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Construction of CLEAs-lipase on magnetic graphene oxide nanocomposite: An efficient nanobiocatalyst for biodiesel production
Bioresource Technology 278 (2019) 473–476
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Construction of CLEAs-lipase on magnetic graphene oxide nanocomposite: An efficient nanobiocatalyst for biodiesel production
T
⁎
Arastoo Badoei-dalfarda,b, , Zahra Karamia, Saied Malekabadia a b
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
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Lipase Immobilization CLEAs Biodiesel Magnetic Nanobiocatalyst
In the present work, cross linked enzyme aggregates of Lipase (CLEAs-lip) was synthesized and immobilized on the magnetic amino functionalized graphene oxide (maGO-CLEAs-lip) nanocomposites. The immobilized lipase showed a broad range of temperature activity about 40–60 °C, as compared to free enzyme. In the case of maGOCLEAs-lip nanocomposite, the observed lower Km value state 2.25 folds affinity for the p-nitrophenyl palmitate. Enzyme activity of maGO-CLEAs-lip nanocomposite was the highest up to 5 cycles. Storage stability results displayed maGO-CLEAs-lip retained about 75% of its original activity after 30 days of incubation. Remarkably, maGO-CLEAs-lip formed the highest biodiesel construction (78%) from R. communis oil after 24 h of incubation. The biodiesel yield of this nanocomposite was 3.0 folds higher than free enzyme, making it talented as an excellent nanobiocatalyst for efficient production of biodiesel.
1. Introduction Despite numerous advantages of lipases, biotechnological application of them has some disadvantages such as high cost and time-consuming production, lack of long term stability, hard recovery and recyclability of the enzyme. Recently, lipase has been successfully immobilized on different nanocomposites to increase recyclability,
⁎
thermal stability, feasible continuous operations, tolerance against high pH and non-aqueous solvents, simple product purification, contribution more capable and cleaner catalytic methods (Zhao et al., 2015; Xie and Zang, 2016; Xie and Zang, 2018). But, there are still some problems such as avoidable loss in the separation procedure and boring recycling by filtration from the liquid phase. Therefore, emerging a more efficient system for enzyme
Corresponding author at: 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.biortech.2019.01.050 Received 15 November 2018; Received in revised form 9 January 2019; Accepted 11 January 2019 Available online 14 January 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
Bioresource Technology 278 (2019) 473–476
A. Badoei-dalfard et al.
linking. The CLEAs of lipase were centrifuged, and washed four times by the mentioned buffer and stored in the same buffer at 4 °C. The CLEAs of lipase (3 mg/mL) were mixed with 3-aminopropyl triethoxysilane functionalized mGO in phosphate buffer solution (100 mM, pH 7.5) and stirred for 30 min at 30 °C. Next, the glutaraldehyde solution (40 mM) was accompanied into the resulting blend and stirred for 180 min at 30 °C (Talekar et al., 2012; Xie and Huang, 2018). maGOCLEAs-lip nanocomposite was gathered using a magnet, washed four times by 100 mM phosphate buffer (pH 7.5) and stored in the mentioned buffer at 4 °C. The protein amounts in the washing samples were dignified by using Bradford technique (Bradford, 1976).
