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

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

ChE - 218

by Priyanka Tiwari February 13, 2012

1

.............. 6 Evaluation of the study ................................................................................................................................................................................ 3 Methods (Mathematical and Experimental Approach) ................. 3 Assumptions and Variables .......................................................................... 6 Conclusions .................. 5 Experimental and Theoretical Results.................................................................................2 2.................................................1 4.....................5 3 4 Summary .. 9 5 Reference .................................................................................... 3 2............................................ 3 Mathematical Method employed .......................................................................................4 Was the study worth doing? ............2 4................................................................................................... 10 2 ...................... 8 Are conclusions reasonable? ................................3 2........................................................................................................................................................................................................................ 8 Credibility of findings ............................... 7 Analysis of the experimental data ...................................................... 4 Experimental Methods employed.....................3 4.............................................................................................Contents 1 2 Purpose of Study....................................1 2..................................................................................... 7 4..4 2.............

3 . vm is the maximum reaction rate μmolmin-1mg-1. [S] is substrate concentration mol/dm3. This set up results in easy separation of products from the enzyme. Fatty acids act as a feedstock for several chemicals such as lubricants. v is the reaction rate of product formation . Because of its application in food industry.  The values of Km and vm are estimated using the Lineweaver-Burk Plot (obtained from linear transformation of Michaelis-Menten equation). These are also used as raw materials for food and cosmetic products. Hydrolysis of oil leads to the production of fatty acids and glycerol. E+S v vm ES  E + P. Authors have used lecithin/isooctane reversed micellar scheme for extraction.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. the hydrolysis utilizes natural micellar system called lecithin which is non-toxic in nature [1]. etc.1 Purpose of Study This report analyses reaction kinetics explained for enzyme catalyzed hydrolysis process of palm oil. 2 2. soaps. Therefore we get the following relationship m where. 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. Km is called as MichaelisMenten constant (mol/dm3).

[P] and [Q] are concentrations of products. the following equation is obtained: [ ] -----------------------------------. researchers obtained the following equation: [ -----------------------------------------------------------. one gets [ ][ At equilibrium value of [ ] --------. [ ] [ ] 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).(6) 4 . single substrate reversible kinetic model.(3) ]. Also [P] = [Q] = [ ] Using (1) and (2). So on calculating and substituting its value in equation (3).(4) and X = Xe (degree of hydrolysis at equilibrium).(5) Integrating equation (5). the degree of hydrolysis is calculated as: ------------------------------------------------------------------[ ] is the initial substrate concentration. [W] is the concentration of water. 2. Therefore the reaction mechanism can be represented as: S+W P + Q where. [S] is concentration of palm oil (ester bonds).3 Mathematical Method employed From the reaction mechanism. The rate equation for the process S + W v Where. It is also assumed that the reaction is first order. ----------------------------------------------.

from equation (6). buffer solution of lipase with pH 7.At t = tm . reaction time Formation of products ( Free fatty acids) Vs. To start the reaction.0 is added to the reaction mixture.g. reaction time 5 . The concentration of substrate is varied from 0. The degree of hydrolysis is calculated by finding the acid value and saponification value of the oil in the reaction mixture. [ Solving for [ Where 2.(7) . is calculated as ] ------------------------------------------------------.(8) Experimental Methods employed Researchers have derived a mathematical model for hydrolysis of palm oil (equation 8). Following parameters are obtained with respect to time:   Degree of hydrolysis Vs.480mol/dm3. The reaction is formed as a batch process. iso-octane and n-hexane. the researchers arrived at following equation ] ------------------------------------------------------------------..059mol/dm3 to 0.4 ] [ . The process is carried out at a fixed temperature of 37˚C. The authors have performed palm oil hydrolysis in laboratory to obtain experimental data. Two organic solvents are tested e. 100cm3 of bottles are filled with 12 cm3 of lecithin/organic solution containing varying amount of substrate concentration (palm oil).

Therefore the data doesn‟t fit the model equation well at large values of substrate concentrations (Figure 1).02 μmol min-1mg-1 and the values of are 0.746mol/dm3). 3 Conclusions Parameters such as m vm are obtained from Lineweaver-Burk plot. This shows a significant difference in the two values of m. lower reaction rates are obtained. Thus it was considered as more efficient solvent for the process.. Figure 1.6mol/dm3 for isooctane and n-hexane respectively.5 Experimental and Theoretical Results The experimental results show that the kinetics of the hydrolysis follows Michaelis-Menten kinetics for low initial substrate concentrations[ ]. The values of vm are m 1. they inferred that use of lecithin isooctane solvent increases the enzyme affinity for the substrate during reaction. 6 . It is found that for high [ ] (for e. Theoretical results (obtained from mathematical model.g.243mol/dm3 and 0.2. Shows the deviation of experimental and theoretical data at high substrate concentration (0. equation (8) predict a higher rate for high[ ]. 0.480 dm3). As the authors obtained a low value of m for isooctane system.29 μmol min-1mg-1 and 1.

