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Magnetic cross-linked enzyme aggregates of Km12 lipase: A stable nanobiocatalyst for biodiesel synthesis from waste cooking oil
Renewable Energy 141 (2019) 874e882
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Magnetic cross-linked enzyme aggregates of Km12 lipase: A stable nanobiocatalyst for biodiesel synthesis from waste cooking oil Arastoo Badoei-dalfard a, b, *, Saeid Malekabadi a, Zahra Karami a, Ghasem Sargazi c a
Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, Iran Research and Technology Institute of Plant Production (RTIPP), Shahid Bahonar University of Kerman, Kerman, Iran c Department of Nanotechnology, Graduate University of Advance Technology, Kerman, Iran b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 May 2018 Received in revised form 19 March 2019 Accepted 13 April 2019 Available online 14 April 2019
Enzymatic production of biodiesel from waste cooking oils (WCOs) is expected as an efficient procedure for resolving the problems of energy demand and environment pollutions. But, high cost of lipases has been found as a main obstacle to commercialize the enzymatic transesterification. In the present study, cross-linked enzyme aggregates of Km12 lipases have been coupled with amino coated magnetite nanoparticles. SEM analysis showed that mCLEAs-lipase (mCLEAs-lip) nanocomposites have spherical structures. The mCLEAs-lip displayed a shift in optimal pH towards the alkaline, whereas optimal temperature was also shift towards low temperature. mCLEAs-lip nanocomposite reserves its total activity up to 6 cycles of enzyme re-using. Furthermore, it reserves 60% of its initial activity more than free enzyme after 24 days of incubation at 4 C. Biodiesel production from waste cooking oils by immobilized enzyme increased about 20% more than free enzyme. Therefore, the present study displays great practical latent to produce renewable fuel such as biodiesel. © 2019 Elsevier Ltd. All rights reserved.
Keywords: mCLEAs Lipase Biodiesel Waste cooking oil Renewable fuel
1. Introduction Due to restricted energy reserves and gradually serious problems with green-house gases coming from the fossil fuels, the application of biofuels has become a hot subject of recent years [1]. Biodiesel, which is an easily biodegradable, renewable, environmentally friendly, and non-toxic energy resource has fascinated extensive consideration [2,3]. Compared to petrodiesel, it is a clean burning fuel which emits lower carcinogenic compounds such as polycyclic aromatic hydrocarbons, sulfur, and metals to the atmosphere. Biodiesel is the methyl ester of fatty acid prepared from oils and animal fats through transesterification. Furthermore, huge volumes of waste cooking oils (WCOs) are formed annually throughout the world [4]. Dispose of waste cooking oils make ecological problems due to which oily films make over aquatic surfaces, which causes disruption in oxygen diffusion and clogging [5]. Therefore, the WCOs should be removed in a sustainable approach, otherwise, they would be discarded and cause environmental pollution problems [6,7]. Consuming WCOs as
* Corresponding author. Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, Iran. E-mail address: [email protected] (A. Badoei-dalfard). https://doi.org/10.1016/j.renene.2019.04.061 0960-1481/© 2019 Elsevier Ltd. All rights reserved.
cheap and starting material for biodiesel production is an attractive approach to make high-value added and ecologically benign compounds [8]. Usually, alkaline catalysts are used for the synthesis of biodiesel owing to their faster reaction rate [9,10], but these catalysts have some disadvantages, such as soap formation, high energy consumption, difficulties in the glycerol recovery, the need for removal of catalyst, neutralization of alkaline wastewater and latent pollution to the environment [10e12]. In spite of that, high content of water concentrations and free fatty acids (FFAs) of waste cooking oils make them improper for the homogeneous alkalinecatalyzed transesterification [4]. Recently, some thermochemical transformation methods such as pyrolysis, combustion and gasification to construct alternative fuel from waste resources have been investigated [13,14]. But, the obtained fuels have some adverse properties such as high acidity and oxygenated compounds, high water content, less burning efficiency and time-consuming [15]. Because of the limitation presented by conventional pyrolysis procedures, microwave pyrolysis has been offered as a substitute to improve the heating and cracking mechanism in pyrolysis development [16e18]. The pyrolysis methodology was also studied using diverse materials as the reaction bed, activated carbon, comprising particulate carbon and mesoporous aluminosilicate (MCM-41). They are
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recognized to have low in cost, high microwave absorbency and heat tolerance [19]. Lam and co-workers (2016) mentioned that an activated carbon prepared fast heating and a restricted reaction hot area that thermally stimulated wide pyrolysis cracking of the waste oil at 450 C, leading to amplified construction of a biofuel invention [20]. In addition, Mahari and co-workers (2018) reported that the microwave copyrolysis of used frying oil and plastic waste established positive synergistic effects in preparing fast heating frequency and a lower reaction time, and produced up to 81 wt% yield of liquid oil [15]. In spite of that, biodiesel production using enzymatic transesterification has numerous benefits including mild reaction conditions, low energy consumption, lower reaction temperatures, complete conversion of free fatty acids to methyl/ethyl esters, and production of very high purity biodiesel even from low quality feedstocks [21e24]. In addition, high catalytic efficiency and substrate specificity make them a smart choice to the chemical procedures. However, high cost, low enzyme stability, and difficult recyclability are the major obstacles of using enzymes in large-scale reactors. Enzyme immobilization is a high impact strategy to dissolve these problems. Re-usage, higher reaction rates, better thermal stability, and reducing the catalyst cost are some advantages of enzyme immobilization [23,25e29]. Ideal immobilization supports are chemically stable and give good dispersion and high enzyme loading. Because of keeping enzyme mobility and mass transfer characteristics in solution, improving enzyme activity and stability, nanomaterials become an excellent support for enzyme immobilization. Cross linked enzyme aggregate (CLEA) is a cheap and efficient strategy for enzyme immobilization [30]. In addition, magnetic nanoparticles were also shown excellent properties in order to improve reusability of enzyme without centrifuge [30]. Although, magnetic cross linked enzyme aggregates of lipase from Candida antarctica lipase B has been reported recently [31], but our strategy was different since we made an active CLEA-lipase separately and then it was covalently coupled to the coated magnetic nanoparticles. It is mentioned that, Km12 lipase is an organic solvent tolerant, cold active and talented lipase mainly for the biotransformation reactions in which recently reported in our group [32,33]. Finally, the application of magnetic cross linked enzyme aggregates of Km12 lipase (mCLEAs-lip) on the biodiesel production was also investigated by waste cooked oils as waste, cheap and abounded feedstocks. 2. Materials and methods 2.1. Materials
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remaining ions, the gained precipitate was centrifuged and washed four times with deionized water up to a pH value of 7.0 was attained; the resulting powder was dried at 100 C for 120 min. Then, the surface of these nanoparticles was functionalized with 3-aminopropyl triethoxysilane (APTES) by a silanization procedure in order to acquire amino activated magnetic nanoparticles. For this reaction magnetite nanoparticles (0.02 g), 3-aminopropyl trimetoxysilane (100 mL), and deionized water (25 mL) in methanol (2.5 mL) was dissolved. The resulting mixture was sonicated for 30 min. Afterward, glycerol (1.5 mL) was added to the mixture, and the solution was heated at 90 C for 6 h with maximum agitation. The acquired precipitate was washed with methanol and deionized water for three times in each case and dried (Fig. 1). 2.3. Preparation of mCLEAs of Km12 lipase Protein purification was performed by 85% (W/V) ammonium sulphate and Q-Sepharose column based on our previous reports [35e38]. For precipitation of lipase, saturated ammonium sulphate solution (5 mL) was mixed gradually to the lipase solution in phosphate buffer (5 mg/mL, 100 mM and pH 7.0) and stirred for 20 min at 30 C. After that, the mixture was also stirred at 4 C for 30 min to complete the precipitation. For making the CLEAs of Lipase, glutaraldehyde solution (final concentration of 40 mM) was added into the resulting suspension and stirred for 3 h at 30 C. mCLEAs of Km12 lipase were also obtained as follows. The amino coated magnetite nanoparticles (5 mg) were gradually mixed with CLEAs-lipase and glutaraldehyde solution (final concentration of 40 mM). The resulting suspension stirred for 3 h at 30 C. mCLEAs of Km12 lipase were separated using magnet, washed for four times by phosphate buffer (100 mM, pH 7.0) and stored in the mentioned buffer at 4 C (Fig. 1). 2.4. Lipase activity assay Hydrolysis of the p-nitrophenyl palmitate was investigated by the colorimetric method. The substrate (pNPP) was dissolved in the isopropanol solution with the final concentration of 25 mM. In the standard assay, 20 mL of a substrate solution was added to 960 mL of phosphate buffer (100 mM, pH 7.0). The reaction was initiated by adding 2 mg of the free enzyme solution/immobilized enzyme. The reaction was incubated for 30 min at 35 C. The absorbance of the product (pNP) was investigated at 410 nm [32,33]. One unit of lipase activity was considered as the amount of enzyme that produced one micromole of product per minute under the standard assay conditions. Spontaneous hydrolysis of p-NPP was also considered using the same of substrate solution in the absence of
3-aminopropyl triethoxysilane (APTES) and p-nitrophenyl palmitate were purchased from Sigma (St. Louis, USA). Glutaraldehyde (25%, v/v), FeSO4$7H2O, FeCl3$6H2O were acquired from Merck (Germany). Other reagents used were of analytical grade and gained either from Sigma or Merck. The water used in this work was Millipore water. 2.2. Preparation of functionalized magnetite nanoparticles Magnetite nanoparticles were synthesized and activated with amino groups by the method of Reza et al. (2010) and CruzIzquierdo et al. (2014) with slight modification [31,34]. These particles were prepared by addition of ammonium hydroxide (NH4OH) into a mixed solution of 0.72 g of ferrous sulphate and 1.43 g of ferric chloride in 30 mL deionized water pending gaining a brown precipitate at room temperature. In order to eliminate the
Fig. 1. Schematic illustration of the preparation of magnetic Cross-Linked Enzyme Aggregates (mCLEAs) of Bacillus licheniformis Km12 Lipase (mCLEA-lip).