recovery has been found more attention for researchers. Recently, enzyme immobilization on magnetic nanoparticles has numerous benefits such as high surface to volume ratio, simplicity and low cost efficiency of a bio-production, high biocompatibility, great stability against oxidation, high adsorption ability, and low toxicity (Vaghari et al., 2016). In addition, easily recovery of enzymes from the assay reaction by using an external magnetic field with minimal loss cusses the magnetic nanoparticles to be a fascinating candidates for enzyme immobilization (Xie and Wang, 2014; Chen et al., 2018; Xie and Huang, 2018). In spite of that, great specific surface capacity and high chemical reactivity of magnetic nanoparticles create them greatly sensitive to acidic and oxidative situations and agglomeration. Therefore, the immobilized enzymes poorly distributed in the assay reaction and decreased their catalytic activities (Xie and Zang, 2017; Cui et al., 2018). To improve this problem, diverse biocompatible composites like alginate, chitosan, silica, dextran, metal-organic framework and graphene have been effectively used for shielding of magnetic nanoparticles to construct the highly active and stable immobilized enzyme (Xie and Wang, 2014; Xie and Huang, 2018; Sargazi et al., 2018; Soozanipour et al., 2019). Because of high biocompatibility, great surface capacity, and rich oxygen-containing functionalities, graphene oxide (GO) can be used as a perfect support for high yield of enzyme immobilization and coating of magnetic nanoparticles. The enzymes can be immobilized on graphene oxide through non-covalent interactions such as π–π stacking, Vander Waals, and hydrophobic interactions and also covalent attachment using a cross-linking agent (Patel et al., 2017; Kashefi et al., 2019). Furthermore, an efficient and simple strategy to improve the enzyme efficiency and reusability has been established as Cross-Linked Enzyme Aggregates (CLEAs). The CLEAs nanohybrids exhibited some advantages such as high thermal stability, high enzyme efficiency, high organic solvents stability, and tolerance to autoproteolysis (Torabizadeh and Mikani, 2018; Lucena et al., 2019). Therefore, in the present study at first the screening of an efficient lipase (thermophilic, inducible, high methanol-tolerant) has been performed from Gehver hot spring. Then, CLEAs of this potent lipase was successfully prepared and was covalently immobilized onto the aminofunctionalized magnetic graphene oxide (maGO) and finally its application in the heterogeneous synthesis of biodiesel from non-edible oil feedstocks has been evaluated. The resulting nanobiocatalyst (maGOCLEAs-lip) magnetically recycled from reaction medium after applying a magnetic aside.
2.2. Characterization of immobilized lipase Scanning electron micrographs (SEMs) of magnetic graphene oxide (mGO), amino functionalized magnetic graphene oxide (maGO) nanocomposites and amino functionalized magnetic graphene oxide (maGO) CLEAs lipase (maGO-CLEAs-lip) were acquired on JEOL JSM6360 (Germany) scanning electron microscope (SEM) run at 5 kV. Elemental EDX analysis from selected part of SEM of maGO-CLEA-lip was also done for elemental mapping. FT-IR spectra were identified on a Shimadzu IR-Prestige-21 spectrometer in the spectrum of 400–4000 cm−1 subsequent the KBr pellet methods. 2.3. Biochemical characterization of lipase To exam the irreversible thermal inactivation of both forms of lipase, the lipase solution was incubated at optimal temperature for 3 h. At different time interval, aliquots were picked up and scanned for remaining activity. Kinetic factors of both free and maGO-CLEAs-lip were examined using several concentrations of p-nitrophenyl palmitate in 100 mM phosphate buffer (pH 7.0) at 45 °C. In both forms, 2 mg of lipase was used in assay reaction. The amounts of Vmax, Km factors for free and maGO-CLEAs-lip were considered from Lineweaver Burk plot of the initial reaction rates equivalent to different substrate concentrations. 2.4. Biodiesel construction Enzymatic transesterification reactions were carried out by free and maGO-CLEAs-lip nanocomposite and maintained for 48 h with a stirring speed of 160 rpm. The reaction consists of 0.4 g oil (oil from Ricinus communis), methanol (1:3 M ratio between R. communis oil and methanol) and 0.2% enzyme (free or correspond lipase on support) (w/w, based on the oil weight, g). At diverse time intervals (6, 12, 18 and 24 h), 100 µL of reaction blend was picked up and diluted with the same volume of n-hexane solvent. Afterward, the sample was gathered and the upper layer (10 µL) was performed to gas chromatography (GC) investigation for biodiesel measurement (Wang et al., 2017; Malekabadi et al., 2018).
