Saboura Ashkevarian, Jalil Badraghi, Fatemeh Mamashli, Behdad Delavari, Ali Akbar Saboury,3,*

1 Research Institute of Applied Sciences, ACECR, Shahid Beheshti University, Tehran, Iran

2 Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran

3 Center of Excellence in Biothermodynamics, University of Tehran, Tehran, Iran

Keywords:Lipase immobilization CoFe2O4 magnetic nanoparticles Steady state anisotropy Biodiesel Biocatalysis Protein stability

ABSTRACT Rhizopus oryzae lipase (ROL) was immobilized on the surface of silica coated amino modified CoFe2O4 nanoparticles and applied for biodiesel production.The results indicated more affinity of the ROL toward its substrate upon immobilization,as revealed by a lower Km value for the immobilized ROL compared to its free counterpart.Intrinsic fluorescence spectroscopy indicated a lower intensity for ROL immobilized on CoFe2O4 nanoparticles.Besides, immobilized ROL steady state anisotropy measurements presented lower values,which implied assembly of ROL molecules on magnetic nanoparticles upon immobilization as well as their restricted rotation upon covalent attachment.Thermal stability analysis revealed improved activity at higher temperatures for the immobilized enzyme compared to its free counterpart.Accordingly, Pace analysis to determine protein thermal stability revealed preservation of the protein conformation in the presence of increasing temperatures upon immobilization on nanoparticles.Finally,ROL immobilized on CoFe2O4 nanoparticles exhibited improved efficiency of biodiesel production in agreement with thermal activity profile.Therefore,the authors suggest application of the lipase molecules immobilized on CoFe2O4 nanoparticles for more efficient biodiesel production.

1.Introduction

Biodiesel has been introduced as an attractive alternative to the declining fossil fuels owing to its environmental advantages such as emitting lower carbon monoxide and sulfur compounds upon combustion.It is a mixture of alkyl esters of long chain fatty acids obtained from renewable feedstock sources such as edible or nonedible vegetable and animal fats by transesterification reaction[1-4].The edible sources might arise the fuel versus food concern and the problem of deforestation.Therefore, application of non-edible sources like waste cooking oil seems to be more promising compared to edible oils.Presence of high contents of free fatty acids and water in waste cooking oils causes difficulties in its application as a feedstock for biodiesel production through conventional chemical catalysis.Employing lipases to catalyze the transesterification reaction for biodiesel production can overcome these problems [1].

Lipases or triacylglycerol acylhydrolases (EC 3.1.1.3) catalyze the transesterification reaction at the water-lipid interface and produce mono- and di-glycerides and free fatty acids [5,6].Using lipases as a biocatalyst for biodiesel production provides a multitude of advantages such as high degree of efficiency, less energy utilization, high purity of the products, soft reaction conditions,and easy separation of glycerol, as a value added co-product,from the reaction mixture[1,4].However,industrial application of lipase for biodiesel production faces challenges due to its high cost and low stability [3], which can be overcome by immobilizing the lipases on solid supports.Immobilization increases the enzyme stability and facilitates its reusability[4].Lipases have been immobilized using various techniques such as adsorption, covalent bonding,cross-linking,entrapment,and encapsulation on different supports and matrices,which have been studied for biodiesel production [2].Immobilization through covalent attachment can prevent leaching of the enzyme from the support and is appropriate for industrial application [7].Besides, it offers the tailor-made nanobiocatalysts owing to potential application of various organic linkers and established strategies of the carriers’ modification and functionalization [1,8].

Magnetic nanoparticles have been used as the support for lipase immobilization because of their high surface area,biocompatibility, and potentially easy recovery of the biocatalyst from the reaction media [9].Furthermore, the grafting of silica layer on the magnetic nanoparticles prevents their aggregation in liquid media and also provides the silanol groups with the ability to react with organosilane compounds for functionalization purposes [1,4].There are no reports in literature on immobilization of lipase on superparamagnetic cobalt ferrite(CoFe2O4)nanoparticles and its application in biodiesel production.