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Km12 lipase as control. Enzyme activity of free and immobilized was also investigated at different concentration of pNPP, as substrate. Activity recovery of Km12 lipase in mCLEAs was considered using:
Reusability of mCLEAs-lip was investigated in the standard assay condition as described previously. After each cycle of 30 min sub-
Total lipase activity in magneticCLEAs ðUÞ 100 Total lipase activity used for magnetic CLEAs preparation ðUÞ
2.5. Characterization of mCLEAs of Km12 lipase Structural characterization of the magnetite nanoparticles and mCLEAs were considered by scanning electron microscope (SEM). Scanning electron micrographs were acquired on JEOL JSM6360 (Germany) scanning electron microscope (SEM) operated at 5 kV. Samples were dried by rinsing with anhydrous acetone, placed on a sample holder, coated with platinum before being scanned under vacuum. Fourier transform infrared (FT-IR) spectra were identified on a Shimadzu IR-Prestige-21 spectrometer in the spectral range of 400e4000 cm1 following the KBr pellet techniques. 2.6. Enzyme characterization 2.6.1. Optimal conditions for enzyme activity The optimum temperature of the free Km12 lipase and mCLEAslip was considered by adding the enzyme (2 mg of each form) into the substrate (pNPP) solution in phosphate buffer (100 mM, pH 7.0) at different temperatures (10e70 C). The optimum pH was also determined by adding the enzyme (2 mg of each form) into the substrate (pNPP) solutions of different pHs (3.0e11.0) at 35 C. In the range of 3.0e5.0, 100 mM acetate buffer, in the range of 6.0e8.0, 100 mM phosphate buffer, for pH 9.0, 100 mM bicarbonate buffer and in the range of 10.0e11.0, 100 mM glycine were utilized. The activities of free and immobilized lipase were determined as mentioned previously. 2.6.2. Thermal stability of free and mCLEAs-lip Thermal stabilities of the free and mCLEAs-lip were investigated by incubating them in phosphate buffer (100 mM, pH 7.0) without substrate at 40 and 50 C. After regular time intervals, samples taken up and lipase activity was determined as mentioned in standard assay. The residual activity at time zero was considered as 100%. Storage stabilities of the free and mCLEAs-lip were also investigated by incubating enzyme samples in phosphate buffer (100 mM, pH 7.0) without substrate at 4 C. Every 2 days, mCLEAslip was separated from the buffer by a magnetic and washed by distilled water. Then, the lipase activity in free and immobilized samples was measured as described previously. The residual activities were measured by taking the initial lipase activity before incubation as 100%. 2.6.3. Determination of kinetic parameters Kinetic parameters of free and mCLEAs-lip were examined using different pNPP solution concentrations in the range of 0.2e3.0 mg/ mL in phosphate buffer (100 mM, pH 7.0) at 35 C. In each form, 2 mg of lipase was used. Km, Vmax values of free enzyme and mCLEAs were calculated from LineweavereBurk plot of the initial reaction rates corresponding to different substrate concentrations by Graph Pad Prism software.
strate hydrolysis reaction, mCLEAs-lip were separated using magnetic separation, washed with buffer and then suspended again in a fresh reaction mixture to measure lipase activity. The enzyme activity of each cycle was determined in terms of residual activity by taking the enzyme activity of the first cycle as 100% [39]. 2.8. Biodiesel production from waste cooking oil Trans-esterification reactions by free and mCLEAs-lip have been done in 10 mL screw-capped vials containing 2.2 g of waste cooking oil, anhydrous methanol (1:3 M ratio between oil and methanol) and 0.3% free or immobilized lipase (w/w, based on the oil weight, g). The reaction was incubated in a shaking incubator with stirring rate of 170 rpm for 72 h at 35 C. At different time intervals, 100 ml of reaction were picked up and mixed with 100 ml of n-hexane. Then, the mixture was vortexed, centrifuged and the upper layer (10 ml) was applied to gas chromatography (GC) analysis for biodiesel measurement [40e42]. 3. Results and discussion 3.1. Lipase immobilization and characterization Km12 Lipase is a valuable enzyme for the biotransformation reactions in which recently reported in our group [24]. Results of protein concentration with Bradford assay method showed that protein loading on the functionalized magnetite nanoparticles was achieved successfully. Additionally, the results of quantitative measurements of protein loading on this magnetic nanoparticles showed that the immobilization efficiency was gained about 75% (Fig. 2).
6
Protein concentration (mg/ml)
Activity recovery ð%Þ ¼
2.7. Reusability of mCLEAs-lip
5 4 3 a
b
2 1 0 Km12 Lipase
Supernatant
mCLEA-lip
Fig. 2. Protein loading of Km12 Lipase on magnetic Cross-Linked Enzyme Aggregates (mCLEAs) of Bacillus licheniformis Km12 Lipase (mCLEA-lip). The insert picture shows the image of mCLEA-lip particles that are dispersed in buffer solution (A) and mCLEAlip particles after a magnet was placed aside (B).
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mCLEAs-Lip particles were distributed in phosphate buffer solution. After a magnet was placed sidewise, mCLEAs-Lip showed fast response (60 s) to the external magnetic field (Fig. 2). It indicated that the mCLEAs-Lip particles were revealed respectable magnetic responsibility even if layers of CLEAs lipase were coated on their surfaces, in which it is serious in term of protein immobilization. 3.2. Analytical characterization Km12 Lipase was immobilized on the surface of amino functionalized magnetite nanoparticles, in which amine (in the lipase) can react with aldehyde group of glutaraldehyde linker and another aldehyde group of glutaraldehyde also react with amino coated magnetite nanoparticles [29]. Fig. 3a and b show the morphology of amino functionalized magnetite-nanoparticles and mCLEAs-Lip in SEM image. The SEM investigations on Fig. 3a revealed that the amino functionalized magnetite nanoparticles were spherical and preserved its structure after several post-synthetic steps. Results of SEM image of lipase immobilization on the coated magnetite nanoparticles (Fig. 3b) shown that, these particles appear to be aggregated. These results indicated that the reaction should have been happened on the surface of magnetite nanoparticles. In addition, this image show separated complexes which indicated that the CLEAs-Lip immobilization on the surfaces of coated
A)
B)
Fig. 3. SEM images of (a) Fe3O4 nanoparticles and (b) mCLEAs of Km12 lipase.