2. Methods 2.1. Lipase production and immobilization Soil and water samples were collected from Gehver hot spring (63 °C and pH 5.7) located in Jiroft (Iran). The best lipase producing bacteria was screened and identified based on our previous reports (Malekabadi et al., 2018). Protein purification was also performed by 85% (W/V) ammonium sulphate and Q-Sepharose column based on our previous reports (Azadian et al., 2016; Ramezani-Pour et al., 2015; Azadian et al., 2017). Graphene oxide (GO) was produced by oxidizing graphite substrate according to the Hummers procedure with slight modification (Hummers and Offeman, 1958). Fe3O4 nanoparticles were also produced and activated with amino functional groups (Reza et al., 2010). Then, these nanoparticles were functionalized with 3-aminopropyl triethoxysilane (APTES) to acquire amino activated magnetic nanoparticles (Talekar et al., 2012; Xie and Huang, 2018). For synthesis of cross linked enzyme aggregates of lipase (CLEAs-lip), lipase MG10 (3.0 mg/mL) was dissolved in 100 mM of phosphate buffer (pH 7.5). After that, saturated ammonium sulphate (5.0 ml) was supplemented with the stirring for 1 h at 4 °C. Glutaraldehyde solution (40 mM) was supplemented and stirred for 4 h at 30 °C to obtain enzyme cross
3. Results and discussion 3.1. Lipase production and immobilization Lipase producing bacteria were screened in enrichment culture medium supplemented with olive oil as a sole source of carbon. Furthermore, methanol (30%, v/v) was also used to acquire the methanol tolerant lipase. The 16S rDNA gene of MG10 isolate was amplified and sequenced (Genbank Accession No. MF927590.1) and compared by BLAST investigation to other bacteria in the NCBI database. The results proposed a near relationship between MG10 isolate and the other members of the Enterobacter genus with an extreme sequence homology (99%) to Enterobacter cloacae. The phylogenetic tree (Fig. 1) designated that the strain MG10 was associated with Enterobacter species and used for the following study. 474
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Fig. 1. Phylogenetic tree of Entrobacter sp. MG10 based on 16S rDNA gene.
functionalized graphene oxide, in which aldehyde groups of glutaraldehyde making linkage between amine of lipase and amino functionalized magnetite nanomaterials (Xie and Huang, 2018). The SEM analysis of graphene oxide shown an irregular circular structure which was similar to the earlier reports, given that a bulky specific surface zone of the nanomaterials. Results of SEM image shown that lipase immobilization seem to diminish the construction of stacked GO structures. Elemental EDX investigation from particular part of SEM image of maGO-CLEAs-lip for elemental plotting obviously specifies the existence of associated atoms of support containing C, N, O, Si, P, S and Fe which displays the effective functionalization of APTES, particularly by noticing Si atom. Furthermore, the remarkable attendance of phosphorous atom can intensely endorse the effective lipase immobilization. Presence of functional groups on graphene oxide and lipase immobilization onto these nanoparticles were investigated by FTIR spectroscopy. The peak around 532–614 cm−1 could be evaluated to the stretching vibration of Fe–O in Fe3O4 nanoparticles, representing the presence of Fe3O4 in the graphene oxide which focused that the preparation of Fe3O4-graphene oxide nanoparticles was effective (Xie and Huang, 2018). Moreover, peaks at 1635 and 1636 cm−1 resemble C]O vibrations of the present carboxyl and carbonyl functional groups on the mGO and presence of amide linkage between glutaraldehyde with Fe3O4 nanoparticles and CLEAs (Xie and Huang, 2018). Additionally, a characteristic adsorption band achieved at 3447 cm−1 equivalent to the adsorbed H2O and OH group on the surface of mGO, which shown excessive absorbance in all of these nanoparticles and the amino functionalized magnetic graphene oxide-CLEA. FTIR spectrum of amino functionalized magnetic graphene oxide shows the presence of a peak in 2922 cm−1 spreads to aliphatic chain of coated APTES. After lipase immobilization on the maGO, the 614 cm−1 band owing to the stretching vibration of Fe–O in Fe3O4 nanoparticle was practically vanished, which signifying the covering of Fe3O4 by lipase. Moreover, FTIR spectrum of maGO-CLEAs-lip also shown two
Table 1 Kinetic parameters of free and immobilized enzymes.