In the present study,we focused on the effect of immobilization of Rhizopus oryzae lipase (ROL) on CoFe2O4nanoparticles in terms of the catalytic activity, thermal stability, and efficiency of biodiesel production.The silica-coated amine modified CoFe2O4nanoparticles were prepared and characterized through electron microscopy and X-ray diffraction.Then, by the help of glutaraldehyde as the coupling agent, lipase was covalently attached to the surface of the prepared functionalized magnetic nanoparticles.In order to characterize the CoFe2O4- ROL interactions and studying the catalytic activity, structural, and functional changes of immobilized ROL, different techniques including fluorescence and UV/visible spectroscopy were employed.To compare biodiesel producing ability of immobilized ROL, transesterification of waste cooking oil (WCO) to biodiesel was performed using free and immobilized ROL followed by analysis by gas chromatography.

2.Materials and Methods

2.1.Materials

Rhizopus oryzae lipase (ROL), p-nitrophenyl butyrate (pNPB),tetraethyl orthosilicate (TEOS), 3-aminopropyltrimethoxysilane(APTS), and glutaraldehyde (GA) were purchased from Sigma-Aldrich.FeCl3·6H2O, CoCl2·6H2O, KH2PO4, K2HPO4, Bradford reagent,Nile red,bovine serum albumin(BSA),acetic acid,ethanol,and methanol were obtained from Merck.Waste cooking oil was taken from the Institute of Biochemistry and Biophysics (IBB)’s restaurant.

2.2.Synthesis of CoFe2O4 nanoparticles

Chemical co-precipitation method was used to prepare the superparamagnetic CoFe2O4nanoparticles in the current study[8,10].At first, 1.72 g FeCl3·6H2O and 0.62 g CoCl2.6H2O (molar ratio of Fe3+/Co2+as 2:1) were dissolved in 25 ml deionized water using a mechanical stirrer (1000 r·min-1) to which, sodium hydroxide solution was added drop-wise to make the solution pH higher than 12.The temperature of the solution was raised to 80 °C before adding the sodium hydroxide solution and stirred for 30 min.The formed nanoparticles were washed using ethanol and de-ionized water.

2.3.Surface modification and functionalization of nanoparticles

The prepared nanoparticles were surface-modified by adding silica and amine groups.In order to coat the nanoparticles with a silica shell, 75 mg CoFe2O4nanoparticles was suspended in 5 ml toluene and dispersed by ultra-sonication for 10 min.Next,a mixture of 80 ml ethanol, 20 ml deionized water, and 2.5 ml ammonium hydroxide solution were added to the suspension and stirred.Then, 1 ml TEOS was added drop-wise to the suspension during 30 min followed by incubation for 24 h.The obtained silica-coated nanoparticles (CoFe2O4@SiO2) were washed four times with ethanol and water and dried under vacuum at room temperature [8].

Subsequently, the CoFe2O4@SiO2was amine functionalized using 3-aminopropyltrimethoxy silane (APTS) by first dispersing 5 mg of the CoFe2O4@SiO2in 5 ml ethanol.Next, 100 μl APTS,200 deionized water μl, and 100 μl acetic acid were added to the mixture at room temperature followed by stirring for 1 h.Afterwards, the amine functionalized nanoparticles were isolated employing an external magnet and washed by PBS buffer (pH 7.4).At the final step, the obtained modified nanoparticles were reacted with 40 μl of 25% glutaraldehyde (500 μl of modified nanoparticle, 1 mg·ml-1) to enable them to bind to lipase molecules.The mixture was stirred gently at 4°C for 1 h,and after that washed five times by PBS and kept in refrigerator for next uses[8].

