Preparation of a biocatalyst via physical adsorption of lipase from Thermomyces lanuginosus on hydrophobic support to catalyze biolubricant synthesis by esterification reaction in a solvent-free system

Accepted Manuscript Title: Preparation of a biocatalyst via physical adsorption of lipase from Thermomyces lanuginosus on hydrophobic support to catalyze biolubricant synthesis by esterification reaction in a solvent-free system Author: Fl´avia A.P. Lage Jaquelinne J. Bassi Maria C.C. Corradini Larissa M. Todero Jaine H.H. Luiz Adriano A. Mendes PII: DOI: Reference:

S0141-0229(15)30090-9 http://dx.doi.org/doi:10.1016/j.enzmictec.2015.12.007 EMT 8845

To appear in:

Enzyme and Microbial Technology

Received date: Revised date: Accepted date:

15-9-2015 15-11-2015 17-12-2015

Please cite this article as: Lage Fl´avia AP, Bassi Jaquelinne J, Corradini Maria CC, Todero Larissa M, Luiz Jaine HH, Mendes Adriano A.Preparation of a biocatalyst via physical adsorption of lipase from Thermomyces lanuginosus on hydrophobic support to catalyze biolubricant synthesis by esterification reaction in a solvent-free system.Enzyme and Microbial Technology http://dx.doi.org/10.1016/j.enzmictec.2015.12.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Preparation of a biocatalyst via physical adsorption of lipase from Thermomyces

lanuginosus

on

hydrophobic

support

to

catalyze

biolubricant synthesis by esterification reaction in a solvent-free system

Flávia A.P. Lage, Jaquelinne J. Bassi, Maria C.C. Corradini, Larissa M. Todero, Jaine H.H. Luiz, Adriano A. Mendes*

Institute of Chemistry, Federal University of Alfenas, 37130-000, Alfenas, MG, Brazil

* Corresponding author. Tel.: +55 35 3299 1477 E-mail address:[email protected]; [email protected] (A.A. Mendes)

GRAPHICAL ABSTRACT

Highlights ► Lipase from Thermomyces lanuginosus was immobilized on poly-methacrylate particles. The biocatalysts prepared were tested in isoamyl oleate (biolubricant) synthesis. ► A satisfactory combination of high catalytic activity and reusability was reached. Ester conversion ≈85% was reached after 30 min of reaction in a solvent-free system. The nature of the ester was characterized by spectroscopy analyses (ATR-FTIR and NMR).

Abstract Lipase from Thermomyces lanuginosus (TLL) was immobilized on mesoporous hydrophobic poly-methacrylate (PMA) particles via physical adsorption (interfacial activation of the enzyme on the support). The influence of initial protein loading (5–200 mg/g of support) on the catalytic properties of the biocatalysts was determined in the hydrolysis of olive oil emulsion and synthesis of isoamyl oleate (biolubricant) by esterification reaction. Maximum adsorbed protein loading and hydrolytic activity were respectively ≈100 mg/g and ≈650 IU/g using protein loading of 150 mg/g of support. The adsorption process followed the Langmuir isotherm model (R2=0.9743). Maximum ester conversion around 85% was reached after 30 min of reaction under continuous agitation (200 rpm) using 2500 mM of each reactant in a solvent-free system, 45°C, 20% m/v of the biocatalyst prepared using 100 mg of protein/g of support. Apparent thermodynamic parameters of the esterification reaction were also determined. Under optimal experimental conditions, reusability tests of the biocatalyst prepared (TLL-PMA) after thirty successive cycles of reaction were performed. TLL-PMA fully retained its initial activity up to twenty two cycles of reaction, followed by a slight decrease around 8.6%. The nature of the product (isoamyl oleate) was confirmed by attenuated total reflection Fourier transform infrared (ATR-FTIR), proton (1H NMR) and carbon (13C NMR) nuclear magnetic resonance spectroscopy analyses.

Keywords: Lipase immobilization, Interfacial activation, Poly-methacrylate particles, Optimization, Biolubricant synthesis, Spectroscopy analyses.

1. Introduction Lipases (triacylglycerol ester acylhydrolases, EC 3.1.1.3) are hydrolases that cleavage carboxylic ester bonds in tri-, di-, and monoacylglycerols to glycerol and free fatty acids at the water-lipid interface. In environments with low water content, these enzymes also catalyze other biotransformation reactions such as esterification, interesterification and transesterification [1–3]. They are produced by several plants, animal tissues, and microorganisms such as fungi and bacteria [2,3]. From the industrial point of view, microbial lipases are the most preferred source due to their great versatility to environmental conditions, simplicity in genetic manipulation and in cultivation conditions [3]. The application of lipases, including microbial lipase from Thermomyces lanuginosus (TLL) – a single chain protein consisting of 269 amino acids with a molecular weight of 31.7 kDa, optimal at pH around 9 and optimal temperature ranging from 55 to 60 °C [2], in a large-scale process is often limited due to their high cost, and sensitivity to high temperature and organic solvents. Moreover, it is difficult to separate them from the reaction system, which limits its recovery and may lead to contamination of the final product [1,2,4,5]. In order to overcome these problems, lipases have been immobilized by several protocols [1,7–14]. Physical adsorption on hydrophobic supports is an attractive protocol from the industrial point of view because its allows the reuse of the support by desorption of inactive enzyme molecules from the biocatalyst surface [11,14]. This protocol promotes the stabilization of the lipases in open conformation (interfacial activation), thus making it especially suitable for lipase immobilization [1,4–7,10–14]. The aim of the present study was to prepare active biocatalysts by immobilizing TLL on PMA particles via physical adsorption to catalyze isoamyl oleate synthesis by esterification reaction. Fatty acid alkyl esters having 22 to 26 carbon atoms, including isoamyl oleate (23 carbon atoms), have been used as plasticizers and lubricant compounds for different industrial and automotive applications such as hydraulic and metal working

fluids, hydraulic of harvesters, drilling oils, slab and gear oils and lubricants for power saw chains [15–18]. These esters present high biodegradability and low toxicity, thus exhibiting an excellent combination of technical and eco-friendly properties [15–17]. In the present study, mesoporous poly-methacrylate (PMA) particles were tested as support in the immobilization of TLL via physical adsorption. The selection of this support was based on previous reports that describe its promising application in the preparation of robust biocatalysts due to its suitable features such as high hydrophobicity, mechanical resistance and large surface area which allows high enzyme loading and stability and good diffusion of reactant and/or product molecules in the biocatalyst microenvironment [7,14].

2. Materials and methods

2.1. Materials Lipase from Thermomyces lanuginosus (TLL) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further treatment. It is a liquid enzymatic preparation with a specific activity of 1100.3 IU/mg of protein and 17.7 mg protein/mL of enzyme solution. Mesoporous PMA particles (Diaion® HP 2-MG), with average particle diameter of 300–700 µm, surface area of 500 m2/g and porous size of 170 Å (Supelco technical information), were purchased from Supelco (Bellefonte, PA, USA). Olive oil (low acidity) from Carbonell (Córdoba, Spain) was purchased at a local market (Alfenas, MG, Brazil). Arabic Gum was acquired from Synth® (São Paulo, SP, Brazil). Isoamyl alcohol (purity ≥ 99.5% m/m) and oleic acid (purity > 99.5% m/m) were acquired from Vetec Química Ltd. (São Paulo, SP, Brazil) and Sigma-Aldrich, respectively. All other chemical reagents were of analytical grade acquired from Vetec Química Ltd. and Synth®.

