have provided data on degree of hydrolysis versus reaction time at varying initial substrate concentration [ ] . There is no information though on the repeated runs performed for

obtaining this data. The plot of degree of hydrolysis Vs. temperature (figure1) follows Michaelis-Menten kinetics consistently at different lower initial substrate concentrations [ ]. Thus the data looks good. However at high range of this concentrations [ ], deviations from Michaelis-Menten is observed. Thus the data doesnt fit the equation well at higher values of [ ] The analysis figures out that the mathematical model defining the system requires modifications to fit the entire range of experimental data. This hints that the authors have made too simplistic and ideal assumptions for the process. Overall we can say that the data from experiment is good. 4.3 Credibility of findings Analysis of results can be summed up as: The authors have made highly simplistic assumptions. The model is not in a generalized form. First, the authors have assumed that the process of hydrolysis follows single substrate, first order kinetics. The process of hydrolysis is however complex enough for such an assumption. Triglycerides present in oil can be broken down to diglycerides and monoglycerides and may follow complex parallel reaction path. Hence the mathematical model obtained is not reliable.

In addition to this, the values of Km and vm are estimated using Lineweaver Burk plot. The plot is a linearized form of Michaelis-Menten equation. It is known that linearization adds a lot of error to a model. Therefore the value of Km obtained is erroneous.

Authors have not discussed the effect of agitation speed of water bath on the hydrolysis rate. Also the effect of varying pH, temperature, enzyme activity and concentration of reversed micellar solution (surfactant) are not analyzed to determine the degree of hydrolysis.

The authors have concluded that the deviation of model from Michaelis-Menten is either due to substrate/product inhibition or as a result of reduction of micellar radii. No attempt is made to modify the model for substrate/product inhibition.

4.4

Are conclusions reasonable? First the researchers have concluded that iso-octane is a better solvent than n-hexane since Km

has a low value for iso-octane than n-hexane. As we know that a low value of Km depicts high affinity of enzyme for the substrate, hence the conclusion is fairly decent. The value of Km obtained is however suspicious because of the use of Lineweaver Burk plot for its determination. Secondly, the authors suspect substrate/product inhibition as a reason for deviation of experimental data from Michaelis-Menten kinetics. This is fairly reasonable stated proposition. The effect of substrate inhibition on reaction kinetics is also correct inference derived by the authors. Finally the authors have also proposed enzyme deactivation as a probability for this deviation. Enzymes denaturation is result of high temperatures and fluctuating pH conditons. ince the process is carried out at a low temperature (37C) and optimum pH (pH 7.0), the proposition doesnt sound appropriate. 9

Reference 1. Z. D. nezevic, . . Marinkovic and L. V. Mojovic, Kinetics of lipase-catalyzed hydrolysis of palm oil in lecithin/isooctane reversed micelles, Applied Microbiology Biotechnology, 49, 267-271 (1998). 2. S. A. Zuhair, M. Hasan and .B. Ramachandran, inetics of the enzymatic hydrolysis of palm oil by lipase, Process Biochemistry, 38, 1155-/1163 (2003). 3. N.A. Serri, A.H. Kamarudin and S.N.A. Rahaman Preliminary Studies for Production of Fatty Acids from Hydrolysis of Cooking Palm Oil Using C. rugosa Lipase, Journal of Physical Science, 19, 7988 (2008).

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