Thermodynamic Evaluation Of Hydrolysis And Esterification Reactions

Published Date: 02 Nov 2017

The present paper investigates about the thermodynamic properties for the two major reaction catalyzed by lipases viz. hydrolysis of fish oils and esterification of fatty acids. The hydrolysis reaction of three fish oils such as tuna, salmon and herring with immobilized Candida antarctica lipase-B (CAL-B) and selective esterification of tuna free fatty acids with immobilized lipases from Pseudomonas cepacia (PCL) and Thermomyces lanuginosus (TLL) have been studied. For both the reactions, thermodynamic constants were determined such as change in Gibbs free energy (ΔG), enthalpy (ΔH), entropy (ΔS) to understand the physical significance of these reactions. Additionally, the catalytic efficiency of lipases has been compared with turn-over number (kcat) and thermal deactivation constant (Kd). The negative magnitude of thermodynamic parameters (ΔG<0, ΔH<0 and ΔS<0) have been obtained, corresponds to the feasibility of hydrolysis and esterification reactions with thermal stability of lipases in reaction conditions. A maximum turn over number (Kcat = 636 sec-1) was observed for esterification with immobilized PCL, indicating a good shell life of enzyme whereas a minimum thermal deactivation (Kd = 80.8 KJ/mol) was obtained with immobilized CAL-B for fish oil hydrolysis.

Keywords: Enthalpy, entropy, Gibbs free energy, thermal deactivation constant and turn-over number.

INTRODUCTION

A diverse class of lipases (EC 3.1.1.3) is available in present era, which not only differs in their activity but also show significantly unique mode of action because they are originated from different sources. They can be synthesized and extracted from various sources such as plant [1], animal [2] and microorganisms [3]. The type of reactions, catalyzed by them can be broadly classified as hydrolysis and esterification, depending upon the nature of substrate available. A hydrolysis reaction is one in which ester bonds breaks in the presence of water to form alcohol and fatty acids whereas reverse is esterification, means ester bonds form with alcohol. It has been seen that same set of lipases can play important role in both hydrolysis and esterification reactions but with different rate and mechanism [4-5]. Lipases do not change the position of equilibrium in a reaction but they change the amount of energy required to achieve the same position of equilibrium in both forward and reverse reactions. The magnitude of energy required can be explained in terms of various thermodynamic parameters such as Gibbs free energy (G), enthalpy (H) and entropy (S) for both reactants and products involved in a reaction. In total, the difference of energy states for converting reactant into product is defined in form of change of Gibbs free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) [6-7]. For a lipase catalyzed reaction, the interaction of a protein molecule in lipase with reactant or product can be due to the formation of hydrogen, van der Walls, covalent, hydrophobic and electrostatic bonds. The type of interaction exists between two molecules can also be examined using the data of ΔG, ΔH and entropy ΔS [8-9].

The present study aims to understand the feasibility of a lipase catalyzed reaction in terms of their thermodynamic behaviour to compare amount of energy released or required. Similarly, the catalytic activity of a lipase is also evaluated in terms of two mathematical constants, known as turn over number (kcat) and thermal deactivation constant (Kd).

MATERIALS AND METHODS

Substrate and lipases

Three types of fish oils such as Tuna (refined and deodorized grade; Lot: 0038601), Salmon (blend; Lot: 029/11) and Herring (veterinary grade; Lot: 802-6980), were purchased from Jedwards International, Inc., Quincy MA 02169, USA in a packing of 3 liters each. For the hydrolysis of fish oils triglycerides, lipase-B from Candida antarctica (CAL-B) immobilized on immobead-150 (0.15-0.3mm; <10% loss on drying), recombinant from yeast (activity >2000U/g) were used. For the selective esterification studies, Pseudomonas cepacia lipase (PCL) and Thermomyces lanuginosus lipase (TLL) immobilized on immobead-150 were used. The commercial properties of all the lipases are given in Table 1 as provided by Sigma-Aldrich, Saskatoon, Canada [10].

Chemicals and solvents

Analytical grade chemicals such as methanol (CH3OH), potassium hydroxide (KOH), oxalic acid, phenolphthalein indicator, anhydrous sodium sulfate, sodium chloride, hydrolchloric acid, BF3/methanol (~1.3 M) 10% solution, lauryl alcohol and glycerol, were purchased from Sigma-Aldrich, Canada and used in the experimental work. Potassium di-basic & mono-basic were used for preparing phosphate buffers of different pH. All the chemicals used were of AR grade. Various polar and non-polar solvent have been used in the study for providing bi-phasic solvent environment to the lipase and substrate such as hexane, iso-octane, di-chloro methane and tetra hydrofuron (THF) at different stages of the reaction (CDH make and high purity HPLC grade). Industrial grade N2 gas cylinder has been supplied by Pyrex for maintaining inert atmosphere during the reaction.