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magnetite nanoparticles. Presence of surface functional groups, binding of lipase onto magnetite nanoparticles and mCLEAs were considered by FTIR spectroscopy. FTIR spectra of mCLEAs (A), coated magnetic nanoparticles (B) and coated mCLEA (C) have been shown in Fig. 4. The peak around 530e580 cm1 could be qualified to the stretching vibration of FeeO in Fe3O4, indicating the existence of Fe3O4 in the microspheres which directed that the preparation of mCLEAs nanoparticles was successful. Furthermore, peaks at 1634 cm1 and 1463 cm1 correspond to C]O vibrations were detected in the products of coated mCLEA and approving the existence of amide linkage (eCONH2) between glutaraldehyde with magnetite nanoparticles and CLEAs [38,43]. FTIR spectrum of mCLEA-lip also shows two absorption peaks at 2854 and 2924 cm1 referring to CeH stretching in eCH3 and eCH2-, which confirm the immobilization of lipase on the support. In addition, the presence of a peak in 2900e3000 cm1 relays to aliphatic chain of functionalized APTES. Moreover, a typical adsorption band performed at 3438 cm1 corresponding to the stretching vibration of NeH and partially OH group, which showed great absorbance in the mCLEA-lip [4,44]. 3.3. Enzyme characterization 3.3.1. Optimum pH and temperature activity pH is a critical parameter in enzymatic catalysis. Lipase activity of immobilized and free enzyme was examined from pH 3.0 to 12.0. As shown in Fig. 5A, the optimum pH activity of free enzyme was about 8.0, while those of immobilized enzyme was pH 9.0. It is mentioned that, the immobilized lipase showed slight shift to the alkaline pH, in which lipase activity was improved about 10% in a wide pH range of pH 9.0e12. The same results was also reported by Qi and co-workers (2017) which showed the immobilized lipase displayed better pH tolerance compared to free lipase [45]. A shift in the optimum pH was also detected by CLEAs of alpha amylase [30], tyrosinase [46], subtilisin [47], and lipase [48]. In the mentioned reports, authors indicated that this shift might be produced by the ionization change of basic and acidic amino acid side chain in the active site microenvironment, which was produced by the newly formed interactions between glutaraldehyde and basic residues of enzyme during cross-linking. Lipase activity in different temperatures was also shown in Fig. 5B. The optimal temperature for free enzyme were about 35 C, whereas the immobilized lipase showed maximum enzyme activity between 30 and 45 C. The shift in optimal temperature was also observed by the other immobilized CLEAs of enzymes. This shift in optimum temperature could be as a result of covalent bond development between enzymes produced by glutaraldehyde during CLEAs construction which might decline the conformational flexibility of protein and protects it from denaturation by heat exchange [30,46,47]. Furthermore, the immobilized enzymes displayed the optimum activity in the upper temperature, which could decrease the mass transfer resistance of the substrate [45]. 3.3.2. Thermal stability measurement The thermostability of the free and immobilized enzyme was examined by incubating them at 40 and 50 C. As shown in Fig. 5C, the immobilized lipase has better thermal stability, which reserved 93% of its initial activity after incubation for 180 min at 50 C. It is more than the results of the thermostability of the free and immobilized enzyme by Qi and co-workers (2017). They reported that, the free lipase approximately deactivated after keeping at 60 C for 150 min, whereas the immobilized lipase preserved 50% activity, which revealed that the immobilized lipase demonstrated better stability than the free one [45]. Compared to free enzyme,
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A)
B)
C)
Fig. 4. The FTIR spectra of (a) Fe3O4, (b) coated-magnetic nanoparticles, and mCLEAs of Km12 lipase (c).
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120
120
A)
B)
100
80
Activity (%)
Activity (%)
100
60 40
Free Immobolized
20
80 60 40
3
4
5
6
7
8
9
Free Immobilized
20
0
0
10 11 12
10
20
30
40
50
60
70
Temperature (°C)
pH 110
C)
120 100 80 60 40
Immobilized Free
20 0 0
20 40 60 80 100 120 140 160 180
Remaining activity (%)
140
Remaining actvity (%)
879
D) 90
70
50
30
Immobilized Free 0
20
Time (min)
40
60
80 100 120 140 160 180
Time (h)
Fig. 5. Influence of temperature (a) and pH (b) on enzyme activity of mCLEAs-lip and free enzyme. Assuming the highest activity of mCLEAs-lip and free enzyme as 100%. The thermal stability of mCLEAs-lip and free enzyme at (c) 40 C, and (d) 50 C. The experiments were done in triplicate and the error bar represents the percentage error in each set of determinations.
thermostability of immobilized lipase improved up to 60 min of incubation at 40 C (Fig. 5D). Furthermore, results in Fig. 5C and D, showed that the activity of immobilized form increased about 20 and 10% after incubation 20 min at 40 and 50 C, respectively. These significant results may be associated with the multipoint interactions between enzyme and magnetic support which may strengthen the intramolecular forces and avoid the autolysis of enzyme [50]. In most lipases, a mobile lid shelters the active site. In this closed building, the lipase is expected to be inactive. But, in its open assembly, the active site is accessible and the lipase is active. It has been expected that the activity and thermostability of lipases can be changed by modifications in their lid domains [49]. It is mentioned that, the improvement in the thermal tolerance of mCLEAs might be due to the suitable covalent cross-linking between proteins, glutaraldehyde and amino functionalized magnetite nanoparticles [30]. These covalent interactions delivered a more actual structure stabilization in mCLEAs needing much more energy to disruption this active structure than free enzyme [51e53]. Furthermore, improving enzyme stability after immobilization can be ascribed to rigidification of enzyme conformation which inhibits protein unfolding [4]. It is mentioned that, in our previous study, we reported that the activity of Km12 lipase was remarkably increase after preincubation at 20 C in which the lipase activity was increased about 37% after incubation for 60 min. These results indicated that Km12 lipase was nominated as cold-active enzyme [32]. Pretreatment of the tissues with cold-adapted lipase increases the softness and durability of the tissue and reduces the pill-formation in the textile industries [54]. Consequently, cold adaptive property of
immobilized Km12 lipase indicated that it is an appreciated candidate for bio-washing and stone washing of fabric materials. 3.3.3. Determination of kinetic parameters Kinetic parameters of free and mCLEAs-lip were investigated by calculating initial reaction rates with different substrate concentrations. For both forms, MichaeliseMenten kinetic behavior and LineweavereBurk plot were considered. As shown in Table 1, Vmax values of both forms are approximately equal, which directed the rate of pNPP hydrolysis was not changed after mCLEAs preparation. The same results were also observed for magnetic CLEAs of amylase [30]. In the case of mCLEAs-lip, the observed lower Km value specify a greater lipase affinity for the pNPP. It recommends that conformational changes due to enzyme immobilization assist the protein to appropriately turn its active site concerning the substrate [30,46,47,55]. 3.3.4. Reusability assay Reusability of immobilized enzyme training is a chief parameter for its cost-effective use in term of biotechnological application. The
Table 1 Kinetic parameters of free enzyme and mCLEAs-lip. Form of lipase
Km (mM)
Vmax (mmol/min)
Km12 lipase mCLEAs-lip
0.53 0.39
5.65 5.95
In each form, 2 mg of lipase was used.
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reusability of mCLEAs-lip was considered up to 10 cycles. No decrease in lipase activity of mCLEAs-lip was found after 5 cycles of using while it was diminished continuously over 5 cycles (Fig. 6A). Leaking of proteins was also considered during reusability experiment of mCLEAs-lip. Results showed no activity was observed in reaction medium up to 5 cycles of reuse for mCLEAs-lip. These results propose that satisfactory cross-linking of lipase and amino coated magnetite nanoparticles caused operationally stable mCLEAs [30]. To examine the effect of immobilization procedure on the storage stability of lipase, free and mCLEAs-lip were stored in phosphate buffer at 4 C. Results showed that the free enzyme lost practically its total activity after 24 days of incubation, but mCLEAs reserved about 85% of its initial activity, after 24 days (Fig. 6B). It is more than the immobilized MG10 lipase on magnetic amino functionalized graphene oxide which retained about 75% of its original activity after 30 days of incubation at 4 C [56]. These results proved that mCLEAs had major improvement on the storage stability relating to free enzyme. These results might be associated to a more active cross-linking of the protein in mCLEAs-lip which avoids enzyme leaking into aqueous buffer and enzyme separation from aggregates [57].