2.4.Immobilization of ROL on the functionalized nanoparticles

ROL was immobilized on amino functionalized CoFe2O4@SiO2nanoparticles through first incubating 2 mg of the prepared nanoparticles with 71 mg·ml-1lipase in PBS (pH 7.4) for a period of 17 h at a temperature of 4 °C.Then, using a strong magnetic field, the nanoparticles were separated from the protein solution[10].The collected solutions, containing the un-reacted protein were analyzed using Bradford assay to estimate amount of the immobilized enzymes.The standard curve for Bradford assay was prepared using BSA and determining the absorbance at 595 nm[11].Fig.1 displays the schematic representation of ROL immobilization on CoFe2O4nanoparticles.

2.5.Characterization of the prepared nanoparticles

Nanoparticles were characterized using field emission scanning electron microscopy (FE-SEM), transmission electron microscopy(TEM), Zeta PALS, Fourier-transform infrared spectroscopy (FTIR),X-ray diffraction (XRD), and vibrating sample magnetometer(VSM).After placing and drying a drop of the colloidal suspension of the nanoparticles on a copper grid, the size and shape of the nanoparticles were evaluated using a Hitachi S-4160 FE-SEM(Japan).Furthermore,a drop of the solution was tested by Phillips CM30 TEM (Netherland).The magnetic characteristics of synthesized nanoparticles were tested using a magnet.Zeta potentials were also measured based on electrophoretic mobility of the nanoparticles using Zeta PALS (Zeta Plus - Brookhaven,USA).FTIR analysis was performed using Unicam Mattson 1000 instrument to study the main chemical bonds [13].Furthermore, the crystalline phases in CoFe2O4were identified based on X-ray diffraction using a Philips Analytical X-ray diffractometer (XPert MPD).Magnetization of the nanoparticles was determined using vibrating sample magnetometer (VSM, Mahamax Co., Iran).

2.6.Enzyme activity assay

Using a Shimadzu UV-160 spectrophotometer, a colorimetric assay was applied to determine the activity of free and immobilized lipases by measuring the initial hydrolysis rate of pnitrophenyl butyrate (pNPB) at 40 °C through spectrophotometrically probing phenol butyrate concentration at 410 nm [14].The assay solution including free or immobilized lipase enzymes and PBS buffer was used.When pNPB dissolved in acetonitrile was added as substrate to the prepared assay solution, the catalytic reaction was measured by the absorption of the yellow color product (p-nitrophenol) after 10 min incubation at 40 °C and 300 r·min-1shaking in a thermo shaker.The absorbance data was converted to concentration by applying Beer-Lambert law using an extinction coefficient of the 12,800 mol·L-1·cm-1for pnitrophenol in this buffer system [15].For kinetic measurements,pNPB concentrations of 2-20 μmol·L-1were used.The data obtained from the UV/Visible absorbance were fitted to the Michaelis-Menten equation.Then the kinetic parameters (Vmax,Km, and kcat) were obtained from the Lineweaver-Burk equation[16,17].All tests were performed in triplicate to ensure a high-level of accuracy.

Fig.1.The experimental schematic for synthesis of nanoparticles core shells structures.* The 3D lipase model was obtained from PDB file of Rhizomucor miehei lipase molecule (PDB code: 3tgl) [12].

2.7.Thermal stability of ROL activity

Thermal stability was evaluated by incubating the free or immobilized lipase in a PBS (pH 7.4) at 30, 40, 50, 60, 70, and 80°C for 10 min followed by 1 h incubation at 4°C.Then,the residual activity was measured at assay condition mentioned in the section 2.6.

2.8.Fluorescence spectroscopy

2.8.1.Intrinsic fluorescence

Fluorescence spectra were obtained on an Agilent Technologies spectrofluorometer at the excitation wavelength of 280 nm.The excitation and emission slits were set at 5.0 nm.The intrinsic fluorescence emissions were recorded in the range between 295-500 nm[18].Analysis of thermal stability of free and immobilized enzyme was carried out at 340 nm and the temperature range of 25-90°C by fluorescence spectrometer.The temperature was programmed to raise 1 °C per min.