Free enzyme maGO-CLEAs-lip
Km (mM)
Vmax (µmol/min)
0.148 0.0658
0.235 0.342
In each forms of enzyme, 2 mg of lipase was used. Table 2 Determination of thermal stability, recyclability, and protein leaching of maGOCLEAs-lip nanocomposite.
Free enzyme maGO-CLEAs-lip
Thermal stabilitya (%)
Recyclabilityb (%)
Leachingc (%)
50 85
– 70
– 11
In each forms of enzyme, 2 mg of lipase was used. a For thermal stability consideration, remaining activities (%) were measured after 3 h incubation in 50 °C. b For recyclability, remaining activity (%) was measured after 8 cycles. c For leaching consideration, the protein concentration was measured in the supernatant after 8 cycles.
Results of protein loading on this nanocomposite shown that, immobilization efficiency was achieved about 73%. After a magnet was positioned sidewise, maGO-CLEAs-lip showed fast response (60 s) to the peripheral magnetic field. It incomes that the maGO-CLEAs-lip particles were shown suitable magnetic concern even though layers of CLEAs-Lip were covered on their surfaces, wherein it is significant in term of lipase immobilization. 3.2. Characterization of the immobilized lipase Lipase MG10 was immobilized on the surface of magnetic 475
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absorption peaks at 2840 and 2922 cm−1 mentioning CeH stretching in eCH3 and eCH2e, which demonstrate the immobilization of enzyme on the support. In addition, the appearance two new FTIR absorption bands at 1404 and 1514 cm−1 owing to the lipase immobilization were discovered, which specified that the enzyme was covalently bounded to the maGO nanocomposites via amide linkage.
Appendix A. Supplementary data
3.3. Activity of free and maGO-CLEAs lipase
Azadian, F., Badoei-dalfard, A., Namaki-Shoushtari, A., Hassanshahian, M., 2016. Purification and biochemical properties of a thermostable, haloalkaline cellulase from Bacillus licheniformis AMF-07 and its application for hydrolysis of different cellulosic substrates to bioethanol production. Mol. Biol. Res. Commun. 5 (3), 143–155. Azadian, F., Badoei-dalfard, A., Shoushtari, A., Karami, Z., Hassanshahian, M., 2017. Production and characterization of an acido-thermophilic, organic solvent stable cellulase from Bacillus sonorensis HSC7 by conversion of lignocellulosic wastes. J. Genet. Eng. Biotechnol. 15, 187–196. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Chen, Q., Liu, D., Wu, C., Yao, K., Li, Z., Shi, N., Wen, F., Gates, I.D., 2018. Co-immobilization of cellulase and lysozyme on amino-functionalized magnetic nanoparticles: an activity-tunable biocatalyst for extraction of lipids from microalgae. Bioresour. Technol. 263, 317–324. Cui, J., Sun, B., Lin, T., Feng, Y., Ji, S., 2018. Enzyme shielding by mesoporous organosilica shell on Fe3O4@silica yolkshell nanospheres. Int. J. Biol. Macromol. 117, 673–682. Hummers, W.S., Offeman, R.E., 1958. Preparation of graphitic oxide. J. Am. Chem. Soc. 80 1339 1339. Kashefi, S., Borghei, S.M., Mahmoodi, N.M., 2019. Superparamagnetic enzyme-graphene oxide magnetic nanocomposite as an environmentally friendly biocatalyst: synthesis and biodegradation of dye using response surface methodology. Microchem. J. 145, 547–558. Lucena, G.N., Santos, C.C., Pinto, G.C., Rochaa, C.O., Brandt, J.V., Paulab, A.V., Júniora, M.J., Marquesa, R.F.C., 2019. Magnetic cross-linked enzyme aggregates (mCLEAs) applied to biomass Conversion. J. Solid State Chem. 270, 58–70. Malekabadi, S., Badoei-dalfard, A., Karami, Z., 2018. Biochemical characterization of a novel cold-active, halophilic and organic solvent-tolerant lipase from B. licheniformis KM12 with potential application for biodiesel production. Int. J. Biol. Macromol. 