100 Immobilized lipase Free lipase
80
Conversion (%)
880
60
40
20
0 6
12 18 24 30 36 42 48 54 60 66 72
Time (h) Fig. 7. Biodiesel production from waste cooking oils (WCOs) using different amounts of free and mCLEAs-lip at different time of incubation.
3.4. Biodiesel production Because of reducing fossil fuel resources, pollution problems and growing energy demands bio-based fuel such as biodiesel get more attention as an alternative fuel. It is mentioned that biodiesel production from waste cooking oil can overcome the problems of food verse fuel, economic and environmental problems related with edible vegetable oils [4,23]. But, there are just few reports on biodiesel production using these abundant resources [4,58e61]. The maximum biodiesel production (49%) from waste cooking oil was obtained at 35 C after around 42 h by Km12 Lipase (10 mg) (Fig. 7). Mehrasbi et al., reported using 100 mg of free Candida antarctica lipase B producing 34% and 48% of FAMEs from waste cooking oil at 50 C after 72 h and 96 h of the reaction, respectively [4]. It is mentioned that, Km12 lipase has some distinctive properties such as cold active, tolerant in polar organic solvents (especially methanol) and glycerol by-product, high affinity to long chain triglycerides, and short time transesterification which make it endowed as a talented biocatalyst for transesterification reactions, especially for biodiesel production [32]. Interestingly, mCLEAs-lip produced maximum biodiesel production (71%) from waste cooking oil around 36 h (Fig. 5). It is more than the immobilized Candida antarctica lipase B which produced
biodiesel synthesis about 42% [62]. In addition, immobilized Km12 lipase increase biodiesel production from waste cooking oil about 10e20% at different time of incubation, compared to free enzyme (Fig. 7). De los Ríos and co-workers (2011) indicated that, the biodiesel production by Candida antarctica lipase B has been attributed to methanol inhibition of lipase [62]. It is mentioned that methanol is extensively used as acyl acceptor for the construction of biodiesel and is regularly favored over other alcohols due to its low cost. But, the enzyme activity of many lipases repressed by methanol which can limit the biodiesel production [4,63]. However, immobilization is a good approach to improve lipase activity in the presence of organic solvents, especially methanol. Mehrasbi and co-workers (2017) reported that liquid lipase from Candida antarctica (CALB) reserved 65% of its initial activity in 50% of methanol, after 96 h of incubation at 25 C. In spite of that, the immobilized CALB displayed outstanding stability in methanol, retaining 94% of its original activity in 50% methanol [4]. Furthermore, Yan and co-workers used Geotrichum sp. lipase to construct cross-linked PCMCs (CLPCMCs), which exhibited better catalytic efficiency in biodiesel construction from waste cooking oil compared to PCMCs and free lipase [64]. It is mentioned that, CLPCMCs also preserved 83% of biodiesel
120 Remaining activity (%)
Activity (%)
100
A)
100 80 60 40 20
B)
80 Free Immobilized
60 40 20 0
0 1
2
3 4 5 6 Number Cycle
7
8
0
9
Fig. 6. a) Reusability of mCLEAs-lip. b) The storage stability of mCLEAs-lip and free enzyme at represents the percentage error in each set of determinations.
4
8
12
16
20
24
Day 4 C
in aqueous buffer. The experiments were done in triplicate and the error bar
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production after incubated in organic solvents for 4 h [64]. In overall, the results of biodiesel production by mCLEA-lip nanocomposite indicated that this immobilization strategy might be improved the lipase stability in the long time biodiesel production, compared to free enzyme. Finally, covalent bond formation between enzyme molecules and support, may preserve enzyme in active conformation and increased the lipase rigidity with concomitant creation of a sheltered micro-environment. Additionally, it made a more active cross-linking of the protein in mCLEAs- lip which avoids enzyme leaking from aggregates and protect it against methanol and the other by products [30,42,43]. 4. Conclusion A facile method to immobilize CLEAs of Km12 lipase onto amino-coated magnetite nanoparticles was established to attain enhanced activity and stability. Shift in optimal pH and temperature towards the alkaline pH and low temperatures are the other valuable parameters of mCLEA-lip for biodiesel production which can reduce the cost of biodiesel production. In addition, mCLEA-lip shows high impact for biodiesel production which increases biodiesel production from waste cooking oil about 20% after 30 h of incubation, compared to free enzyme. Taken to gather, unique properties of mCLEAs-lip open an attractive way towards commercialize the enzymatic transesterification of waste cooking oil. Acknowledgements The authors express their gratitude to the Research Council of the Shahid Bahonar University of Kerman, Kerman (Iran) and Research, Technology Institute of Plant Production (RTIPP), Kerman (Iran) for their financial support during the course of this project. References [1] K.R. Jegannathan, S. Abang, D. Poncelet, E.S. Chan, P. Ravindra, Production of biodiesel using immobilized lipase d a critical review, Crit. Rev. Biotechnol. 28 (2008) 253e264. [2] W. Tao, P. Yu, S. Gong, Q. Li, Y. Luo, Application of KF/MgO as a heterogeneous catalyst in the production of biodiesel from rapeseed oil, Kor. J. Chem. Eng. 25 (2008) 998e1003. [3] M.D. Serio, R. Tesser, L. Pengmei, E. Santacesaria, Heterogeneous catalysts for biodiesel production, Energy Fuels 22 (2008) 207e217. [4] M.R. Mehrasbi, J. Mohammadi, M. Peyda, M. Mohammadi, Covalent immobilization of Candida antarctica lipase on core-shell magnetic nanoparticles for production of biodiesel from waste cooking oil, Renew. Energy 101 (2017) 593e602. [5] H.O.D. Clarissa, Z.P. Debora, F. Roselaine, M.O.M. Marcia, S.N. Augusto, Z.C.F. Cello, F.Z. William, F.F. Luciana, Bioremediation of cooking oil waste using lipases from wastes, PLoS One 12 (2017) 1e17. [6] A.B. Chhetri, K.C. Watts, M.R. Islam, Waste cooking oil as an alternate feedstock for biodiesel production, Energies 1 (2008) 3e18. [7] M.K. Lam, K.T. Lee, A.R. Mohamed, Homogeneous, heterogeneous and enzymatic catalysis for transesterification of high free fatty acid oil (waste cooking oil) to biodiesel: a review, Biotechnol. Adv. 28 (2010) 500e518. [8] D. Li, W. Wang, M. Faiza, B. Yang, Y. Wang, A novel and highly efficient approach for the production of biodiesel from high-acid content waste cooking oil, Catal. Commun. 102 (2017) 76e80. [9] S.V. Ranganathan, S.L. Narasimhan, K. Muthukumar, An overview of enzymatic production of biodiesel, Bioresour. Technol. 99 (2008) 3975e3981. [10] L.P. Christopher, H. Kumar, V.P. Zambare, Enzymatic biodiesel: challenges and opportunities, Appl. Energy 119 (2014) 497e520. [11] H. Fukuda, A. Kondo, H. Noda, Biodiesel fuel production by transesterification of oils, J. Biosci. Bioeng. 92 (2001) 405e416. [12] B.K. Barnwal, M.P. Sharma, Prospects of biodiesel production from vegetable oils in India, Renew. Sustain. Energy Rev. 9 (2005) 363e378. [13] S.A. Opatokun, V. Strezov, T. Kan, Product based evaluation of pyrolysis of food waste and its digestate, Energy 92 (2015) 349e354. [14] K. Im-orb, W. Wiyaratn, A. Arpornwichanop, Technical and economic assessment of the pyrolysis and gasification integrated process for biomass conversion, Energy 53 (2018) 592e603.