2.2. Determination of the hydrolytic activity of soluble and immobilized lipase The catalytic activity of soluble and immobilized lipase in aqueous medium was determined in the hydrolysis of olive oil emulsion, according methodology described by Soares et al. [19]. The substrate was prepared by mixing 50 g of olive oil with 150 g of Arabic Gum solution at 3% m/v. The reaction mixture containing 5 g of the emulsion, 5 g of 100 mM buffer sodium phosphate pH 8.0 and soluble (30 µL, 0.26 mg/mL) or immobilized lipase (0.1 g) was incubated for 5 min at 37°C under agitation in an orbital shaker (200 rpm). The reaction was stopped by adding 10 mL of ethanol solution at 95% m/m. The released free fatty acids were then titrated with a standard 20 mM NaOH solution using phenolphthalein as indicator. Blank reactions were performed by adding soluble and immobilized TLL after ethanol solution. One international unit (IU) of hydrolytic activity was defined as the mass of enzyme required to release 1 µmol of free fatty acids per minute of reaction under the experimental conditions above described.

2.3. Determination of protein Protein was determined according to the Bradford´s method [20]. Bovine serum albumin (BSA) was used as standard protein. In this study, all solutions were prepared using Milli-Q water.

2.4. Preparation of biocatalysts via physical adsorption 10 g of PMA particles were initially wetted with 50 mL ethanol solution (95% m/m) under static conditions for 18 h at room temperature. After, the support was filtered in a Buchner funnel under vacuum and thoroughly washed with distilled water in order to remove residual ethanol solution. The wet support (10 g) was then incubated in 190 mL of 5 mM sodium phosphate pH 7.0 containing different protein loadings that permitted to vary the support loading from 5 to 200 mg protein/g of support. The suspensions were kept under

agitation (200 rpm) in an orbital shaker at room temperature for 12 h. The biocatalysts prepared were then filtered (Whatman filter paper 41) under vacuum, washed with distilled water (volume ratio 1:5) and stored at 4 °C for 24 h prior to use.

2.5. Determination of immobilization parameters Immobilization yield (IY) was determined according following equation (Eq. 1):

 EA 0  EA f IY %    EA 0 

(1)

   100 

where EA0 is the hydrolytic activity in the supernatant before immobilization (IU/mL) and EAf is the hydrolytic activity in the supernatant after immobilization (IU/mL). Immobilized protein loading (IP) was calculated as follows (Eq. 2):

IP mg / g  

V enz  C 0  C f



(2)

m PMA

where Venz is the volume of enzyme solution (mL), C0 is the initial concentration of protein in the supernatant (mg/mL), Cf is the concentration of protein in the supernatant after immobilization (mg/mL) and mPMA is the mass of support (g). Specific activity (SA) was calculated as shown in Eq. 3:

SA IU / mg  

HA IP

(3)

where HA is the hydrolytic activity of the biocatalysts prepared (IU/g of support) and IP is the immobilized protein loading (mg/g of support).

2.6. Adsorption isotherm models In this study, Langmuir (Eq. 4) and Freundlich (Eq. 5) isotherm models were used to fit the experimental data from TLL adsorption on PMA particles [21]. The data were

analyzed using Origin Pro software, version 8.0 (OriginLab Corporation, Northampton, MA, USA).

qe 

q max  C e K L  Ce

(4)

1

(5)

qe  K F  C e n

where qe is the adsorption capacity at equilibrium (mg protein/g support), Ce is defined as the residual mass of protein in unit volume of lipase solution (mg protein/mL), qmax is the maximum adsorption capacity (mg protein/g support), KL is the Langmuir constant related to the energy of adsorption (mL/mg protein), KF is the Freundlich isotherm constant (mL/mg support), and n is the Freundlich exponent (dimensionless). The separation factor (RL) is a dimensionless parameter which has been used to investigate the adsorption system feasibility at different initial protein concentrations, and it was calculated according following equation (Eq. 6).

RL 

1

(6)

1  K L  C0

where KL is Langmuir constant (mL/mg of protein) and C0 is the initial protein concentration in the immobilization supernatant (mg/mL). The values of RL indicate the type of the isotherm, irreversible (RL=0), favorable (01) [21].

2.7. Isoamyl oleate synthesis procedure The reaction mixture consisting of isoamyl alcohol and oleic acid at equimolar ratio (1:1) diluted in heptane (6 mL of reaction medium) was used as substrate in isoamyl oleate

synthesis. Esterification reaction in solvent-free system was also performed under similar conditions. The reactant solutions were mixed in 100 mL screw-capped glass bottles and incubated in an orbital shaker under agitation at 200 rpm [4,7]. After, the biocatalysts prepared were then added to the reaction mixtures. The conversion was calculated by measuring the concentration of residual oleic acid in the reaction mixture. Samples were withdrawn, diluted in 10 mL of an ethanol/acetone 1:1 (v/v) mixture and titrated with a standard 20 mM NaOH solution using phenolphthalein as indicator [4,7,15,18]. All the experiments were performed in triplicate. Blank reactions were performed by adding the support (PMA particles) in the reaction mixture and no conversion was observed after 2 h of incubation.

2.7.1. Effect of protein loading on biocatalyst performance The effect of immobilized protein loading, previously prepared using protein loadings between 5 and 200 mg/g of support, on the ester synthesis was evaluated. The reactions were performed at 500 mM of each reactant, reaction temperature of 37°C, biocatalyst concentration of 10% m/v and 15 min of reaction.

2.7.2. Effect of reaction temperature: Determination of apparent thermodynamic parameters The effect of reaction temperature ranging from 25 to 50°C on the ester synthesis was studied. The reactions were performed at 500 mM of each reactant, biocatalyst concentration of 10% m/v prepared using protein loading of 100 mg/g of support and 60 min of reaction. In this study, a second-order kinetic model of esterification of isoamyl alcohol and oleic acid was proposed, according previous studies reported in the literature for enzymatic synthesis of several esters by esterification reaction [22–24]. The reaction rate equation for a second-order kinetic model is expressed as follows (Eq. 7) [23]: 

rOA  

d [ OA ]  k IA  OA  dt

(7)

where rOA or –d[OA]/dt is the consumption rate of oleic acid (mM/min), k is the initial reaction rate constant (1/mM.min) and [IA] and [OA] are the concentrations of isoamyl alcohol and oleic acid at certain reaction time t (mM), respectively. The reactions were performed at equimolar ratio alcohol:acid ([IA]=[OA]), thus the equation will be (Eq. 8) [22,24]:

rOA  

d [ OA ] 2  k OA  dt

(8)

The integrated equation is expressed as follows (Eq. 9):

1 1 1 1   k t    k t [ OA ] [ OA 0 ] [ OA ] [ OA 0 ]

(9)

where [OA0] is the initial concentration of oleic acid and [OA] is the concentration of oleic acid at certain reaction time t (min). The initial reaction rate constants at different temperatures (20–45°C) were then determined from the slope of the plots of the inverse of oleic acid concentration versus time at first 10 min of reaction, as shown in Eq. 9. The apparent energy activation was determined using linearized Arrhenius equation, as shown in Eq. 10 [25,26]:

ln k  ln A 

Ea 1  R T

(10)

where A is the Arrhenius collision factor, Ea is the energy activation (kJ/mol), R is the gas universal constant (8.314×10-3 kJ/mol.K) and T is the absolute temperature (K). In order to determine other thermodynamic parameters, Eyring plot of ln(k/T) versus 1/T was used (Eq. 11) [26]:

k ln  T

  k  S H 1     ln  B   h R R T   

(11)

where kB is the Boltzmann constant (1.38×10-23 J/K), h is Planck constant (6.6256×10-34 J.s), ∆S is the entropy (J/mol.K) and ∆H is the enthalpy (kJ/mol). The values calculated for entropy and enthalpy were then used to calculate Gibbs free energy (∆G – kJ/mol) at different reaction temperature values as follows (Eq. 12):

G  H  T  S

(12)

2.7.3. Effect of biocatalyst concentration The effect of biocatalyst concentration varying from 5 to 25% m/v on the ester synthesis was performed under following conditions: 500 mM of each reactant in heptane medium, reaction temperature of 45°C and 60 min of reaction. In this set of experiments, the biocatalyst used was also prepared using protein loading of 100 mg/g of support.