the researchers 7 . This is because the process occurs at low temperature.g. „lipase‟. 250 ˚C and 50 bars. This makes the whole process a high energy intensive mechanism [2]. Low operating temperatures prevent enzyme denaturation and thus help in reducing process cost.   Due to the reduction in size of micellar radius.The authors concluded that the experimental results of the process fitted the model equation well for low values of[ ]. Under these conditions polymerization of fats occur that results in the formation of extremely dark colored fatty acids. 37˚C 3 . 4 4. This is because the microorganisms producing the enzyme can be genetically manipulated. In addition to this. Therefore distillation is further required for products purification.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.. It‟s also considered as an environment friendly process since it involves mild operating temperature. i. the basic Michaelis-Menten equation doesn‟t hold true. Enzyme catalyzed reactions thus reduce thermal degradation of the products formed. In this case.e.. Due to deactivation of enzyme during the course of the reaction. The industrial process of hydrolysis works at high operating temperatures and pressures e. The process is also economical as the enzyme. is available at low cost. 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.

However at high range of this concentrations [ ]. Overall we can say that the data from experiment is good. 8 .2 Analysis of the experimental data The authors have provided data on degree of hydrolysis versus reaction time at varying initial substrate concentration [ ] .3 Credibility of findings Analysis of results can be summed up as:  The authors have made highly simplistic assumptions.have utilized a reverse micellar system for the process. first order kinetics. This set up requires less energy for increasing interfacial area to perform hydrolysis. The process of hydrolysis is however complex enough for such an assumption. deviations from Michaelis-Menten is observed. There is no information though on the repeated runs performed for obtaining this data. Thus it can be inferred that the study is energy efficient. First. the authors have assumed that the process of hydrolysis follows single substrate. The model is not in a generalized form. „temperature‟ (figure1) follows Michaelis-Menten kinetics consistently at different lower initial substrate concentrations [ ]. low cost and environment friendly process that makes it a worth studying process. Triglycerides present in oil can be broken down to diglycerides and monoglycerides and may follow complex parallel reaction path. Thus the data doesn‟t 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. Hence the mathematical model obtained is not reliable. This hints that the authors have made too simplistic and ideal assumptions for the process. 4. Thus the data looks good. The plot of „degree of hydrolysis‟ Vs. 4.

9 . The value of Km obtained is however suspicious because of the use of Lineweaver Burk plot for its determination. It is known that linearization adds a lot of error to a model.  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. the proposition doesn‟t sound appropriate. Therefore the value of Km obtained is erroneous. Finally the authors have also proposed enzyme deactivation as a probability for this deviation. Also the effect of varying pH.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. the values of Km and vm are estimated using Lineweaver Burk plot. The effect of substrate inhibition on reaction kinetics is also correct inference derived by the authors. Secondly. enzyme activity and concentration of reversed micellar solution (surfactant) are not analyzed to determine the degree of hydrolysis. ince the process is carried out at a low temperature (37˚C) and optimum pH (pH 7. No attempt is made to modify the model for substrate/product inhibition.  Authors have not discussed the effect of agitation speed of water bath on the hydrolysis rate. The plot is a linearized form of Michaelis-Menten equation. hence the conclusion is fairly decent. In addition to this.0). 4. This is fairly reasonable stated proposition. Enzymes denaturation is result of high temperatures and fluctuating pH conditons. As we know that a low value of Km depicts high affinity of enzyme for the substrate. temperature. the authors suspect substrate/product inhibition as a reason for deviation of experimental data from Michaelis-Menten kinetics.

V. 2. “ inetics of the enzymatic hydrolysis of palm oil by lipase. 10 . 49.” Journal of Physical Science. Ramachandran. “Kinetics of lipase-catalyzed hydrolysis of palm oil in lecithin/isooctane reversed micelles.N. Hasan and . 79–88 (2008). 1155-/1163 (2003).A. Serri. Zuhair.B. 267-271 (1998).” Applied Microbiology Biotechnology. 3. . Mojovic. S. Rahaman “Preliminary Studies for Production of Fatty Acids from Hydrolysis of Cooking Palm Oil Using C. D. Kamarudin and S. nezevic.” Process Biochemistry. A. rugosa Lipase.A. N. 38. 19. Marinkovic and L.5 Reference 1. M.H. Z. A. .

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