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Renewable Energy journal homepage: www.elsevier.com/locate/renene
Magnetic cross-linked enzyme aggregates of Km12 lipase: A stable nanobiocatalyst for biodiesel synthesis from waste cooking oil Arastoo Badoei-dalfard a, b, *, Saeid Malekabadi a, Zahra Karami a, Ghasem Sargazi c a
Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, Iran Research and Technology Institute of Plant Production (RTIPP), Shahid Bahonar University of Kerman, Kerman, Iran c Department of Nanotechnology, Graduate University of Advance Technology, Kerman, Iran b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 May 2018 Received in revised form 19 March 2019 Accepted 13 April 2019 Available online 14 April 2019
Enzymatic production of biodiesel from waste cooking oils (WCOs) is expected as an efficient procedure for resolving the problems of energy demand and environment pollutions. But, high cost of lipases has been found as a main obstacle to commercialize the enzymatic transesterification. In the present study, cross-linked enzyme aggregates of Km12 lipases have been coupled with amino coated magnetite nanoparticles. SEM analysis showed that mCLEAs-lipase (mCLEAs-lip) nanocomposites have spherical structures. The mCLEAs-lip displayed a shift in optimal pH towards the alkaline, whereas optimal temperature was also shift towards low temperature. mCLEAs-lip nanocomposite reserves its total activity up to 6 cycles of enzyme re-using. Furthermore, it reserves 60% of its initial activity more than free enzyme after 24 days of incubation at 4 C. Biodiesel production from waste cooking oils by immobilized enzyme increased about 20% more than free enzyme. Therefore, the present study displays great practical latent to produce renewable fuel such as biodiesel. © 2019 Elsevier Ltd. All rights reserved.
Keywords: mCLEAs Lipase Biodiesel Waste cooking oil Renewable fuel
1. Introduction Due to restricted energy reserves and gradually serious problems with green-house gases coming from the fossil fuels, the application of biofuels has become a hot subject of recent years [1]. Biodiesel, which is an easily biodegradable, renewable, environmentally friendly, and non-toxic energy resource has fascinated extensive consideration [2,3]. Compared to petrodiesel, it is a clean burning fuel which emits lower carcinogenic compounds such as polycyclic aromatic hydrocarbons, sulfur, and metals to the atmosphere. Biodiesel is the methyl ester of fatty acid prepared from oils and animal fats through transesterification. Furthermore, huge volumes of waste cooking oils (WCOs) are formed annually throughout the world [4]. Dispose of waste cooking oils make ecological problems due to which oily films make over aquatic surfaces, which causes disruption in oxygen diffusion and clogging [5]. Therefore, the WCOs should be removed in a sustainable approach, otherwise, they would be discarded and cause environmental pollution problems [6,7]. Consuming WCOs as
* Corresponding author. Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, Iran. E-mail address: [email protected] (A. Badoei-dalfard). https://doi.org/10.1016/j.renene.2019.04.061 0960-1481/© 2019 Elsevier Ltd. All rights reserved.
cheap and starting material for biodiesel production is an attractive approach to make high-value added and ecologically benign compounds [8]. Usually, alkaline catalysts are used for the synthesis of biodiesel owing to their faster reaction rate [9,10], but these catalysts have some disadvantages, such as soap formation, high energy consumption, difficulties in the glycerol recovery, the need for removal of catalyst, neutralization of alkaline wastewater and latent pollution to the environment [10e12]. In spite of that, high content of water concentrations and free fatty acids (FFAs) of waste cooking oils make them improper for the homogeneous alkalinecatalyzed transesterification [4]. Recently, some thermochemical transformation methods such as pyrolysis, combustion and gasification to construct alternative fuel from waste resources have been investigated [13,14]. But, the obtained fuels have some adverse properties such as high acidity and oxygenated compounds, high water content, less burning efficiency and time-consuming [15]. Because of the limitation presented by conventional pyrolysis procedures, microwave pyrolysis has been offered as a substitute to improve the heating and cracking mechanism in pyrolysis development [16e18]. The pyrolysis methodology was also studied using diverse materials as the reaction bed, activated carbon, comprising particulate carbon and mesoporous aluminosilicate (MCM-41). They are
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recognized to have low in cost, high microwave absorbency and heat tolerance [19]. Lam and co-workers (2016) mentioned that an activated carbon prepared fast heating and a restricted reaction hot area that thermally stimulated wide pyrolysis cracking of the waste oil at 450 C, leading to amplified construction of a biofuel invention [20]. In addition, Mahari and co-workers (2018) reported that the microwave copyrolysis of used frying oil and plastic waste established positive synergistic effects in preparing fast heating frequency and a lower reaction time, and produced up to 81 wt% yield of liquid oil [15]. In spite of that, biodiesel production using enzymatic transesterification has numerous benefits including mild reaction conditions, low energy consumption, lower reaction temperatures, complete conversion of free fatty acids to methyl/ethyl esters, and production of very high purity biodiesel even from low quality feedstocks [21e24]. In addition, high catalytic efficiency and substrate specificity make them a smart choice to the chemical procedures. However, high cost, low enzyme stability, and difficult recyclability are the major obstacles of using enzymes in large-scale reactors. Enzyme immobilization is a high impact strategy to dissolve these problems. Re-usage, higher reaction rates, better thermal stability, and reducing the catalyst cost are some advantages of enzyme immobilization [23,25e29]. Ideal immobilization supports are chemically stable and give good dispersion and high enzyme loading. Because of keeping enzyme mobility and mass transfer characteristics in solution, improving enzyme activity and stability, nanomaterials become an excellent support for enzyme immobilization. Cross linked enzyme aggregate (CLEA) is a cheap and efficient strategy for enzyme immobilization [30]. In addition, magnetic nanoparticles were also shown excellent properties in order to improve reusability of enzyme without centrifuge [30]. Although, magnetic cross linked enzyme aggregates of lipase from Candida antarctica lipase B has been reported recently [31], but our strategy was different since we made an active CLEA-lipase separately and then it was covalently coupled to the coated magnetic nanoparticles. It is mentioned that, Km12 lipase is an organic solvent tolerant, cold active and talented lipase mainly for the biotransformation reactions in which recently reported in our group [32,33]. Finally, the application of magnetic cross linked enzyme aggregates of Km12 lipase (mCLEAs-lip) on the biodiesel production was also investigated by waste cooked oils as waste, cheap and abounded feedstocks. 2. Materials and methods 2.1. Materials
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remaining ions, the gained precipitate was centrifuged and washed four times with deionized water up to a pH value of 7.0 was attained; the resulting powder was dried at 100 C for 120 min. Then, the surface of these nanoparticles was functionalized with 3-aminopropyl triethoxysilane (APTES) by a silanization procedure in order to acquire amino activated magnetic nanoparticles. For this reaction magnetite nanoparticles (0.02 g), 3-aminopropyl trimetoxysilane (100 mL), and deionized water (25 mL) in methanol (2.5 mL) was dissolved. The resulting mixture was sonicated for 30 min. Afterward, glycerol (1.5 mL) was added to the mixture, and the solution was heated at 90 C for 6 h with maximum agitation. The acquired precipitate was washed with methanol and deionized water for three times in each case and dried (Fig. 1). 2.3. Preparation of mCLEAs of Km12 lipase Protein purification was performed by 85% (W/V) ammonium sulphate and Q-Sepharose column based on our previous reports [35e38]. For precipitation of lipase, saturated ammonium sulphate solution (5 mL) was mixed gradually to the lipase solution in phosphate buffer (5 mg/mL, 100 mM and pH 7.0) and stirred for 20 min at 30 C. After that, the mixture was also stirred at 4 C for 30 min to complete the precipitation. For making the CLEAs of Lipase, glutaraldehyde solution (final concentration of 40 mM) was added into the resulting suspension and stirred for 3 h at 30 C. mCLEAs of Km12 lipase were also obtained as follows. The amino coated magnetite nanoparticles (5 mg) were gradually mixed with CLEAs-lipase and glutaraldehyde solution (final concentration of 40 mM). The resulting suspension stirred for 3 h at 30 C. mCLEAs of Km12 lipase were separated using magnet, washed for four times by phosphate buffer (100 mM, pH 7.0) and stored in the mentioned buffer at 4 C (Fig. 1). 2.4. Lipase activity assay Hydrolysis of the p-nitrophenyl palmitate was investigated by the colorimetric method. The substrate (pNPP) was dissolved in the isopropanol solution with the final concentration of 25 mM. In the standard assay, 20 mL of a substrate solution was added to 960 mL of phosphate buffer (100 mM, pH 7.0). The reaction was initiated by adding 2 mg of the free enzyme solution/immobilized enzyme. The reaction was incubated for 30 min at 35 C. The absorbance of the product (pNP) was investigated at 410 nm [32,33]. One unit of lipase activity was considered as the amount of enzyme that produced one micromole of product per minute under the standard assay conditions. Spontaneous hydrolysis of p-NPP was also considered using the same of substrate solution in the absence of
3-aminopropyl triethoxysilane (APTES) and p-nitrophenyl palmitate were purchased from Sigma (St. Louis, USA). Glutaraldehyde (25%, v/v), FeSO4$7H2O, FeCl3$6H2O were acquired from Merck (Germany). Other reagents used were of analytical grade and gained either from Sigma or Merck. The water used in this work was Millipore water. 2.2. Preparation of functionalized magnetite nanoparticles Magnetite nanoparticles were synthesized and activated with amino groups by the method of Reza et al. (2010) and CruzIzquierdo et al. (2014) with slight modification [31,34]. These particles were prepared by addition of ammonium hydroxide (NH4OH) into a mixed solution of 0.72 g of ferrous sulphate and 1.43 g of ferric chloride in 30 mL deionized water pending gaining a brown precipitate at room temperature. In order to eliminate the
Fig. 1. Schematic illustration of the preparation of magnetic Cross-Linked Enzyme Aggregates (mCLEAs) of Bacillus licheniformis Km12 Lipase (mCLEA-lip).