109, 389–398. Patel, S.K., Choi, S.H., Kang, Y.C., Lee, J.K., 2017. Eco-friendly composite of Fe3O4-reduced graphene oxide particles for efficient enzyme immobilization. ACS Appl. Mater. Interfaces 9, 2213–2222. Ramezani-Pour, N., Badoei-Dalfard, A., Namaki-Shoushtari, A., Karami, Z., 2015. Nitrilemetabolizing potential of Bacillus cereus strain FA12; Nitrilase production, purification, and characterization. Biocatal. Biotransform. 33, 156–166. Reza, R.T., Martínez-Pérez, C.A., Rodríguez-González, C.A., Romero, H.M., GarcíaCasillas, P.E., 2010. Effect of the polymeric coating over Fe3O4 particles used for magnetic separation. Cent. Eur. J. Chem. 8, 1041–1046. Sargazi, G., Afzali, D., Khajeh-Ebrahimi, A., Badoei-dalfard, A., Malekabadi, S., Karami, Z., 2018. Ultrasound assisted reverse micelle efficient synthesis of new Ta-MOF@ Fe3O4 core/shell nanostructures as a novel candidate for lipase immobilization. Mater. Sci. Eng. C 93, 768–775. Soozanipour, A., Taheri-Kafrani, A., Barkhori, M., Nasrollahzadeh, M., 2019. Preparation of a stable and robust nanobiocatalyst by efficiently immobilizing of pectinase onto cyanuric chloride-functionalized chitosan grafted magnetic nanoparticles. J Colloid Interface Sci. 536, 261–270. Talekar, S., Ghodake, V., Ghotage, T., Rathod, P., Deshmukh, P., Nadar, S., Mulla, M., Ladole, M., 2012. Novel magnetic cross-linked enzyme aggregates (magnetic CLEAs) of alpha amylase. Bioresour. Technol. 123, 542–547. Torabizadeh, H., Mikani, M., 2018. Nano-magnetic cross-linked enzyme aggregates of naringinase an efficient nanobiocatalyst for naringin hydrolysis. Int. J. Biol. Macromol. 117, 134–143. Vaghari, H., Jafarizadeh-Malmiri, H., Mohammadlou, M., Berenjian, A., Anarjan, N., Jafari, N., Nasiri, S., 2016. Application of magnetic nanoparticles in smart enzyme immobilization. Biotechnol. Lett. 38 (2), 223–233. Wang, X., Qin, X., Li, D., Yang, B., Wang, Y., 2017. One-step synthesis of high-yield biodiesel from waste cooking oils by a novel and highly methanol-tolerant immobilized lipase. Bioresour. Technol. 235, 18–24. Xie, W., Zang, X., 2016. Immobilized lipase on core–shell structured Fe3O4–MCM-41 nanocomposites as a magnetically recyclable biocatalyst for interesterification of soybean oil and lard. Food Chem. 194, 1283–1292. Xie, W., Huang, M., 2018. Immobilization of Candida rugosa lipase onto graphene oxide Fe3O4 nanocomposite: characterization and application for biodiesel production. Energy Convers Manage. 159, 42–53. Xie, W., Wang, J., 2014. Enzymatic production of biodiesel from soybean oil by using immobilized lipase on Fe3O4/poly(styrene-methacrylic acid) magnetic microsphere as a biocatalyst. Energy Fuels 28, 2624–2631. Xie, W., Zang, X., 2017. Covalent immobilization of lipase onto aminopropyl-functionalized hydroxyapatite-encapsulated-γ-Fe2O3 nanoparticles: a magnetic biocatalyst for interesterification of soybean oil. Food Chem. 227, 397–403. Yong, Y., Bai, Y.-X., Li, Y.F., Lin, L., Cui, Y.-J., Xia, C.G., 2008. Characterization of candida rugose lipase immobilized onto magnetic microspheres with hydrophilicity. Process Biochem. 43, 1179–1185. Zhao, X., Qi, F., Yuan, C., Du, W., Liu, D., 2015. Lipase-catalyzed process for biodiesel production: enzyme immobilization, process simulation and optimization. J. Renew. Sustain. Energy 44, 182–197.