2.7.4. Effect of reactants concentration The effect of reactants concentration from 500 to 2500 mM of each reactant on the ester synthesis was performed under following conditions: equimolar ratio alcohol:acid, reaction temperature of 45°C, 20% m/v of biocatalyst prepared using protein loading of 100 mg/g of support and 45 min of reaction. In this study, esterification reactions between 500 and 2000 mM were performed in heptane medium, whereas the reaction at 2500 mM was conducted in a solvent-free system.

2.7.5. Effect of molecular sieve concentration The effect of molecular sieve concentration from 0 to 30% m/v on the ester synthesis was performed under the following conditions: 2500 mM of each reactant in a solvent-free system, reaction temperature of 45°C, 20% m/v of biocatalyst prepared using protein

loading of 100 mg/g of support and 45 min of reaction. In this study, molecular sieves were previously dehydrated by heating in an oven at 250 °C for 24 h prior to use. Under these experimental conditions, both reactants were not adsorbed on the dehydrating agent after 2 h of incubation.

2.7.6. Ester synthesis catalyzed by soluble and immobilized TLL prepared by several protocols Comparative study of isoamyl oleate synthesis under optimal experimental conditions catalyzed by TLL-PMA (selected biocatalyst prepared in this study) and immobilized TLL by physical adsorption on hydrophobic poly-hydroxybutyrate particles (TLL-PHB) and multipoint covalent attachment on glyoxyl-agarose beads activated with glycidol (TLL-Gly-agarose) was also performed. These biocatalysts were prepared using protein loading of 80 mg/ g of support, which presented maximum immobilization protein loading of 26.0 mg/g of support (TLL-PHB) [4], and 21.2 mg/g of support (TLL-Glyagarose) [27], respectively. The results were also compared with soluble TLL. In this set of experiments, all the reactions were performed using the same immobilized protein concentration – 1.2 g of TLL-PMA (or 92.76 mg of immobilized protein loading), which corresponds to 4.37 g of TLL-Gly-agarose, 3.57 g of TLL-PHB and 5.24 mL of soluble TLL, respectively.

2.8. Reusability test Reusability tests were performed under optimal experimental conditions (2500 mM of each reactant in a solvent-free system, reaction temperature of 45°C and 20% m/v of biocatalyst prepared using protein loading of 100 mg/g of support). At the end of each reaction (thirty successive cycles of 30 min each), the biocatalyst was removed from the

reaction mixture, washed with chilled heptane in excess to remove reactant and/or product molecules retained in the biocatalyst microenvironment and filtered under vacuum for 2 h in order to remove water molecules. After, the biocatalyst was then introduced into a fresh reaction mixture.

2.9. Purification of the product The biocatalyst was separated from the reaction system by filtration in a Buchner funnel under vacuum. Residual oleic acid was removed by neutralization with Na2CO3 solution at 15% m/v (volume ratio 1:1), according methodologies previously described [28,29], with slight modifications. The soap formed in the reaction of residual oleic acid and Na2CO3 solution and isoamyl alcohol were removed by washing with distilled water at 40°C (volume ratio 1:5) by five times. After, the mixture consisting of ester and water was evaporated with a rotary evaporator under vacuum at 50°C. Then, the purified product was dried for 24 h of incubation at room temperature over molecular sieves (30 % m/v) thermally activated for 24 h at 250°C.

2.10. ATR-FTIR and NMR analyses In order to check the esterification reaction, ATR-FTIR spectrophotometer (Nicolet iS50 FTIR – Thermo Scientific, Madison, WI, USA) with a diamond single bounce accessory (GladiATR, PIKE Technologies) was used. The samples were directly applied on the crystal cell. ATR-FTIR spectra for isoamyl alcohol, oleic acid and isoamyl oleate were acquired after 64 scans between 4000 and 400 cm−1 with spectral resolution of 4 cm−1. 1H (300 MHz) and 13C (75 MHz) nuclear magnetic resonance (NMR) spectra were recorded on a Bruker 300 MHz apparatus DRX spectrometer (Bruker BioSpin, Germany). Approximately 120 mg of purified ester was dissolved in 0.6 mL of CDCl3 and the resulting solution was placed in a 5 mm diameter NMR tube.

3. Results and discussion

3.1. Characterization of the biocatalysts prepared in hydrolysis reaction TLL is a globular protein with a molecular volume of 35 Å × 45 Å × 50 Å [2], and molecular diameter 53.2 Å which was determined as follows (Eq. 13) [30]:

R

3

3V  D  2R 4

(13)

where R is the molecular radius, V is the molecular volume and D is the molecular diameter of TLL molecules. The average porous size of PMA particles (170 Å) was found to be around 3.2-times higher than the molecular diameter of TLL molecules. These results indicate that the enzyme may be adsorbed on both external and internal support surfaces. In order to evaluate the loading capacity of PMA particles, different initial protein loadings varying from 5 to 200 mg protein/g of support were offered. These values respectively correspond to 5,501.3 and 220,052.0 units of activity (IU) per gram of support, previously determined in the hydrolysis of olive oil emulsion (Section 2.2). The adsorption assays were performed at low ionic strength (5 mM) pH 7.0, 25°C and 18 h of incubation under agitation (200 rpm). As it can be seen in Table 1, immobilization procedure performed up to 25 mg protein/g of support presented immobilization yield above 90%. A decrease of immobilization yield by increasing the initial protein loading was observed due to possible support saturation [4,10]. Maximum immobilization yield around 50% using highest initial protein load (200 mg/g of support) was observed, which corresponds to around 110,000 IU/g of support.

The increase of initial protein loading increased the adsorption of lipase on the support surface (Table 1). Maximum adsorbed protein loading around 100 mg/g of support was reached using initial protein loading of 150 mg/g of support. After, similar immobilized protein loading due to support saturation was observed, as described above. According to Table 2, PMA particles exhibited higher immobilized protein loading when compared to other mesoporous and macroporous supports broadly applied in the immobilization of several lipases via physical adsorption such as inorganic (silica, ferric silica nanocomposite, organobentonite) and organic (Sepabeads, poly-styrene–divinylbenzene – poly(STY-DVB), poly-hydroxybutyrate (PHB), acrylic resin, foam, etc.) materials. Maximum adsorbed protein loading reached for PMA particles was found to be very similar to other supports as macroporous poly-styrene–divinylbenzene beads (MCI GEL CHP20P) and mesoporous silica particles used in the immobilization of Lipase B from Candida antarctica (CALB) and Candida rugosa lipase (CRL), respectively [10,37]. These results show that PMA is a promising support to prepare biocatalysts with high catalytic activity due to its large specific surface area (500 m2/g) and porous size (170 Å) which allows the adsorption of high protein loading. Adsorption isotherms are important tools to explain how adsorbate molecules are distributed between the liquid and solid phases when the equilibrium is reached. Moreover, these models are usefully used to describe how adsorbate molecules interact with the adsorbent surface. The investigation on adsorption isotherm was performed by fitting the experimental data to the Langmuir (Eq. 4) and Freundlich (Eq. 5) models. These models have been the most commonly used to explain the interaction between lipase molecules and hydrophobic support surfaces and to predict their equilibrium parameters [4,31,33,39]. The plots of the non-linear fit and the determination of isotherm parameters are shown in Fig. 1a,b. The adsorption isotherm for TLL molecules on PMA particles followed the Langmuir model with a very high correlation coefficient (R2=0.9743) which was higher than