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Km12 lipase as control. Enzyme activity of free and immobilized was also investigated at different concentration of pNPP, as substrate. Activity recovery of Km12 lipase in mCLEAs was considered using:
Reusability of mCLEAs-lip was investigated in the standard assay condition as described previously. After each cycle of 30 min sub-
Total lipase activity in magneticCLEAs ðUÞ 100 Total lipase activity used for magnetic CLEAs preparation ðUÞ
2.5. Characterization of mCLEAs of Km12 lipase Structural characterization of the magnetite nanoparticles and mCLEAs were considered by scanning electron microscope (SEM). Scanning electron micrographs were acquired on JEOL JSM6360 (Germany) scanning electron microscope (SEM) operated at 5 kV. Samples were dried by rinsing with anhydrous acetone, placed on a sample holder, coated with platinum before being scanned under vacuum. Fourier transform infrared (FT-IR) spectra were identified on a Shimadzu IR-Prestige-21 spectrometer in the spectral range of 400e4000 cm1 following the KBr pellet techniques. 2.6. Enzyme characterization 2.6.1. Optimal conditions for enzyme activity The optimum temperature of the free Km12 lipase and mCLEAslip was considered by adding the enzyme (2 mg of each form) into the substrate (pNPP) solution in phosphate buffer (100 mM, pH 7.0) at different temperatures (10e70 C). The optimum pH was also determined by adding the enzyme (2 mg of each form) into the substrate (pNPP) solutions of different pHs (3.0e11.0) at 35 C. In the range of 3.0e5.0, 100 mM acetate buffer, in the range of 6.0e8.0, 100 mM phosphate buffer, for pH 9.0, 100 mM bicarbonate buffer and in the range of 10.0e11.0, 100 mM glycine were utilized. The activities of free and immobilized lipase were determined as mentioned previously. 2.6.2. Thermal stability of free and mCLEAs-lip Thermal stabilities of the free and mCLEAs-lip were investigated by incubating them in phosphate buffer (100 mM, pH 7.0) without substrate at 40 and 50 C. After regular time intervals, samples taken up and lipase activity was determined as mentioned in standard assay. The residual activity at time zero was considered as 100%. Storage stabilities of the free and mCLEAs-lip were also investigated by incubating enzyme samples in phosphate buffer (100 mM, pH 7.0) without substrate at 4 C. Every 2 days, mCLEAslip was separated from the buffer by a magnetic and washed by distilled water. Then, the lipase activity in free and immobilized samples was measured as described previously. The residual activities were measured by taking the initial lipase activity before incubation as 100%. 2.6.3. Determination of kinetic parameters Kinetic parameters of free and mCLEAs-lip were examined using different pNPP solution concentrations in the range of 0.2e3.0 mg/ mL in phosphate buffer (100 mM, pH 7.0) at 35 C. In each form, 2 mg of lipase was used. Km, Vmax values of free enzyme and mCLEAs were calculated from LineweavereBurk plot of the initial reaction rates corresponding to different substrate concentrations by Graph Pad Prism software.
strate hydrolysis reaction, mCLEAs-lip were separated using magnetic separation, washed with buffer and then suspended again in a fresh reaction mixture to measure lipase activity. The enzyme activity of each cycle was determined in terms of residual activity by taking the enzyme activity of the first cycle as 100% [39]. 2.8. Biodiesel production from waste cooking oil Trans-esterification reactions by free and mCLEAs-lip have been done in 10 mL screw-capped vials containing 2.2 g of waste cooking oil, anhydrous methanol (1:3 M ratio between oil and methanol) and 0.3% free or immobilized lipase (w/w, based on the oil weight, g). The reaction was incubated in a shaking incubator with stirring rate of 170 rpm for 72 h at 35 C. At different time intervals, 100 ml of reaction were picked up and mixed with 100 ml of n-hexane. Then, the mixture was vortexed, centrifuged and the upper layer (10 ml) was applied to gas chromatography (GC) analysis for biodiesel measurement [40e42]. 3. Results and discussion 3.1. Lipase immobilization and characterization Km12 Lipase is a valuable enzyme for the biotransformation reactions in which recently reported in our group [24]. Results of protein concentration with Bradford assay method showed that protein loading on the functionalized magnetite nanoparticles was achieved successfully. Additionally, the results of quantitative measurements of protein loading on this magnetic nanoparticles showed that the immobilization efficiency was gained about 75% (Fig. 2).
6
Protein concentration (mg/ml)
Activity recovery ð%Þ ¼
2.7. Reusability of mCLEAs-lip
5 4 3 a
b
2 1 0 Km12 Lipase
Supernatant
mCLEA-lip
Fig. 2. Protein loading of Km12 Lipase on magnetic Cross-Linked Enzyme Aggregates (mCLEAs) of Bacillus licheniformis Km12 Lipase (mCLEA-lip). The insert picture shows the image of mCLEA-lip particles that are dispersed in buffer solution (A) and mCLEAlip particles after a magnet was placed aside (B).
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mCLEAs-Lip particles were distributed in phosphate buffer solution. After a magnet was placed sidewise, mCLEAs-Lip showed fast response (60 s) to the external magnetic field (Fig. 2). It indicated that the mCLEAs-Lip particles were revealed respectable magnetic responsibility even if layers of CLEAs lipase were coated on their surfaces, in which it is serious in term of protein immobilization. 3.2. Analytical characterization Km12 Lipase was immobilized on the surface of amino functionalized magnetite nanoparticles, in which amine (in the lipase) can react with aldehyde group of glutaraldehyde linker and another aldehyde group of glutaraldehyde also react with amino coated magnetite nanoparticles [29]. Fig. 3a and b show the morphology of amino functionalized magnetite-nanoparticles and mCLEAs-Lip in SEM image. The SEM investigations on Fig. 3a revealed that the amino functionalized magnetite nanoparticles were spherical and preserved its structure after several post-synthetic steps. Results of SEM image of lipase immobilization on the coated magnetite nanoparticles (Fig. 3b) shown that, these particles appear to be aggregated. These results indicated that the reaction should have been happened on the surface of magnetite nanoparticles. In addition, this image show separated complexes which indicated that the CLEAs-Lip immobilization on the surfaces of coated
A)
B)
Fig. 3. SEM images of (a) Fe3O4 nanoparticles and (b) mCLEAs of Km12 lipase.