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2019.01.050. References
Results showed that, the maximum activity of both forms of enzyme was obtained at pH 8.0 and 9.0, respectively. Moreover, relative lipase activity of maGO-CLEAs-lip was faintly lower than free enzyme in acidic pH, but marginally greater than in basic pH. Results of thermal stability shown that, the lasting activity of the free lipase is 50% while the maGO-CLEAs-lip reserved 85% of its initial activity after 3 h of incubation at 50 °C. These results evidently designate that the immobilization of lipases onto maGO can avoid their conformation transition at high temperature, and improving their thermal tolerance. As shown in Table 1, Vmax of maGO-CLEAs-lip was slightly upper than free enzyme about 0.1 µmol/min, which directed the rate of pNPP hydrolysis was not significantly changed after maGO-CLEAs-lip preparation. In the case of maGO-CLEAs-lip, the detected lower Km value state a better lipase affinity for the pNPP substrate, about 2.25 folds. It approves that conformational changes by the reason of enzyme immobilization assistance the protein to appropriately turn its active site concerning the substrate (Talekar et al., 2012). Reusability results of maGO-CLEAs-lip shown that enzyme activity of maGO-CLEAs-lip was the highest up to 5 cycles, but it continuously decreased over 5 cycles. In addition, results showed that the cumulative leaching of 11% of the protein concentration in the supernatant after 8 cycles (Table 2). These results recommend that suitable cross-linking of enzyme and mGO nanomaterials produced stable maGO-CLEAs-lip (Talekar et al., 2012). Results of storage stability displayed maGOCLEAs-lip reserved about 75% of its original activity after 30 days of incubation, wherein free enzyme missed its preliminary activity at the similar time. These results verified that maGO-CLEAs-lip had chief protection on the storage stability of lipase (Yong et al., 2008). 3.4. Biodiesel production from Ricinus communis oil The highest biodiesel synthesis (26%) from R. communis oil was gained at room temperature after 24 h of incubation by Entrobacter Lipase MG10 (10 mg). Remarkably, maGO-CLEAs-lip formed the highest biodiesel construction (78%) from R. communis oil after 24 h. Besides, the immobilized MG10 lipase enriched biodiesel construction from R. communis oil about 3.0 folds at diverse time of incubation, as compared to free lipase. 4. Conclusion Lipase MG10 is a high potent lipase which was isolated from Gehver hot spring. The CLEAs of this lipase was immobilized on the magnetic amino functionalized graphene oxide. The observed lower Km value state 2.25 folds affinity for the pNPP substrate, as compared to free enzyme. The biodiesel yield of this nanocomposite was 3.0 folds higher than free enzyme, creation it capable as an outstanding nanobiocatalyst for effective production of biodiesel. Evaluation of this nanobiocatalyst for efficient biodiesel production from the other non-edible oil feedstocks and waste cocking oils is also in progress. Acknowledgements The authors express their gratitude to the Research Council of the Shahid Bahonar University of Kerman (Iran) and Research, Technology Institute of Plant Production (RTIPP).