Freundlich model (R2=0.9459), as shown in Fig. 1a and 1b, respectively. Langmuir isotherm model is based on the assumption of monolayer adsorption on the support surface containing a finite number of sites of uniform energies of adsorption [21]. The theoretical values of Langmuir constant (KL) and the maximum adsorbed protein loading on PMA particles (qmax) were 0.783 mL/mg and 124.0 mg/g of support, respectively. However, the experimental qmax value was 102.4 ± 3.0 mg protein/g of support (see Table 1). The difference between experimental and theoretical values of qmax could be attributed to the adsorption of stabilizing compounds and/or impurities present in crude TLL extract (Lipolase 100L) on the support surface [40], which could reduce the available surface for the enzyme molecules. In fact, the support has some brown color after immobilization procedure. The RL values for the adsorption of TLL molecules on PMA particles varied from 0.108 (highest initial protein loading – 200 mg/g of support) to 0.829 (lowest initial protein loading – 5 mg/g of support), as shown in Fig. 2. The RL values obtained are found to decrease with the increase of initial protein concentration and as 0 < RL < 1, thus indicating that the adsorption of TLL molecules on the support was a favorable process [4]. The hydrolytic activity of the biocatalysts prepared was increased from 120.1 ± 2.0 to 657.2 ± 18.7 IU/g of support when the initial protein loading was increased from 5 to 200 mg/g of support. Immobilization procedure performed above 150 mg/g of support did not promote a significant increase on the hydrolytic activity. As it can be observed, an increment of initial protein loading by a factor 40-fold (from 5 to 200 mg/g of support) resulted in an increase of hydrolytic activity around 5.5-fold. According to the results displayed in Table 1, the immobilization process drastically reduced the specific activity value of soluble TLL (1100.3 IU/mg protein – see Section 2.1). Additionally, a decrease of specific activity by increasing the initial protein loading was also observed. Specific activity values ranged from 24.5 ± 0.1 to 6.5 ± 0.2 IU/mg of immobilized protein loading which correspond to the biocatalysts prepared using lowest and highest

initial protein loading, respectively. These results may be caused for diffusion limitations due to the high activity of the biocatalyst (both substrate and pH gradients may be produced in its microenvironment).

3.2. Isoamyl oleate synthesis by direct esterification reaction In this set of experiments, the effects of various parameters, including the immobilized protein loading, reaction temperature, biocatalyst concentration, reactants concentration and molecular sieve concentration, on the synthesis of isoamyl oleate by direct esterification reaction of oleic acid and isoamyl alcohol in organic medium were studied. When using a solvent, heptane (a hydrophobic organic solvent with Log P value = 4.57) was chosen due to high stability of TLL in this organic solvent [2]. Moreover, previous studies performed in our lab aiming the synthesis of esters by esterification reactions such as shortchain esters from oleic acid, hexyl esters and isoamyl butyrate catalyzed by immobilized TLL on hydrophobic supports were successfully performed using heptane as reaction medium [4–7].

3.2.1. Effect of immobilized protein loading The effect of immobilized protein loading on the ester synthesis was studied aiming to select the most active biocatalyst prepared. According to results shown in Fig. 3, the synthesis of ester was improved by increasing the initial protein loading up to 100 mg protein/g of support that exhibited immobilized protein loading of 77.3 ± 1.3 mg/g of support (Table 1). The biocatalysts prepared by offering initial protein loading above 100 mg/g of support reached similar performance due to possible diffusional limitation of reactant molecules (oleic acid and isoamyl alcohol) to the internal surface of the biocatalyst,

as previously described for hydrolysis reactions. These results show that the biocatalyst prepared at 100 mg protein/g of support was considered the most efficient, thus selected for subsequent studies.

3.2.2. Effect of reaction temperature: Determination of energy activation In general, initial reaction rate increases with temperature due to the increase of the solubility of reactants in the reaction system and the frequency of collisions between biocatalyst and reactant molecules. Moreover, a better access of reactant molecules to the internal surface of the biocatalyst is reached due to reduction of the viscosity of the reaction mixture [41]. On the other hand, the stability of enzyme molecules tends to decline when T increases by possible distortion of their native conformation (inactivation of the biocatalyst) [4,31,33]. In this study, the effect of reaction temperature was evaluated in the range of 2050°C (Fig. 4a). As it can be observed, the increase of temperature between 20 and 45°C increased the values of initial reaction rate constant (k) from 1.22×10-4 to 3.39×10-4 1/mM.min, as shown in Table 3. Interestingly, high correlation coefficients (R2) for the determination of k values in the range of 20 to 45°C at first 10 min of reaction were observed (see Fig. 4b and Table 3). These results confirm better access of reactant molecules to the microenvironment of the biocatalyst by increasing the reaction temperature, as expected. According to Fig. 4a, the increase of reaction temperature up to 45 °C led to a respective increase of the rate of synthesis of ester. Increasing to 50°C, no significance increment on the reaction was observed due to possible inactivation of some enzyme molecules. Thus, reaction temperature of 45°C was selected to perform further studies. The apparent activation energy (Ea) of the esterification reaction was determined by plotting the logarithmic of k values versus the inverse of absolute temperature which resulted in a high correlation coefficient (R2=0.9864), as shown in Fig. 4c. These results show that the ln(k) values appear to change linearly with 1/T ranging from 20 to 45 °C as

expected for a single rate-limited thermally activated process [42]. The apparent activation energy for isoamyl oleate synthesis was found to be 32.8 kJ/mol. This value is in agreement with those studies previously reported for the enzymatic synthesis of several esters. Apparent activation energy of 37.31 kJ/mol was estimated for the synthesis of L-ascorbyl acetate by esterification reaction catalyzed by Lipozyme TLIM, a commercial biocatalyst prepared from T. lanuginosus lipase immobilized on silica gel [43]. Similar result was also reported by Converti et al. [44] in the synthesis of ethyl acetate (31 kJ/mol) and geranyl acetate (35 kJ/mol) using lyophilized cells of Aspergillus oryzae as biocatalyst. Eyring plot of ln(k/T) versus 1/T was used to estimate apparent ∆H value from the slope of the curve and apparent ∆S from the intercept (Fig. 4d), which high correlation coefficient was also observed (R2=0.9845). These thermodynamic parameters as well as ∆G values for the esterification reactions performed between 20 and 45°C are summarized in Table 3. It can be seen that the reaction was endothermic with ∆H value of 29.7 kJ/mol. The value determined obtained for entropy (∆S) was negative (–197.4 J/mol.K) which indicates rapid formation of the enzyme-substrate complex due to high catalytic activity of the biocatalyst prepared, thus leading to loss in entropy. Previous study reported in the literature shows that the formation of the product with the progress of the reaction is an important factor for the loss in entropy [45]. Positive value of ΔH and negative value of ΔS indicates that the esterification reaction did not proceed spontaneously at any temperature. Indeed, the increase of reaction temperature from 20 to 45°C also increased the apparent ∆G values from 87.5 to 92.5 kJ/mol (see Table 3). These positive values of Gibb's free energy indicate that the esterification reaction was clearly a non-spontaneous process, as above described. Similar results were recently reported by Badgujar and Bhanage [26] for the enzymatic

synthesis of several levulinate esters via esterification reaction catalyzed by immobilized C. antarctica lipase on Immobead 150.

3.2.3. Effect of biocatalyst concentrations The effect of biocatalyst concentration on the synthesis of ester was studied. The plots of conversion percentage versus reaction time for esterification reactions performed at different biocatalyst concentrations are shown in Fig. 5. It is possible to note that the conversion increased with the increase of biocatalyst concentration from 5 to 25% m/v, thus indicating that the reaction was intrinsically kinetically controlled. However, esterification reactions performed with high biocatalyst concentration (20 and 25% m/v) reached similar conversion percentage values after 30 min of reaction (around 85%). These results could be attributed to possible agglomeration of the biocatalyst in the reaction mixture or to the lack of substrate molecules to access the active sites of the enzyme at highest concentration (25% m/v) [7,46]. Considering the production cost and efficiency, 20% m/v of biocatalyst was chosen to evaluate the effect of reactants concentration on the ester synthesis.

3.2.4. Effect of reactants concentration The effect of the reactants concentration varying from 500 to 2500 mM of each reactant on the ester synthesis was evaluated. In this study, esterification reactions between 500-2000 mM were performed in heptane medium, whereas at 2500 mM was conducted in a solvent-free system. As it can be observed in Fig. 6, similar initial reaction rate values and conversion percentage was observed. After 30 min of reaction, maximum conversion percentage around 85% for the different reaction systems tested was reached. These results suggest a good accessibility of reactant molecules to the biocatalyst microenvironment due to its large internal surface. From the industrial point of view, esterification reactions performed in solvent-free systems are more attractive due to toxicity and flammability of organic solvents and/or costs of their recovery and recycling. Based on these results, esterification reaction performed in a solvent-free system was selected. These results are in agreement with previous studies reported for the enzymatic synthesis of esters with lubricant

properties such as alkyl oleate from fusel oil [15], hexyl laurate [47], and 2-ethylhexyl palmitate [48], which were successfully synthetized via esterification reaction in solvent-free systems.