877
magnetite nanoparticles. Presence of surface functional groups, binding of lipase onto magnetite nanoparticles and mCLEAs were considered by FTIR spectroscopy. FTIR spectra of mCLEAs (A), coated magnetic nanoparticles (B) and coated mCLEA (C) have been shown in Fig. 4. The peak around 530e580 cm1 could be qualified to the stretching vibration of FeeO in Fe3O4, indicating the existence of Fe3O4 in the microspheres which directed that the preparation of mCLEAs nanoparticles was successful. Furthermore, peaks at 1634 cm1 and 1463 cm1 correspond to C]O vibrations were detected in the products of coated mCLEA and approving the existence of amide linkage (eCONH2) between glutaraldehyde with magnetite nanoparticles and CLEAs [38,43]. FTIR spectrum of mCLEA-lip also shows two absorption peaks at 2854 and 2924 cm1 referring to CeH stretching in eCH3 and eCH2-, which confirm the immobilization of lipase on the support. In addition, the presence of a peak in 2900e3000 cm1 relays to aliphatic chain of functionalized APTES. Moreover, a typical adsorption band performed at 3438 cm1 corresponding to the stretching vibration of NeH and partially OH group, which showed great absorbance in the mCLEA-lip [4,44]. 3.3. Enzyme characterization 3.3.1. Optimum pH and temperature activity pH is a critical parameter in enzymatic catalysis. Lipase activity of immobilized and free enzyme was examined from pH 3.0 to 12.0. As shown in Fig. 5A, the optimum pH activity of free enzyme was about 8.0, while those of immobilized enzyme was pH 9.0. It is mentioned that, the immobilized lipase showed slight shift to the alkaline pH, in which lipase activity was improved about 10% in a wide pH range of pH 9.0e12. The same results was also reported by Qi and co-workers (2017) which showed the immobilized lipase displayed better pH tolerance compared to free lipase [45]. A shift in the optimum pH was also detected by CLEAs of alpha amylase [30], tyrosinase [46], subtilisin [47], and lipase [48]. In the mentioned reports, authors indicated that this shift might be produced by the ionization change of basic and acidic amino acid side chain in the active site microenvironment, which was produced by the newly formed interactions between glutaraldehyde and basic residues of enzyme during cross-linking. Lipase activity in different temperatures was also shown in Fig. 5B. The optimal temperature for free enzyme were about 35 C, whereas the immobilized lipase showed maximum enzyme activity between 30 and 45 C. The shift in optimal temperature was also observed by the other immobilized CLEAs of enzymes. This shift in optimum temperature could be as a result of covalent bond development between enzymes produced by glutaraldehyde during CLEAs construction which might decline the conformational flexibility of protein and protects it from denaturation by heat exchange [30,46,47]. Furthermore, the immobilized enzymes displayed the optimum activity in the upper temperature, which could decrease the mass transfer resistance of the substrate [45]. 3.3.2. Thermal stability measurement The thermostability of the free and immobilized enzyme was examined by incubating them at 40 and 50 C. As shown in Fig. 5C, the immobilized lipase has better thermal stability, which reserved 93% of its initial activity after incubation for 180 min at 50 C. It is more than the results of the thermostability of the free and immobilized enzyme by Qi and co-workers (2017). They reported that, the free lipase approximately deactivated after keeping at 60 C for 150 min, whereas the immobilized lipase preserved 50% activity, which revealed that the immobilized lipase demonstrated better stability than the free one [45]. Compared to free enzyme,
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A)
B)
C)
Fig. 4. The FTIR spectra of (a) Fe3O4, (b) coated-magnetic nanoparticles, and mCLEAs of Km12 lipase (c).
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120
120
A)
B)
100
80
Activity (%)
Activity (%)
100
60 40
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20
80 60 40
3
4
5
6
7
8
9
Free Immobilized
20
0
0
10 11 12
10
20
30
40
50
60
70
Temperature (°C)
pH 110
C)
120 100 80 60 40
Immobilized Free
20 0 0
20 40 60 80 100 120 140 160 180
Remaining activity (%)
140
Remaining actvity (%)
879
D) 90
70
50
30
Immobilized Free 0
20
Time (min)
40
60
80 100 120 140 160 180
Time (h)
Fig. 5. Influence of temperature (a) and pH (b) on enzyme activity of mCLEAs-lip and free enzyme. Assuming the highest activity of mCLEAs-lip and free enzyme as 100%. The thermal stability of mCLEAs-lip and free enzyme at (c) 40 C, and (d) 50 C. The experiments were done in triplicate and the error bar represents the percentage error in each set of determinations.
thermostability of immobilized lipase improved up to 60 min of incubation at 40 C (Fig. 5D). Furthermore, results in Fig. 5C and D, showed that the activity of immobilized form increased about 20 and 10% after incubation 20 min at 40 and 50 C, respectively. These significant results may be associated with the multipoint interactions between enzyme and magnetic support which may strengthen the intramolecular forces and avoid the autolysis of enzyme [50]. In most lipases, a mobile lid shelters the active site. In this closed building, the lipase is expected to be inactive. But, in its open assembly, the active site is accessible and the lipase is active. It has been expected that the activity and thermostability of lipases can be changed by modifications in their lid domains [49]. It is mentioned that, the improvement in the thermal tolerance of mCLEAs might be due to the suitable covalent cross-linking between proteins, glutaraldehyde and amino functionalized magnetite nanoparticles [30]. These covalent interactions delivered a more actual structure stabilization in mCLEAs needing much more energy to disruption this active structure than free enzyme [51e53]. Furthermore, improving enzyme stability after immobilization can be ascribed to rigidification of enzyme conformation which inhibits protein unfolding [4]. It is mentioned that, in our previous study, we reported that the activity of Km12 lipase was remarkably increase after preincubation at 20 C in which the lipase activity was increased about 37% after incubation for 60 min. These results indicated that Km12 lipase was nominated as cold-active enzyme [32]. Pretreatment of the tissues with cold-adapted lipase increases the softness and durability of the tissue and reduces the pill-formation in the textile industries [54]. Consequently, cold adaptive property of
immobilized Km12 lipase indicated that it is an appreciated candidate for bio-washing and stone washing of fabric materials. 3.3.3. Determination of kinetic parameters Kinetic parameters of free and mCLEAs-lip were investigated by calculating initial reaction rates with different substrate concentrations. For both forms, MichaeliseMenten kinetic behavior and LineweavereBurk plot were considered. As shown in Table 1, Vmax values of both forms are approximately equal, which directed the rate of pNPP hydrolysis was not changed after mCLEAs preparation. The same results were also observed for magnetic CLEAs of amylase [30]. In the case of mCLEAs-lip, the observed lower Km value specify a greater lipase affinity for the pNPP. It recommends that conformational changes due to enzyme immobilization assist the protein to appropriately turn its active site concerning the substrate [30,46,47,55]. 3.3.4. Reusability assay Reusability of immobilized enzyme training is a chief parameter for its cost-effective use in term of biotechnological application. The
Table 1 Kinetic parameters of free enzyme and mCLEAs-lip. Form of lipase
Km (mM)
Vmax (mmol/min)
Km12 lipase mCLEAs-lip
0.53 0.39
5.65 5.95
In each form, 2 mg of lipase was used.
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reusability of mCLEAs-lip was considered up to 10 cycles. No decrease in lipase activity of mCLEAs-lip was found after 5 cycles of using while it was diminished continuously over 5 cycles (Fig. 6A). Leaking of proteins was also considered during reusability experiment of mCLEAs-lip. Results showed no activity was observed in reaction medium up to 5 cycles of reuse for mCLEAs-lip. These results propose that satisfactory cross-linking of lipase and amino coated magnetite nanoparticles caused operationally stable mCLEAs [30]. To examine the effect of immobilization procedure on the storage stability of lipase, free and mCLEAs-lip were stored in phosphate buffer at 4 C. Results showed that the free enzyme lost practically its total activity after 24 days of incubation, but mCLEAs reserved about 85% of its initial activity, after 24 days (Fig. 6B). It is more than the immobilized MG10 lipase on magnetic amino functionalized graphene oxide which retained about 75% of its original activity after 30 days of incubation at 4 C [56]. These results proved that mCLEAs had major improvement on the storage stability relating to free enzyme. These results might be associated to a more active cross-linking of the protein in mCLEAs-lip which avoids enzyme leaking into aqueous buffer and enzyme separation from aggregates [57].