2.8.2.Steady state anisotropy measurements

Free ROL and CoFe2O4@SiO2@ROL steady state anisotropies were measured on the Agilent Technologies Spectrofluorometer,equipped with manual polarizers.G factor correction was done manually and Nile red was used as the reference fluorophore.The labeled protein was then excited at 550 nm and the parallel and perpendicular emissions were collected at 640-680 nm, followed by calculating the free and immobilized ROL anisotropy values based on the following formula [19]:

Where IVVand IVHrepresent the fluorescence intensities of the vertically and horizontally polarized emissions,respectively.G factor is the ratio of responsiveness of the detection system to vertically and horizontally polarized light and is measured by the following formula [19]:

2.9.Biodiesel production

In the current study, the waste cooking oil (WCO) for production of biodiesel was received from the Institute of Biochemistry and Biophysics (IBB)’s restaurant.The suspended food particles were removed by filtering through a normal sieve followed by heating at 90 °C for 15 min to eliminate remains of water prior to the production of biodiesel.The fatty acid content of the WCO was extracted using BF3 method and identified through gas chromatography [20,21].

The transesterification reaction was performed to convert the WCO to fatty acid methyl esters (FAMEs) in presence of free and immobilized ROL as well as CoFe2O4nanoparticles to test its probable catalyzing activity.For this purpose,20%(mass)free or immobilized ROLs or CoFe2O4nanoparticles and 3 mmol WCO were mixed followed by incubation at 45 °C and stirring at 250 r·min-1for 17 h.To avoid ROL deactivation, an equivalent amount of 3-6 mmol methanol was added in reaction time steps of 0, 8 and 12 h during the biodiesel production process[2].Two-phase media was observed at the end of the reaction for each reaction mixture.The lower(aqueous)phase contained free or immobilized ROL and CoFe2O4nanoparticles were collected applying a magnet from the aqueous (lower) phase mainly consisting of glycerol and other components soluble in water.The upper phase composed of FAMEs were collected and centrifuged at 10,000 r·min-1for 10 min to remove the residual water followed by analyzing the mixture by gas chromatography.

The fatty acid compositions of the WCO and the prepared biodiesel were analyzed by gas chromatography(Claus 580,PerkinElmer).The inert temperature of GC was set at 250 °C.Besides,99.99%pure helium gas with a flow rate of 1 ml·min-1was applied as the mobile phase.An internal standard, nonadecanoic acid(C19:0),was added to each sample before GC measurements.Based on the following equations, the reaction yield and FAME contents for each sample were calculated:

where Aiand A denote the peak areas of standard and total samples in the gas chromatograms, respectively.Moreover, M and miare the masses of sample and standard in the samples,respectively[22].

3.Results and Discussion

3.1.Characterization of the prepared CoFe2O4 nanoparticles

Formation,size,and morphology of CoFe2O4and CoFe2O4@SiO2core/shell was observed through FE-SEM.Fig.2a and b display FESEM images of the nanoparticles before and after silica coating,respectively.According to the SEM images, CoFe2O4nanoparticles were about 66 nm in diameter.The nanoparticles were spherical or quasi-spherical in shape and presented a uniform size distribution.SEM images of silica coated CoFe2O4nanoparticles indicated sizes of nearly 247 nm in diameter after silica coating.The larger sizes observed for the silica coated nanoparticles could be due to the presence of silica layers that covered the surfaces of the nanoparticles.The appeared aggregates in SEM images could be as a result of drying process [23].

Fig.2.SEM images of the CoFe2O4 nanoparticle before (a) and after (b) modification with silica.

Magnetic properties of the prepared nanoparticles were measured using vibrating sample magnetometer.According to the results presented in Fig.3,the S-shape curve obtained for the sample over the applied magnetic field at room temperature pointed out that the sample was superparamegnetic.The magnetic response for the synthesized CoFe2O4nanoparticles was determined to be 10.53 emu·g-1(1 emu = 10-3A·m2), which was in agreement with the published results on magnetization of CoFe2O4nanoparticles [24,25].