3.2.5. Effect of molecular sieve concentration The use of dehydrating agents is convenient to capture the water molecules and reduce the adsorption of water molecules in the biocatalyst microenvironment [49–51]. In the present study, water content in the reaction system was controlled using molecular sieves, an important class of synthetic adsorbents which possess large porous of uniform size and essentially molecular dimensions [50]. The effect of molecular sieve concentration ranging from 0 to 30% m/v on the ester synthesis was then evaluated. In this study, the addition of molecular sieves in the reaction mixture did not exhibit significant influence on the initial reaction rates and conversion percentage (data not shown). Water molecules could act as a co-solvent which facilitates the miscibility or diffusion of reactant molecules to the internal microenvironment of the biocatalyst [51]. Thus, molecular sieves were not used in subsequent experiments concerning the reusability of the biocatalyst after successive cycles of reaction.

3.2.6. Comparative study of isoamyl oleate synthesis catalyzed by soluble and immobilized TLL prepared by several protocols Under optimal experimental conditions, isoamyl oleate was also synthetized using soluble enzyme and immobilized TLL prepared by several protocols such as multipoint covalent attachment on glyoxyl-agarose beads (TLL-Gly-agarose) and physical adsorption on poly-hydroxybutyrate particles (TLL-PHB). These biocatalysts were selected due to their high catalytic activities in esterification and transesterification reactions in solvent-free systems [4,27]. Under the experimental conditions tested, a bad dispersion of the reaction

medium using either TLL-PHB or TLL-Gly-agarose was observed. These results could be attributed to high mass of biocatalyst required, thus increasing the viscosity of the reaction medium. With respect to reaction catalyzed by TLL-PHB, the volume of biocatalyst required was around 1.5-fold higher than the volume of reaction medium (isoamyl alcohol + oleic acid) due to its low specific mass. Thus, esterification reaction catalyzed by TLL-PHB was excluded from this study. According results summarized in Fig. 7, maximum conversion percentage of 26.7% was reached using TLL-Gly-agarose as biocatalyst – around 3-fold lower than TLL-PMA (around 85% after 30 min of reaction). Similar initial reaction rate values were observed for soluble TLL and TLL-PMA (up to 5 min of reaction), however maximum conversion percentage around 68.2% was reached using soluble enzyme as biocatalyst. These results could be attributed to better dispersion of enzyme molecules in the support surface which allowed good accessibility of reactant molecules from the reaction medium to the biocatalyst microenvironment. These results are in agreement with previous studies performed in our lab for the synthesis of butyl butyrate catalyzed by porcine pancreatic lipase (PPL) [31], and short-chain alkyl esters from oleic acid catalyzed by TLL [4]. Moreover, its application is not suitable because it is very difficult to separate it from the reaction medium for reuse. On the other hand, immobilized lipases have been easily recovered and recycled for subsequent application [4,7,31]. These results clearly show the promising application of PMA in the immobilization of lipase because it allows prepare biocatalysts with high immobilized protein loading per unit volume of support due to its large surface area and porous size.

3.2.6. Reusability studies The reusability of immobilized enzymes is very important for their application, especially on industrial scale. The reusability of the selected biocatalyst prepared was studied in a solvent-free system after 30 successive cycles of esterification reaction of 30

min each. As it can be observed in Fig. 8, the biocatalyst prepared fully retained its initial activity up to twenty two cycles of reaction. After, a slight decrease of its activity around 8.6% was observed due to possible accumulation of water molecules in the internal microenvironment of the support and/or possible inactivation and desorption of some enzyme molecules preferentially adsorbed in the external microenvironment of PMA particles [4,7,31]. These results suggest strong interaction of the enzyme with the support microenvironment and the correct elimination of the remaining compounds during the washing steps. The biocatalyst prepared in this study was more stable than those ones used in the enzymatic synthesis of other esters, including biolubricants. Khan et al. [29] synthetized cetyl oleate (wax ester) by esterification reaction under ultrasonic irradiation catalyzed by Fermase CALBTM 10000, a commercial Candida antarctica lipase B immobilized on polyacrylate beads. These authors observed a slight decrease of its initial activity after three cycles of reaction. After seven cycles of reaction, the biocatalyst retained 20% of its initial catalytic activity. In another study, TLL adsorbed on mesoporous PHB particles was used in the synthesis of short-chain alkyl esters from oleic acid (methyl and ethyl oleate) [4]. The biocatalyst retained 70% of its initial activity after five successive cycles of reaction of 15 min each.

3.2.7. ATR-FTIR and NMR spectroscopy analyses Fig. 9 shows the ATR-FTIR spectra of isoamyl alcohol, oleic acid and purified isoamyl oleate. According to first spectrum, isoamyl alcohol presents an intense band at 3320 cm-1 which is attributed to the O–H stretching absorption. The bands between 2955 and 2871 cm-1 correspond to the vibration of symmetrical and asymmetrical stretching of CH methyl and methylene groups and the band at 1055 cm-1 is attributed to the C-O single bond stretching vibration of a primary alcohol. In the second spectrum (oleic acid), the intense band around at 2971 cm-1 is attributed to stretching of C–H overlapped to O–H bond,

whereas the stretching vibrational of carbonyl group from the carboxylic moiety was observed at 1707 cm-1. The band due to the OH group presents in isoamyl alcohol was not detected in the ATR-FTIR spectrum of isoamyl oleate (third spectrum), which confirms the high purity of the product. Moreover, the intense band at 1737 cm−1 corresponds to carbonyl group of the ester formed after esterification reaction and the bands at 1244 and 1167 cm-1 to the stretching vibrational of –C(=O)O– group [52], thus confirming the synthesis of isoamyl oleate. The synthesis of isoamyl oleate was also confirmed by proton and carbon NMR spectra, as shown in Fig. 10a,b. According to the 13C NMR spectrum of the ester (Fig. 10a), the intense peak at 173.53 ppm corresponds to carbonyl group [–CH2(C=O)OCH2–] from the ester structure [53]. As can it be observed in Fig. 10b, 1H NMR spectrum showed specific peaks of two-hydrogen triplet at 2.27 ppm for the methylene group bound to carbonyl group from the ester moiety [–CH2(C=O)OCH2–], and two-hydrogen triplet at 4.09 ppm for the methylene group attached to the oxygen atom [–CH2(C=O)OCH2–] [7,53]. Spectroscopy analyses (ATR-FTIR and NMR) confirmed the successfully synthesis of isoamyl oleate.

Conclusion Isoamyl oleate was produced via esterification reaction catalyzed by immobilized TLL on mesoporous PMA particles. The support used exhibited high lipase adsorption capacity due to its large internal surface and maximum adsorbed protein loading around 100 mg/g of support. Under optimal conditions, maximum conversion percentage around 85% was reached after 30 min of reaction performed in a solvent-free system. The biocatalyst prepared in this study exhibited not only excellent catalytic activity but also satisfactory reusability in esterification reaction cycles. These results could be attributed to strong

interaction of TLL with the internal support surface, thus reducing its possible desorption from the biocatalyst microenvironment. The nature of the product was confirmed by different spectroscopy analyses such as ATR-FTIR and NMR (1H NMR and

13

C NMR).

These results indicate that the application of the biocatalyst prepared is highly promising in the synthesis of important compounds from the industrial point of view as isoamyl oleate (biolubricant).