100 Immobilized lipase Free lipase
80
Conversion (%)
880
60
40
20
0 6
12 18 24 30 36 42 48 54 60 66 72
Time (h) Fig. 7. Biodiesel production from waste cooking oils (WCOs) using different amounts of free and mCLEAs-lip at different time of incubation.
3.4. Biodiesel production Because of reducing fossil fuel resources, pollution problems and growing energy demands bio-based fuel such as biodiesel get more attention as an alternative fuel. It is mentioned that biodiesel production from waste cooking oil can overcome the problems of food verse fuel, economic and environmental problems related with edible vegetable oils [4,23]. But, there are just few reports on biodiesel production using these abundant resources [4,58e61]. The maximum biodiesel production (49%) from waste cooking oil was obtained at 35 C after around 42 h by Km12 Lipase (10 mg) (Fig. 7). Mehrasbi et al., reported using 100 mg of free Candida antarctica lipase B producing 34% and 48% of FAMEs from waste cooking oil at 50 C after 72 h and 96 h of the reaction, respectively [4]. It is mentioned that, Km12 lipase has some distinctive properties such as cold active, tolerant in polar organic solvents (especially methanol) and glycerol by-product, high affinity to long chain triglycerides, and short time transesterification which make it endowed as a talented biocatalyst for transesterification reactions, especially for biodiesel production [32]. Interestingly, mCLEAs-lip produced maximum biodiesel production (71%) from waste cooking oil around 36 h (Fig. 5). It is more than the immobilized Candida antarctica lipase B which produced
biodiesel synthesis about 42% [62]. In addition, immobilized Km12 lipase increase biodiesel production from waste cooking oil about 10e20% at different time of incubation, compared to free enzyme (Fig. 7). De los Ríos and co-workers (2011) indicated that, the biodiesel production by Candida antarctica lipase B has been attributed to methanol inhibition of lipase [62]. It is mentioned that methanol is extensively used as acyl acceptor for the construction of biodiesel and is regularly favored over other alcohols due to its low cost. But, the enzyme activity of many lipases repressed by methanol which can limit the biodiesel production [4,63]. However, immobilization is a good approach to improve lipase activity in the presence of organic solvents, especially methanol. Mehrasbi and co-workers (2017) reported that liquid lipase from Candida antarctica (CALB) reserved 65% of its initial activity in 50% of methanol, after 96 h of incubation at 25 C. In spite of that, the immobilized CALB displayed outstanding stability in methanol, retaining 94% of its original activity in 50% methanol [4]. Furthermore, Yan and co-workers used Geotrichum sp. lipase to construct cross-linked PCMCs (CLPCMCs), which exhibited better catalytic efficiency in biodiesel construction from waste cooking oil compared to PCMCs and free lipase [64]. It is mentioned that, CLPCMCs also preserved 83% of biodiesel
120 Remaining activity (%)
Activity (%)
100
A)
100 80 60 40 20
B)
80 Free Immobilized
60 40 20 0
0 1
2
3 4 5 6 Number Cycle
7
8
0
9
Fig. 6. a) Reusability of mCLEAs-lip. b) The storage stability of mCLEAs-lip and free enzyme at represents the percentage error in each set of determinations.
4
8
12
16
20
24
Day 4 C
in aqueous buffer. The experiments were done in triplicate and the error bar
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production after incubated in organic solvents for 4 h [64]. In overall, the results of biodiesel production by mCLEA-lip nanocomposite indicated that this immobilization strategy might be improved the lipase stability in the long time biodiesel production, compared to free enzyme. Finally, covalent bond formation between enzyme molecules and support, may preserve enzyme in active conformation and increased the lipase rigidity with concomitant creation of a sheltered micro-environment. Additionally, it made a more active cross-linking of the protein in mCLEAs- lip which avoids enzyme leaking from aggregates and protect it against methanol and the other by products [30,42,43]. 4. Conclusion A facile method to immobilize CLEAs of Km12 lipase onto amino-coated magnetite nanoparticles was established to attain enhanced activity and stability. Shift in optimal pH and temperature towards the alkaline pH and low temperatures are the other valuable parameters of mCLEA-lip for biodiesel production which can reduce the cost of biodiesel production. In addition, mCLEA-lip shows high impact for biodiesel production which increases biodiesel production from waste cooking oil about 20% after 30 h of incubation, compared to free enzyme. Taken to gather, unique properties of mCLEAs-lip open an attractive way towards commercialize the enzymatic transesterification of waste cooking oil. Acknowledgements The authors express their gratitude to the Research Council of the Shahid Bahonar University of Kerman, Kerman (Iran) and Research, Technology Institute of Plant Production (RTIPP), Kerman (Iran) for their financial support during the course of this project. References [1] K.R. Jegannathan, S. Abang, D. Poncelet, E.S. Chan, P. Ravindra, Production of biodiesel using immobilized lipase d a critical review, Crit. Rev. Biotechnol. 28 (2008) 253e264. [2] W. Tao, P. Yu, S. Gong, Q. Li, Y. Luo, Application of KF/MgO as a heterogeneous catalyst in the production of biodiesel from rapeseed oil, Kor. J. Chem. Eng. 25 (2008) 998e1003. [3] M.D. Serio, R. Tesser, L. Pengmei, E. Santacesaria, Heterogeneous catalysts for biodiesel production, Energy Fuels 22 (2008) 207e217. [4] M.R. Mehrasbi, J. Mohammadi, M. Peyda, M. Mohammadi, Covalent immobilization of Candida antarctica lipase on core-shell magnetic nanoparticles for production of biodiesel from waste cooking oil, Renew. Energy 101 (2017) 593e602. [5] H.O.D. Clarissa, Z.P. Debora, F. Roselaine, M.O.M. Marcia, S.N. Augusto, Z.C.F. Cello, F.Z. William, F.F. Luciana, Bioremediation of cooking oil waste using lipases from wastes, PLoS One 12 (2017) 1e17. [6] A.B. Chhetri, K.C. Watts, M.R. Islam, Waste cooking oil as an alternate feedstock for biodiesel production, Energies 1 (2008) 3e18. [7] M.K. Lam, K.T. Lee, A.R. Mohamed, Homogeneous, heterogeneous and enzymatic catalysis for transesterification of high free fatty acid oil (waste cooking oil) to biodiesel: a review, Biotechnol. Adv. 28 (2010) 500e518. [8] D. Li, W. Wang, M. Faiza, B. Yang, Y. Wang, A novel and highly efficient approach for the production of biodiesel from high-acid content waste cooking oil, Catal. Commun. 102 (2017) 76e80. [9] S.V. Ranganathan, S.L. Narasimhan, K. Muthukumar, An overview of enzymatic production of biodiesel, Bioresour. Technol. 99 (2008) 3975e3981. [10] L.P. Christopher, H. Kumar, V.P. Zambare, Enzymatic biodiesel: challenges and opportunities, Appl. Energy 119 (2014) 497e520. [11] H. Fukuda, A. Kondo, H. Noda, Biodiesel fuel production by transesterification of oils, J. Biosci. Bioeng. 92 (2001) 405e416. [12] B.K. Barnwal, M.P. Sharma, Prospects of biodiesel production from vegetable oils in India, Renew. Sustain. Energy Rev. 9 (2005) 363e378. [13] S.A. Opatokun, V. Strezov, T. Kan, Product based evaluation of pyrolysis of food waste and its digestate, Energy 92 (2015) 349e354. [14] K. Im-orb, W. Wiyaratn, A. Arpornwichanop, Technical and economic assessment of the pyrolysis and gasification integrated process for biomass conversion, Energy 53 (2018) 592e603.
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