Experimental details

The enrichment of free fatty acids containing DHA has been conducted by the hydrolysis of fish oils fish oil with immobilized CAL-B. The optimization of parameters was carried out at fixed pH 7.0 in orbital shaker (supplied by VWR, Canada; model: 1570) for 1 h with tuna fish oil only and then same conditions have been used in case of three fish oils. For studying the hydrolysis reaction, fish oils (0.25-1.5 g) were dissolved in one g solvent. 0.13 g of immobilized CAL-B along with the distilled water (3 g) and 1 g of pH 7 phosphate buffer was added to prepare the reaction mixture in a 125 ml Erlenmeyer flask. Standard AOCS and ASTM test methods were used for estimating the acid value [11-12] of product samples at different reaction times to determine moles of free fatty acids (FFAs) formed per ml of reaction mixture.

The tuna free fatty acids (TFFAs) have been extracted and purified from tuna fish oil by the enzymatic hydrolysis with immobilized Candida antarctica lipase-B (CAL-B). Therefore, the extracted tuna free fatty acids have been further utilized as substrate for the selective esterification reaction. For studying the esterification reaction, TFFAS (1 g) were dissolved in 1 g of iso-octane, 0.53 g of lipase along with the lauryl alcohol (3g), 0.5 g of buffer (pH 8) at 50 ºC and 800 rpm speed up to a duration of 72 h. The reaction was conducted continuously in a small glass reactor, placed in an oil bath on hot plate which was equipped with electrically driven high rpm motor, thermometer for monitoring stable temperature of reaction mixture and sampling port for constant sample collection (1 ml) at defined time intervals. The activity of lipase for esterification has been expressed as moles of DHA esters formed per g of immobilized lipase per min. The experiments were performed in repeated sets and a variation in TFFAs ester formation <±5 % was observed.

Standards and Analysis

An Agilent make Gas Chromatography system (model 7890A) has been used, equipped with flame ionization detector (FID, 260C) and capillary column DB-23 (dimensions: 60 m length, 0.25 mm ID, 0.25 m film) for analysis of free fatty acids in methyl ester form (FAMEs). The initial composition of TFFAs was analyzed by converting the free fatty acids into methyl ester form with BF3/methanol solution under inert N2 purging atmosphere. Throughout the experimentation, the GC was operated at constant conditions (Carrier: hydrogen gas with flow rate 20 cm/min & 23.148 psi pressure; Oven: 140 to 240 C at 4C/min. and Injection: 1l sample, 260 C & split: 20:1). Supelco 37 component FAME mixture (Catalog No. 47885 Supelco and Lot No. LB-85810; 10 mg/ml in methylene chloride) was purchased from Sigma-Aldrich, Canada for GC calibration and analysis [10].

RESULTS AND DISCUSSION

The thermodynamics is concerned with the change in energy and similar factors as biochemical process takes place. Thermodynamic study for both the reactions hydrolysis and selective esterification were carried out at optimized reaction conditions, estimated separately. In connection with this, initially dependency of rate constant on temperature was studied at 25, 30 & 35 ºC and 30, 40 & 50 ºC for hydrolysis and esterification respectively. After that, various thermodynamic constants were determined such as change in Gibbs free energy (ΔG), enthalpy (ΔH), entropy (ΔS) to understand the physical significance of these reactions. Additionally, some thermodynamic constants for the lipases used, have also been evaluated such as turn-over number (kcat) and dissociation constant (Kd). For a hydrolysis and esterification reactions, the rate dependency on temperature was determined using Arrhenius law as given below in Eq. I.

Where, k = rate constant; Ao = pre-exponential factor; E = activation energy (J/mol); R = gas constant and T = temperature (Kelvin). The linear form of Arrhenius equation is given in Eq. [II].

The percentage conversion of triglycerides into free fatty acids and tuna free fatty acids into esters have been plotted with time (up to 2 h) to determine the value of rate constant (k) at varying temperatures for hydrolysis of fish oils (see Table 2) and esterification of TFFAs (see Table 3) respectively. Therefore, ln k was plotted with respect to the reciprocal of temperature as given in Figure 1 and 2 with data in Table 2 and 3 respectively.

Thermodynamic functions such as Gibbs free energy, enthalpy and entropy, are functions of state. This means that they depend only on the state of the system being considered and not on how that system came into being. Changes in the functions of state between two states depend only on the initial and final states and not on the route between them [7]. For a chemical reaction, the change in the Gibbs free energy function (ΔG) is the energy which is available to do work as the reaction proceeds from the given concentrations of reactant and products to chemical equilibrium. The enthalpy change (ΔH) is defined as the quantity of heat adsorbed by the system under the given conditions. Whereas the increase in the entropy of surroundings is represented by (-ΔH/T) and the increase in the entropy of system is (ΔH/T). For any spontaneous process at constant temperature and pressure, J. Willard Gibbs in 1878 defined the increase in the free energy of the system (ΔG) as

According to Eq. [IV], a plot of ln k against 1/T, will give a straight line whose slope will be equal to ΔH/RT and intercept will be ΔS/R [13]. The thermodynamic catalytic constant of an enzyme which is a measure of its catalytic efficiency can be defined as given below:

This quantity is also known as the turnover number (Kcat) of an enzyme because it is the number of reactions processes that each site catalyses per unit time [14]. When using expensive catalyst, the turnover number should be as high as possible so as to reduce the cost of the product [15]. Immobilized enzymes lose their catalytic activity upon reuse over time. The constant of deactivation (Kd) was calculated with Eq. [VI] where Tmax is the maximum reaction temperature (in kelvin). The dissociation constant value shows the deactivation nature. The magnitude of Kd can also be determined according to a thermodynamic study conducted by Gitin et al. in 2006 for the thermal deactivation of Novozym 435 assuming enzyme may be reversible, irreversible or a combination of the two. In this study, it was concluded that the smaller values of Kd is a indicative for the more active enzyme [16-17].