Acknowledgements This paper was financially supported by FAPEMIG (Process number APQ–00968– 12), CNPq (Process number 475289/2012–9), CAPES and FINEP (Brazil). F.A.P. Lage, M.C.C. Corradini, J.J. Bassi, and L.M. Todero would like to thank to CNPq and FAPEMIG for their student fellowships. The authors are grateful to MSc. Kris Simone T. Dias (LFQM – UNIFAL) and Jonas C. Cruz (LAQF – UNIFAL) by the NMR and ATR-FTIR spectroscopy analyses, respectively. The help and comments from Dr. R. FernándezLafuente (ICP-CSIC, Madrid, Spain) are gratefully recognized.

References [1] P. Adlercreutz, Immobilisation and application of lipases in organic media, Chem. Soc. Rev. 42 (2013) 6406–6436. [2] R. Fernández-Lafuente, Lipase from Thermomyces lanuginosus: uses and prospects as an industrial biocatalyst, J. Mol. Catal. B: Enzym. 62 (2010) 197–212. [3] F. Hasan, A.A. Shah, A. Hameed, Industrial applications of microbial lipases, Enzyme Microb. Technol. 39 (2006) 235–251. [4] J.S. Miranda, N.C.A. Silva, J.J. Bassi, M.C.C. Corradini, F.A.P. Lage, D.B. Hirata, A.A. Mendes, Immobilization of Thermomyces lanuginosus lipase on mesoporous polyhydroxybutyrate particles and application in alkyl esters synthesis: isotherm, thermodynamic and mass transfer studies, Chem. Eng. J. 251 (2014) 392–403. [5] A.A. Mendes, P.C. Oliveira, A.M. Velez, R.C. Giordano, R.L.C. Giordano, H.F. Castro, Evaluation of immobilized lipases on poly-hydroxybutyrate beads to catalyze biodiesel synthesis, Int. J. Biol. Macromol. 50 (2012) 503–511. [6] A.P.P. Bressani, K.C.A. Garcia, D.B. Hirata, A.A. Mendes, Production of alkyl esters from macaw palm oil by a sequential hydrolysis/esterification process using heterogeneous biocatalysts: optimization by response surface methodology, Bioprocess Biosyst. Eng. 38 (2015) 287–297. [7] L.M. Todero, J.J. Bassi, F.A.P. Lage, M.C.C. Corradini, J.C.S. Barboza, D.B. Hirata, A.A. Mendes, Enzymatic synthesis of isoamyl butyrate catalyzed by immobilized lipase on poly-methacrylate particles: optimization, reusability and mass transfer studies, Bioprocess Biosyst. Eng. 38 (2015) 1601–1613. [8] R.L. de Souza, E.L.P. de Faria, R.T. Figueiredo, LS. Freitas, M. Iglesias, S. Mattedi, G.M. Zanin, O.A.A. dos Santos, J.A.P. Coutinho, A.S. Lima, C.M.F. Soares, Protic ionic liquid as additive on lipase immobilization using silica sol–gel, Enzyme Microb. Technol. 52 (2013) 141–150. [9] K.C. Badgujar, B.M. Bhanage, Application of lipase immobilized on the biocompatible ternary blend polymer matrix for synthesis of citonellyl acetate in non-aqueous media: kinetic modelling study, Enzyme Microb. Technol. 57 (2014) 16–25. [10] K. Hernández, C. Garcia-Galan, R. Fernández-Lafuente, Simple and efficient immobilization of lipase B from Candida antarctica on porous styrene–divinylbenzene beads, Enzyme Microb. Technol. 49 (2011) 72–78. [11] Z. Cabrera, G. Fernández-Lorente, R. Fernández-Lafuente, J.M. Palomo, J.M. Guisán, Novozym 435 displays very different selectivity compared to lipase from Candida antarctica B adsorbed on other hydrophobic supports, J. Mol. Catal. B: Enzym. 56 (2009) 171–176.

[12] E.A. Manoel, J.C.S. dos Santos, D.M.G. Freire, N. Rueda, R. Fernández-Lafuente, Immobilization of lipases on hydrophobic supports involves the open form of the enzyme, Enzyme Microb. Technol. 71 (2015) 53–57. [13] J.M. Palomo, G. Muñoz, G. Fernández-Lorente, C. Mateo, R. Fernández-Lafuente, J.M. Guisán, Interfacial adsorption of lipases on very hydrophobic support (octadecyl– Sepabeads): immobilization, hyperactivation and stabilization of the open form of lipases, J. Mol. Catal. B: Enzym. 19–20 (2002) 279–286. [14] A.G. Cunha, M.D. Besteti, E.A. Manoel, A.A.T. Silva, R.V. Almeida, A.B.C. Simas, R. Fernández-Lafuente, J.C. Pinto, D.M.G. Freire, Preparation of core-shell polymer supports to immobilize lipase B from Candida antarctica – effect of the support nature on catalytic properties, J. Mol. Catal. B: Enzym. 100 (2014) 59–67. [15] N. Dörmo, K. Belafi-Bako, L. Bartha, U. Ehrenstein, L. Gubicza, Manufacture of an environmental-safe biolubricant from fusel oil by enzymatic esterification in solventfree system, Biochem. Eng. J. 21 (2004) 229–234. [16] J. Salimon, N. Salih, E. Yousif, Biolubricants: raw materials, chemical modifications and environmental benefits, Eur. J. Lipid Sci. Technol. 112 (2010) 519–530. [17] A. Willing, Lubricants based on renewable resources – an environmentally compatible alternative to mineral oil products, Chemosphere 43 (2001) 89–98. [18] C.O. Åkerman, A.E.V. Hagström, M.A. Mollaahmad, S. Karlsson, R. Hatti-Kaul, biolubricant synthesis using immobilised lipase: process optimization of trimethylolpropane oleate production, Process Biochem. 46 (2011) 2225–2231. [19] C.M.F. Soares, H.F. de Castro, G.M. Zanin, F.F. de Moraes, Characterization and utilization of Candida rugosa lipase immobilized on controlled pore silica, Appl. Biochem. Biotechnol. 77/79 (1999) 745–757. [20] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [21] K.Y. Foo, B.H. Hameed, Insights into the modeling of adsorption isotherm systems, Chem. Eng. J. 156 (2010) 2–10. [22] J. Giacometti, F. Giacometti, C. Milin, D. Vasic-Racki, Kinetic characterisation of enzymatic esterification in a solvent system: adsorptive control of water with molecular sieves, J. Mol. Catal. B: Enzym. 11 (2001) 921–928. [23] Y. Liu, H. Tan, X. Zhang, Y. Yan, B.H. Hameed, Effect of monohydric alcohols on enzymatic transesterification for biodiesel production, Chem. Eng. J. 157 (2010) 223– 229. [24] K.N.P. Rani, T.S.V.R. Neeharika, T.P. Kumar, B. Satyavathi, C. Sailu, R.B.N. Prasad, Kinetics of enzymatic esterification of oleic acid and decanol for wax ester and evaluation of its physico-chemical properties, J. Taiwan Inst. Chem. Eng. 55 (2015) 12– 16.