3.2.Immobilization of ROL on CoFe2O4 nanoparticles

Immobilization of ROL was performed by incubating the modified CoFe2O4nanoparticles in the ROL solution at room temp erature.The immobilization yield of ROL on the prepared nanoparticles was obtained to be 77.43% using Bradford assay[11].However,ROL activity decreased after immobilization,which can be due to changes induced in the enzyme conformation.It should be noted that it is the first report of ROL immobilization on CoFe2O4nanoparticles.Bohara et al.reported the immobilization of cellulase on amine functionalized CoFe2O4nanoparticles[26].

3.2.1.Transmission electron microscopy

Fig.3.The VSM analysis of the synthetized CoFe2O4 nanoparticles.(1 Oe=79.5775 A·m-1, 1 emu = 10-3 A·m2)

Fig.4 displays the TEM images of the silica coated CoFe2O4nanoparticles.Appearance of a gray layer around the nanoparticles implies formation of the silica shell.This is consistent with FE-SEM images.The TEM images also showed some particles of about 5 nm that are supposed to be lipase molecules immobilized on the nanoparticles surfaces.3.2.2.Zeta potential (ζ) measurement

Zeta potential (ζ) measurements were performed to determine the nanoparticles surface charge at pH 7.4 and 25 °C.Functionalization of CoFe2O4with amino groups and coating with silica and ROL resulted in higher zeta potentials for CoFe2O4nanoparticles which means a decreased particle potential and/or surface charges.In this study, the zeta potentials of - 10.69 and - 16.61 mV were obtained for CoFe2O4in presence and absences of the immobilized ROL, respectively.The measurements were performed in PBS pH 7.4, which is considered as the isoelectric pH for ROL [27].It was noticeable that by amine modification of CoFe2O4, their surface charges changes to more positive values(-3.81 mV)and by immobilizing ROL on CoFe2O4, the positive changes in zeta potential return to negative again, which can be indicative of the successful modification of the nanoparticles and immobilization of ROL on CoFe2O4[28].These results revealed that the immobilization of ROL on CoFe2O4can partially neutralize the surface charges of CoFe2O4.

3.2.3.Fourier-transformed infrared spectroscopy

FTIR spectra of bare CoFe2O4and CoFe2O4@SiO2@ROL are exhibited in Fig.5.The absorption band around 589.16 cm-1corresponds to Fe-O bond, which can be detected for bare CoFe2O4nanoparticle.The peak at 1090.17 cm-1is attributed to the symmetric stretching of Si-O-Si bonds in CoFe2O4@SiO2@ROL.This indicates the successful coating of silica shell on the CoFe2O4nanoparticles.The absorption band at 1632.75 cm-1in CoFe2O4@-SiO2@ROL,as well as the broad band between 3000 and 3500 cm-1can be assigned to—NH2[8].In general,FTIR spectra confirmed the successful functionalization of the CoFe2O4and immobilization of ROL, which corresponded with TEM images and zeta potential measurements.

3.2.4.XRD patterns

The X-ray powder diffraction (XRD) of the prepared CoFe2O4,CoFe2O4@SiO2, and CoFe2O4@SiO2@ROL are presented in Fig.6.The peaks in the diffractogram were in agreement with the expected sample peaks for cobalt ferrite in the JCPDS database and relevant published papers.The Fig.6a shows a series of characteristic peaks at 2θ of 31.15°, 35.39°, 42.67°, 57.11°, and 64.4°associated with crystallographic planes (220), (311), (400), (511),and (440), respectively,in bare CoFe2O4, which pointed to the formation of single cubic spinel crystallographic structure observed in ferrites and corresponded with the iron spinel planes described in the reference pattern JCPDS 22-1086.These results indicated formation of the crystalline structure of CoFe2O4nanoparticles [24].Analysis of the XRD peaks based on Scherrer equation indicated the small size of CoFe2O4crystalline about 5-20 nm, which was in a good agreement with the magnetic properties of CoFe2O4nanoparticles measured through VSM.