To determine the magnitude of various thermodynamic constants for both hydrolysis and esterification reaction Eqs. [III] to [VI] has been used simultaneously. The change in enthalpy (ΔH) and entropy (ΔS) were first determined from the slope and intercept of Figures 1 and 2 using Eq [IV]. Then, change in Gibbs free energy (ΔG) was estimated with Eq [III]. The turnover number (kcat) was further calculated using maximum rate of reaction (Vmax) and total enzyme concentration [Et] which were calculated separately with the kinetic study for these two reactions. The maximum rate of reaction was found to be 0.14, 0.08 and 0.06 (moles FFAs/sec.ml) for hydrolysis of tuna, salmon and herring fish oils respectively at 7.4 x 10-4 (µmoles/ml) concentration of immobilized CAL-B. Similarly, 2.8 and 0.58 moles DHA esters formed per sec per volume (ml) of reaction mixture with immobilized PCL and TLL respectively at 4.4 x 10-3 (µmoles/ml) concentration. The deactivation constant was calculated at optimized maximum reaction temperature i.e. 35 and 50 ºC for hydrolysis and esterification reactions respectively. The results calculated are reported in Table 5 and 6 for hydrolysis of three fish oils with immobilized CAL-B and esterification of TFFAs with immobilized PCL and TLL respectively.

According to the results obtained from the theoretical calculations of thermodynamic parameters for hydrolysis and esterification reaction, it was seen that the values of change in Gibbs free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) were negative in magnitude in all the cases. In the literature various explanations have been given by different researchers in this regard. According to Ferreira et al. 2010 the negative magnitude for ΔG and ΔH correspondence to the spontaneous and exothermic nature of the lipase catalyzed reactions [18-19]. Graber et al. in 2002 have studied the effect of water on the alcoholysis of methyl propionate and n-propanol catalyzed by immobilized Candida antarctica lipase-B in a continuous solid/gas reactor and in organic liquid medium. According to him, the change in the magnitude of entropy (ΔS) for lipase catalyzed reactions may be either positive or negative. A negative value indicates the fast and convenient formation of ES complex when enzyme (E) binds with substrate (S) which causes considerable loss in entropy. The consumption of reactant with the progress of reaction combined with release of translational and rotational energies are mainly responsible for the loss in entropy, which is most common for enzymatic reactions [20-21]. Li et al. 2011 have also reported the negative magnitude of the three parameters (ΔG, ΔH and ΔS) for the interaction of flavonoids with pancreatic lipase, mainly due to the formation of a enzyme-substrate complex [8]. For the hydrolysis of three oils, a maximum the turnover number (kcat) 189.2 sec-1 was found for tuna fish oil than salmon and herring. Whereas among the two lipases used for esterification, kcat was obtained as 636.4 and 131.8 sec-1 for immobilized PCL and TLL respectively, corresponds to the highest catalytic effectiveness of immobilized CAL-B for hydrolyzing tuna fish and immobilized PCL for esterification reaction. Additionally, Gitin et al. in 2006 have shown in their studies that the lower magnitude of thermal deactivation constant is desired, indicative of highly active enzyme. In case of both the reactions including three immobilized lipases such as CAL-B, PCL and TLL. It has been seen from the results that immobilized CAL-B with herring fish oil is most active for hydrolysis and immobilized TLL retains better activity than immobilized PCL for esterification of fatty acids [16].

CONCLUSIONS

The present study concludes that the hydrolysis and the esterification reactions catalyzed by different lipases are thermodynamically favorable according to the magnitude of the thermodynamic constants (ΔG, ΔH and ΔS) obtained. The negative magnitude of these parameters also indicates the thermal stability of lipases involved at maximum reaction temperature. The parameters for measuring catalytic efficiency (kcat) and thermal deactivation (Kd) for immobilized CAL-B and PCL & TLL lipases during hydrolysis and esterification reactions have been calculated. The results indicate that immobilized PCL has highest turnover whereas immobilized CAL-B was showing least thermal deactivation with time.

ACKNOWLEDGEMENT

Ms. Sharma would like to present her sincere gratitude & thanks to Prof. Aditya Shastri, Vice Chancellor, Banasthali University, Rajasthan, India for providing her study leave to perform her research work at Department of Chemical & Biological Engineering, College of Engineering, University of Saskatchewan, Saskatoon, Canada. Also, the funding provided by Canada Research Chair (CRC) Program and by Natural Sciences and Engineering Council (NSERC) of Canada in terms of student assistance ship to Ms. Sharma for this research is acknowledged.