[25] M. Zoumpanioti, P. Parmaklis, P. Domınguez de Maria, H. Stamatis, J.V. Sinisterra, A. Xenakis, Esterification reactions catalyzed by lipases immobilized in organogels. Effect of temperature and substrate diffusion, Biotechnol. Lett. 30 (2008) 1627–1631. [26] K.C. Badgujar, B.M. Bhanage, Thermo-chemical energy assessment for production of energy-rich fuel additive compounds by using levulinic acid and immobilized lipase, Fuel Process. Technol. 138 (2015) 139–146. [27] A.A. Mendes, H.F. Castro, R.L.C. Giordano, Covalent attachment of lipases on glyoxylagarose beads: application in fruit flavor and biodiesel synthesis, Int. J. Biol. Macromol. 70 (2014) 78–85. [28] D. Li, W. Xiaojing, N. Kaili, W. Fang, L. Junfeng, W. Pu, T. Tianwei, Synthesis of wax esters by lipase-catalyzed esterification with immobilized lipase from Candida sp. 99– 125, Chin. J. Chem. Eng. 19 (2011) 978–982. [29] N.R. Khan, S.V. Jadhav, V.K. Rathod, Lipase catalysed synthesis of cetyl oleate using ultrasound: optimisation and kinetic studies, Ultrason. Sonochem. 27 (2015) 522–529. [30] H.P. Erickson, Size and shape of protein molecules at the nanometer level determined by sedimentation, gel filtration, and electron microscopy, Biol. Proced. Online 11 (2009) 32–51. [31] N.C.A. Silva, J.S. Miranda, I.C.A. Bolina, W.C. Silva, D.B. Hirata, H.F. Castro, A.A. Mendes, Immobilization of porcine pancreatic lipase on poly-hydroxybutyrate particles for the production of ethyl esters from macaw palm oils and pineapple flavor, Biochem. Eng. J. 82 (2014) 139–149. [32] H. Dong, J. Li, Y. Li, L. Hu, D. Luo, Improvement of catalytic activity and stability of lipase by immobilization on organobentonite, Chem. Eng. J. 181–182 (2012) 590–596. [33] D.-T. Tran, C.-L. Chen, J.-S. Chang, Immobilization of Burkholderia sp. lipase on a ferric silica nanocomposite for biodiesel production, J. Biotechnol. 158 (2012) 112–119. [34] G. Bayramoglu, B. Karagoz, B. Altintas, M.Y. Arica, N. Bicak, Poly(styrene– divinylbenzene) beads surface functionalized with di-block polymer grafting and multimodal ligand attachment: performance of reversibly immobilized lipase in ester synthesis, Bioprocess Biosyst. Eng. 34 (2011) 735–746. [35] M. Shakeri, K. Kawakami, Enhancement of Rhizopus oryzae lipase activity immobilized on alkyl-functionalized spherical mesocellular foam: influence of alkyl chain length, Microporous Mesoporous Mat. 118 (2009) 115–120. [36] N. Kharrat, Y.B. Ali, S. Marzouk, Y.-T. Gargouri, M. Karra-Chaabouni, Immobilization of Rhizopus oryzae lipase on silica aerogels by adsorption: comparison with the free enzyme, Process Biochem. 46 (2011) 1083–1089. [37] M. Nikolic, V. Srdic, M. Antov, Immobilization of lipase into mesoporous silica particles by physical adsorption, Biocatal. Biotransform. 27 (2009) 254–262.

[38] W.H. Yu, D.S. Tong, M. Fang, P. Shao, C.H. Zhou, Immobilization of Candida rugosa lipase on MSU-H type mesoporous silica for selective esterification of conjugated linoleic acid isomers with ethanol, J. Mol. Catal. B: Enzym. 111 (2015) 43–50. [39] B. Al-Duri, Y.P. Yong, Characterisation of the equilibrium behaviour of lipase PS (from Pseudomonas) and lipolase 100L (from Humicola) onto Accurel EP100, J. Mol. Catal. B: Enzym. 3 (1997) 177–188. [40] D. Hekmat, D. Hebel, D. Weuster-Botz, Crystalline proteins as an alternative to standard formulations, Chem. Eng. Technol. 31 (2008) 911–916. [41] Z. Guo, X. Xu, Lipase-catalyzed glycerolysis of fats and oils in ionic liquids: a further study on the reaction system, Green Chem. 8 (2006) 54–62. [42] A.D. Ferrão-Gonzales, I.C. Véras, F.A.L. Silva, H.M. Alvarez, V.H. Moreau, Thermodynamic analysis of the kinetics reactions of the production of FAME and FAEE using Novozyme 435 as catalyst, Fuel Process. Technol. 92 (2011) 1007–1011. [43] D.H. Zhang, C. Li, G.Y. Zhi, Kinetic and thermodynamic investigation of enzymatic Lascorbyl acetate synthesis, J. Biotechnol. 168 (2013) 416–420. [44] A. Converti, A. Del Borghi, R. Gandolfi, F. Molinari, E. Palazzi, P. Perego, M. Zilli, Simplified kinetics and thermodynamics of geraniol acetylation by lyophilized cells of Aspergillus oryzae, Enzyme Microb. Technol. 30 (2002) 216–223. [45] M. Graber, M.P. Bousquet-Dubouch, N. Sousa, S. Lamare, M.D. Legoy, Water plays a different role on activation thermodynamic parameters of alcoholysis reaction catalyzed by lipase in gaseous and organic media, Biochim. Biophys. Acta 1645 (2003) 56–62. [46] P. Mahapatra, A. Kumari, V.K. Garlapati, R. Banerjee, A. Nag, Enzymatic synthesis of fruit flavor esters by immobilized lipase from Rhizopus oligosporus optimized with response surface methodology. J. Mol. Catal. B: Enzym. 60 (2009) 57–63. [47] S.W. Chang, J.F. Shaw, C.H. Shieh, C.J. Shieh, Optimal formation of hexyl laurate by Lipozyme IM-77 in solvent-free system, J. Agric. Food Chem. 54 (2006) 7125–7129. [48] A. Richetti, S.G.F. Leite, O.A.C. Antunes, L.A. Lerin, R.M. Dallago, D. Emmerich, M. Di Luccio, J.V. Oliveira, H. Treichel, D. de Oliveira, Assessment of process variables on 2-ethylhexyl palmitate production using Novozym 435 as catalyst in a solvent-free system, Bioprocess Biosyst. Eng. 33 (2010) 331–337. [49] L.P. Fallavena, F.H.F. Antunes, J.S. Alves, N. Paludo, M.A.Z. Ayub, R. FernándezLafuente, R.C. Rodrigues, Ultrasound technology and molecular sieves improve the thermodynamically controlled esterification of butyric acid mediated by immobilized lipase from Rhizomucor miehei, RSC Adv. 4 (2015) 8675–8681. [50] J.S. Beck, D.C. Calabro, S.B. McCullen, B.P. Pelrine, K.D. Schmitt, J.C. Vartuli, Method for functionalizing synthetic mesoporous crystalline material, U.S. Patent 2,069,722, 1992.

[51] D.H. Guo, Z. Jin, Y.S. Xu, P. Wang, Y. Lin, S.Y. Han, S.P. Zheng, Scaling-up the synthesis of myristate glucose ester catalyzed by a CALB-displaying Pichia pastoris whole-cell biocatalyst, Enzyme Microb. Technol. 75–76 (2015) 30–36. [52] D.L. Pavia, G.M. Lampman, G.S. Kriz, Introduction to Spectroscopy: A Guide for Students of Organic Chemistry, 2nd ed., Saunders College Publishing, Philadelphia, 1996. [53] H. Horchani, A. Bouaziz, Y. Gargouri, A. Sayari, Immobilized Staphylococcus xylosus lipase-catalysed synthesis of ricinoleic acid esters, J. Mol. Catal. B: Enzym. 75 (2012) 35–42.

Figure Captions

Figure 1. Adsorption isotherm models of immobilization of TLL on PMA particles: Langmuir (a) and Freundlich (b).

Figure 2. Relation between separation factor (RL) and initial protein loading (C0).

Figure 3. Effect of immobilized protein loading in the synthesis of isoamyl oleate. The reactions were performed at 500 mM of each reactant in heptane medium, reaction temperature of 37°C, biocatalyst concentration of 10% m/v and 15 min of reaction. All values are represented as mean ± standard deviation of three replications.

Figure 4. Time courses of esterification reaction performed at different reaction temperatures (a), determination of initial reaction rate constants (b), plot of ln(k) versus 1/T (c), and plot of ln(k/T) versus 1/T (d). The reactions were performed at 500 mM of each reactant in heptane medium, 10% m/v of biocatalyst prepared using protein loading of 100 mg/g of support and 60 min of reaction. All values are represented as mean ± standard deviation of three replications.

Figure 5. Time courses of esterification reaction performed at different biocatalyst concentrations. The biocatalyst used was prepared using protein loading of 100 mg/g of support. The reactions were performed at 500 mM of each reactant in heptane medium, reaction temperature of 45°C and 60 min of reaction. All values are represented as mean ± standard deviation of three replications.