Fig.4.TEM images of CoFe2O4 nanoparticles before (a) and after (b) lipase immobilization.

Fig.5.FTIR spectra of (a) CoFe2O4 and (b) CoFe2O4@SiO2@ROL.

Fig.6.The XRD patterns of (a) CoFe2O4, (b) CoFe2O4@SiO2, and (c) CoFe2O4@SiO2@ROL.

The XRD pattern for SiO2coated CoFe2O4nanoparticles is shown in Fig.6b.As shown, the dominating peaks observed are those of CoFe2O4as discussed above.The broad peak at low angles may reveal the presence of the amorphous SiO2.According to the JCPDS database for SiO2, the peaks at 2θ of 31 and about 35 imply the presence of SiO2; although both of them are masked by the CoFe2O4peaks in the same area [25].

Following immobilization of the ROL, the characteristic XRD peaks for both CoFe2O4and CoFe2O4/SiO2were appeared for the immobilized ROL.Presence of the same peaks in the XRD patterns of CoFe2O4@SiO2@ROL indicated that the modification and ROL immobilization caused no effect on the crystalline phase of the nanoparticles [24,25].Accordingly, the immobilized ROL is expected to maintain its magnetic features; hence, permitting the efficient and facile separation of the biocatalyst from the reaction mixture.It is also interesting to note the decrease on the intensity CoFe2O4peaks of the nanocomposite core, which is commonly observed for core-shell structures [24,25].

3.2.5.ROL kinetic parameters

The kinetic studies for the free and immobilized ROL were performed by measuring the initial activity of the enzymes in various concentrations of pNPB as the substrate.The Lineweaver-Burk(double reciprocal) plots of free and immobilized ROL based on Michaelis-Menten (rate of the reaction as a function of substrate concentration) kinetics are shown in Fig.7.

The Lineweaver-Burk plot was drawn to determine Vmaxand Kmvalues for the free and immobilized enzymes.The slope (Km/Vmax)and Y-intercept (1/Vmax) along with their corresponding standard deviations were obtained for the free and immobilized ROLs [16].According to the results of kinetic studies summarized in Table 1,the Vmaxvalues for ROL immobilized on the CoFe2O4nanoparticles decreased compared to the free ROL.In a parallel trend, Kmvalue for the immobilized ROL declined in comparison with free ROL.Furthermore, the catalytic constant (kcat) and kcat/Kmvalues(Table 1) indicated lower and higher values, respectively, for the immobilized ROL compared to free ROL.

Table 1 ROL kinetic parameters upon immobilization on CoFe2O4 nanoparticles and ΔG° and Tm values of free ROL and CoFe2O4@SiO2@ROL obtained by Pace analysis.

Knowledge about the affinity of an enzyme to its substrate can be acquired based on its Kmvalue.Lower value of Kmimplies the higher affinity of enzyme towards its substrate.The presented Kmresults indicated that the affinity of the ROL to its substrate was increased after immobilization on the nanoparticles.Pashangeh et al.and Zhao et al.reported lower Kmvalues for lipase immobilized on Fe3O4nanoparticles compared to its free counterpart[8,29].Furthermore, the higher kcat/Kmvalue presented by CoFe2-O4@SiO2@ROL implies higher accessibility of pNPB to the ROL active site as a result of enhanced diffusion and mass transfer[29].The decrease in the Kmvalue could be due to the change in microenvironment of the enzyme molecules, which in turn depends on the tertiary structure of the enzyme.Since magnetic nanoparticles were so small, it could be imagined that the lipase molecules might be expanded over the particle surface with a better orientation leading to higher affinity to substrate and more available active site [8].

Fig.7.The Lineweaver-Burk plots of the free and immobilized ROL.

3.2.6.Thermal stability of free and immobilized ROL activity

Thermal stability of the immobilized enzyme activity can be considered as a desirable property for the industrial applications in which high temperature conditions are employed in order to enhance the reaction conversion rate and the solubility of some reactants [13,30].Thermal stability of the free and immobilized ROLs was determined by measuring the residual activity of the prepared samples incubated at various temperatures 30,40,50,60,70 and 80°C in the PBS.Fig.8 shows the relative activities of free and immobilized ROLs.Based on the results, free ROL exhibited its maximum activity at 40 °C along with a sharp reduction.In fact,heating the free ROL from 50-80°C resulted in near zero activity of the enzyme.On the contrary, CoFe2O4@SiO2@ROL presented its maximum activity at 50 °C and preserved its activity until 70 °C.This higher stability of the enzyme upon immobilization can be assigned to higher compactness and less flexibility of ROL upon immobilization.It seems that enzyme attachment via glutaraldehyde to the nanoparticles could strengthen the enzyme skeleton[31].

Fig.8.Thermal stability of immobilized ROLs as compared to free ROL at 30,40,50,60, 70, and 80 °C.The results were normalized based on ROL activity at each temperature.

3.2.7.Fluorescence spectroscopy

3.2.7.1.Intrinsic fluorescence intensity measurements.According to the results presented in Fig.9,fluorescence emission of ROL immobilized on CoFe2O4decreased compared to the free ROL.This was in parallel with reduced activity detected for CoFe2O4@SiO2@ROL,which can be because of changes induced in ROL conformation following immobilization.Furthermore, homo-FRET possibly occurred among the ROLs attached to the CoFe2O4nanoparticles surface owing to placement of the enzyme molecules closer together to allow for excitation energy transfer and consequently unpolarization of their emission[32], which can led to lower fluorescence intensity.

Fig.9.Intrinsic fluorescence intensity of free ROL and CoFe2O4@SiO2@ROL.The measurements were performed at room temperature.

3.2.7.2.Pace analysis to determine ROL stability.Heat denaturation of free ROL and CoFe2O4@SiO2@ROL was analyzed using fluorescence spectroscopy at 340 nm in the temperature range of 25-90°C.According to the results shown in Fig.10,fluorescence intensity of free ROL at 340 nm started a sharp decrease from approximately 50 °C while that of CoFe2O4@SiO2@ROL presented a mild decrease from 55 °C.It seemed that immobilization of ROL on the CoFe2O4nanoparticles resulted in protection of ROL from thermal denaturation.Increasing enzyme stability after immobilization can be attributed to compression of enzyme structure, which prevents unfolding of the protein.

The obtained graphs can be applied to determine denatured fraction of protein (fd) based on Eq.(6) [33].

where Fnand Fddenote fluorescence intensities of native and denatured states of ROL, respectively.Besides, Fobsdenotes the observed fluorescence intensity at 340 nm.In the graph presented in Fig.10, initial points indicate native state of the enzyme while the fluorescence intensities at 90 °C were regarded as the denatured states (Fd).The equations of the line for these points were obtained.Moreover, Fnwas calculated for each temperature.According to the two-state theory(Native ?Denatured),the equilibrium constant (K) values can be calculated using Eq.(7).Next,the standard Gibbs free energy (ΔG°) values for the free ROL and CoFe2O4@SiO2@ROL were determined according to Eqs.(8) and(9) [33,34]:

Fig.10.Pace analysis to determine free ROL and CoFe2O4@SiO2@ROL thermal stability.

According to the ΔG°values presented in Table 1,it seems that immobilization process induced no or minor changes in ΔG° values,implying that immobilization has not destroyed ROL structure and has improved thermal stability of the enzyme conformation.However, ROL Tm value did not seem to undergo significant changes following immobilization.This was in agreement with thermal stability studies,which revealed higher activities of CoFe2-O4@SiO2@ROL compared to free ROL.