Figure 6. Time courses of esterification reaction performed at different reactant concentrations. The reactions were performed at equimolar ratio alcohol:acid, reaction temperature of 45°C, 20% m/v of biocatalyst prepared using protein loading of 100 mg/g of support and 45 min of reaction. All values are represented as mean ± standard deviation of three replications.

Figure 7. Time courses of esterification reaction catalyzed by soluble and immobilized TLL prepared via several protocols. The reactions were performed at 2500 mM of each reactant in a solvent-free system, reaction temperature of 45°C, 92.76 mg of immobilized protein loading and 30 min of reaction. All values are represented as mean ± standard deviation of three replications.

Figure 8. Reusability tests of TLL-PMA in successive cycles of isoamyl oleate synthesis. The reactions were performed at equimolar ratio alcohol:acid, reaction temperature of 45°C, 20% m/v of TLL-PMA prepared using protein loading of 100 mg/g of support and 30 min of reaction. All values are represented as mean ± standard deviation of three replications.

Figure 9. ATR-FTIR spectra of isoamyl alcohol, oleic acid and purified isoamyl oleate.

Figure 10. 1H NMR (a) and 13C NMR (b) spectra of purified isoamyl oleate.

120

120

(a)

(b) 100

qe (mg/g of support)

qe (mg/g of support)

100 80 60 40

qe 

20

124 . 0 C e 0 . 783  C e

0

1

2

3

4

Ce (mg/mL) Figure 1

5

60 40

q e  59 . 9 C e

20

R 2  0 . 9743

0

80

R 2  0 . 9459

0

6

0 . 492

0

1

2

3

4

Ce (mg/mL)

5

6

1,0

0,8

RL

0,6

0,4

0,2

0,0 0

2

4

6

C0 (mg/mL) Figure 2

8

10

12

70 60

Conversion (%)

50 40 30 20 10 0 5

10

25

50

75 100 125 150 175 200

Initial protein loading (mg/g) Figure 3

100

0,006

(b)

(a) 0,005

20°C 25°C 30°C 40°C 45°C

-1

1/[OA] (mM )

Conversion (%)

80

60

20°C 25°C 30°C 40°C 45°C 50°C

40

20

0,004

0,003

0,002

0 0

10

20

30

40

50

0

60

4

6

8

10

Reaction time (min)

Reaction time (min)

-13,6

(c)

-8,0

2

(d) -13,8

-8,2

-14,0

ln(k/T)

ln(k)

-8,4 -8,6

-14,2 -14,4

-8,8 -9,0

 

ln k   4 .42  3947 .62 

1 T

 

-14,6

R 2  0.9869

0,0031

0,0032

0,0033

1/T

Figure 4

0,0034

1 k ln     2 .28  3646 . 72  T T   R 2  0 .9845

-14,8 0,0031

0,0032

0,0033

1/T

0,0034

100

Conversion (%)

80

60

5% m/v 7.5% m/v 10% m/v 12.5% m/v 15% m/v 20% m/v 25% m/v

40

20

0 0

10

20

30

40

Reation time (min) Figure 5  

50

60

100

Conversion (%)

80

60

500 mM 1000 mM 1250 mM 1500 mM 1750 mM 2000 mM 2500 mM

40

20

0 0

10

20

30

40

50

Reaction time (min)  

Figure 6  

100

Conversion (%)

80

60

40

20 TLL-PMA Soluble TLL TLL-Gly-agarose

0 0

10

20

Reaction time (min) Figure 7      

30

100

Residual activity (%)

80

60

40

20

0 5

10

15

20

25

30

Cycles  

Figure 8

100

80

Transmitance (%)

966 1465

1010

60 1056 1384

40

3320 2871 2928

1367 1125

20

2956

Isoamyl alcohol 0 4000

3500

3000

2500

2000

1500

1000

500

-1

Wavenumber (cm ) 100

80

935

Trasmitance (%)

1284 3005

1412

60

1457

40 2852

20

1707 2971

Oleic acid 0 4000

3500

3000

2500

2000

1500

1000

500

Wavenumber (cm-1) 100

3005

Transmitance (%)

80 1167

60

1244 1368 2956

40

1464

2853

1737

20

2923

Isoamyl oleate 0 4000

3500

3000

2500

2000

1500

1000

500

-1

Wavenumber (cm )

Figure 9  

42  

 

(a) 10000

9000

8000

7000

6000

5000

4000

3000

2000

1000

0

-1000 180

170

160

150

140

130

120

110

100

90 f1 (ppm)

80

70

60

50

40

30

20

10

0

(b)

Figure 10  

43  

Table 1. Influence of initial protein loading on the catalytic properties of immobilized TLL on PMA particles. Protein loading

IYa

IPb

HAc

SAd

(mg/g of support)

(%)

(mg/g support)

5

97.0 ± 0.8

4.9 ± 0.1

120.1 ± 2.0

24.5 ± 0.1

10

96.1 ± 1.9

9.3 ± 0.3

125.4 ± 6.8

13.5 ± 1.0

25

89.5 ± 1.4

20.5 ± 1.5

165.7 ± 7.7

8.1 ± 0.8

50

84.1 ± 3.3

39.8 ± 1.4

424.2 ± 21.6

10.1 ± 0.5

75

81.3 ± 0.6

63.7 ± 2.2

521.7 ± 22.4

8.2 ± 0.1

100

76.1 ± 3.2

77.3 ± 1.3

564.3 ± 24.9

7.3 ± 0.3

125

67.1 ± 1.7

90.4 ± 3.9

604.5 ± 37.1

6.4 ± 0.6

150

65.6 ± 0.4

100.4 ± 3.6

655.0 ± 8.2

6.5 ± 0.2

175

62.4 ± 1.8

102.4 ± 3.0

652.8 ± 14.6

6.5 ± 0.3

200

50.5 ± 2.3

101.6 ± 5.5

657.2 ± 18.7

6.5 ± 0.2

of (IU/g of (IU/mgIP) biocatalyst)

a – Immobilization yield b – Immobilized protein loading c – Hydrolytic activity d – Specific activity

44  

Table 2. Maximum immobilized protein loading (IP) of lipases on several supports via physical adsorption. Lipase source

Surface area

Porous size

IP

(m2/g)

(Å)

(mg protein/g support)

PHB

17

31

24.3 ± 1.7

[31]

Organobentonite

32.5

-

15.6

[32]

Candida antarctica Decaoctyl Sepabeads

-

-

48

[10]

(CALB)

Diaion HP20LX

500–600

200–300

41

MCI GEL CHP20P

500–600

400–600

113

Lewatit

-

-

30 ± 2

[11] 

Burkholderia sp.

alkyl-grafted Fe3O4–SiO2

128

32

29.45

[33] 

Mucor miehei

Functionalized

14.6

-

78.1

[34] 

silica-based 475

-

84.43 ± 4

[35] 

Porcine pancreatic

Support

Reference

poly(STY-DVB) beads Rhizopus oryzae

Spherical

mesocellular foam Silica aerogels

110

-

10

[36] 

Silica particles

359

158

100

[37] 

MSU-H silica

176

10

48.2

[38] 

Thermomyces

PHB

17

31

24.7 ± 1.1

[4] 

lanuginosus

PMA

500

170

102.4 ± 3.0

This study

Candida rugosa

45  

Table 3. Determination of thermodynamic parameters of enzymatic synthesis of isoamyl oleate performed at different reaction temperatures. Reaction

k×104a

temperature

(1/mM.min)

R2b

Eac

∆Hd

∆Se

∆Gf

(kJ/mol)

(kJ/mol)

(J/mol.K)

(kJ/mol)

(°C) 20

1.22

0.9905

87.5

25

1.37

0.9821

88.6

30

1.91

0.9982

40

2.81

0.9946

91.4

45

3.39

0.9979

92.5

32.8

29.7

–197.4

89.5

a – Reaction rate constant b – Correlation coefficient (R2) for k values. c – Activation energy d – Enthalpy e – Entropy f – Gibbs free energy  

46