3.2.7.3.Steady state anisotropy.Steady-state anisotropy (A) values of free ROL and CoFe2O4@SiO2@ROL were found to be 0.133 and 0.116, respectively.According to our results, immobilization of ROL on CoFe2O4resulted in decreased value of A.Lower A value of CoFe2O4@SiO2@ROL can be due to this fact that immobilized ROLs are placed closer together compared to free ROL, which allows for incidence of homo FRET among ROL molecules and consequently lower anisotropy value [35].Furthermore, the lower A value of the immobilized ROL can also be owing to the restricted freedom of the enzyme molecules to rotate when immobilized on CoFe2O4[32].

3.3.Biodiesel production

Fatty acid content of the WCO was determined using gas chromatography and presented in Table 2.Based on the results, the WCO was mainly consisted of 39.38% oleic acid, 32.15% palmitic acid, 21.17% linoleic acid, and 4.2% stearic acid.

Table 2 Fatty acid composition of the waste cooking oil.

Transesterification reaction of WCO in presence of free ROL and CoFe2O4@SiO2@ROL resulted in the biodiesel conversions of 36.2%and 47.4%, respectively (Fig.11).CoFe2O4nanoparticles were unable to produce biodiesel, which implied they have presented no catalytic activity in transesterification reaction.The lower biodiesel production by free ROL compared to CoFe2O4@SiO2@ROL can be assigned to deactivation of free ROL due to presence of methanol and the impurities in the reaction mixture as well as prevention of the immobilized ROL denaturation owing to the covalent bonding to the nanoparticles.The increased biodiesel conversion by CoFe2O4@SiO2@ROL can be attributed to its higher catalytic efficiency compared to free ROL.Presence of amine groups on the nanoparticles can decrease the steric hindrance of the enzyme.This along with attachment of the ROL to the nanoparticles through formation of the covalent bond between amine and aldehyde groups (GA) impedes denaturation of ROL conformation during the reaction time [36].Moreover, immobilization of ROL on the nanoparticles can facilitate formation of enzyme-substrate complex through creation of a larger polar area on the nanoparticles surface [37].Besides, higher CoFe2O4@SiO2@ROL catalyticactivity than free ROL was in agreement with thermal stability studies at 45 °C.

Fig.11.GC analysis of (a) ROL; (b) CoFe2O4@SiO2@ROL and, (c) CoFe2O4 produced biodiesel.

4.Conclusions

In the current study,silica coated amino functionalized CoFe2O4nanoparticles were prepared and used to immobilize the Rhizopus oryzae lipase.The FE-SEM, TEM, zeta potential, VSM, XRD pattern and FTIR analyses determined the successful synthesis and modification of the magnetic nanoparticles,size,and successful immobilization of ROL on the nanoparticles.Comparing activities of CoFe2O4@SiO2@ROL with an equal amount of the free enzyme,indicated improvement in catalytic efficiency as demonstrated by lower Kmvalues.The CoFe2O4@SiO2@ROL showed a higher thermal stability compared to its free counterpart as demonstrated also by Pace analysis to determine protein stability.Intrinsic fluorescence studies revealed lower fluorescence emission intensities of CoFe2-O4@SiO2@ROL than free ROL.Moreover, immobilization caused a decrease in steady-state anisotropy value, which can be due to restricted freedom of the immobilized ROL molecules and incidence of homo-FRET because of placement of the enzyme molecules close together.Besides, ROL immobilized on CoFe2O4nanoparticles revealed higher rate of biodiesel production compared to free ROL.This can be attributed to more stability of the enzyme due to immobilization on the nanoparticles through covalent bonding.The results indicated that the immobilized lipase on CoFe2O4nanoparticles can be successfully used for more efficient biodiesel production.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The work, originated from a Ph.D.thesis, was financially supported by Research Institute of Applied Science(RIAS)ACECR,Institute of Biochemistry and Biophysics(IBB)and Iran National Science Foundation (INSF).