476
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Construction of CLEAs-lipase on magnetic graphene oxide nanocomposite: An efficient nanobiocatalyst for biodiesel production
T
⁎
Arastoo Badoei-dalfarda,b, , Zahra Karamia, Saied Malekabadia a b
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
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Lipase Immobilization CLEAs Biodiesel Magnetic Nanobiocatalyst
In the present work, cross linked enzyme aggregates of Lipase (CLEAs-lip) was synthesized and immobilized on the magnetic amino functionalized graphene oxide (maGO-CLEAs-lip) nanocomposites. The immobilized lipase showed a broad range of temperature activity about 40–60 °C, as compared to free enzyme. In the case of maGOCLEAs-lip nanocomposite, the observed lower Km value state 2.25 folds affinity for the p-nitrophenyl palmitate. Enzyme activity of maGO-CLEAs-lip nanocomposite was the highest up to 5 cycles. Storage stability results displayed maGO-CLEAs-lip retained about 75% of its original activity after 30 days of incubation. Remarkably, maGO-CLEAs-lip formed the highest biodiesel construction (78%) from R. communis oil after 24 h of incubation. The biodiesel yield of this nanocomposite was 3.0 folds higher than free enzyme, making it talented as an excellent nanobiocatalyst for efficient production of biodiesel.
1. Introduction Despite numerous advantages of lipases, biotechnological application of them has some disadvantages such as high cost and time-consuming production, lack of long term stability, hard recovery and recyclability of the enzyme. Recently, lipase has been successfully immobilized on different nanocomposites to increase recyclability,
⁎
thermal stability, feasible continuous operations, tolerance against high pH and non-aqueous solvents, simple product purification, contribution more capable and cleaner catalytic methods (Zhao et al., 2015; Xie and Zang, 2016; Xie and Zang, 2018). But, there are still some problems such as avoidable loss in the separation procedure and boring recycling by filtration from the liquid phase. Therefore, emerging a more efficient system for enzyme
Corresponding author at: 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.biortech.2019.01.050 Received 15 November 2018; Received in revised form 9 January 2019; Accepted 11 January 2019 Available online 14 January 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
Bioresource Technology 278 (2019) 473–476
A. Badoei-dalfard et al.
linking. The CLEAs of lipase were centrifuged, and washed four times by the mentioned buffer and stored in the same buffer at 4 °C. The CLEAs of lipase (3 mg/mL) were mixed with 3-aminopropyl triethoxysilane functionalized mGO in phosphate buffer solution (100 mM, pH 7.5) and stirred for 30 min at 30 °C. Next, the glutaraldehyde solution (40 mM) was accompanied into the resulting blend and stirred for 180 min at 30 °C (Talekar et al., 2012; Xie and Huang, 2018). maGOCLEAs-lip nanocomposite was gathered using a magnet, washed four times by 100 mM phosphate buffer (pH 7.5) and stored in the mentioned buffer at 4 °C. The protein amounts in the washing samples were dignified by using Bradford technique (Bradford, 1976).
recovery has been found more attention for researchers. Recently, enzyme immobilization on magnetic nanoparticles has numerous benefits such as high surface to volume ratio, simplicity and low cost efficiency of a bio-production, high biocompatibility, great stability against oxidation, high adsorption ability, and low toxicity (Vaghari et al., 2016). In addition, easily recovery of enzymes from the assay reaction by using an external magnetic field with minimal loss cusses the magnetic nanoparticles to be a fascinating candidates for enzyme immobilization (Xie and Wang, 2014; Chen et al., 2018; Xie and Huang, 2018). In spite of that, great specific surface capacity and high chemical reactivity of magnetic nanoparticles create them greatly sensitive to acidic and oxidative situations and agglomeration. Therefore, the immobilized enzymes poorly distributed in the assay reaction and decreased their catalytic activities (Xie and Zang, 2017; Cui et al., 2018). To improve this problem, diverse biocompatible composites like alginate, chitosan, silica, dextran, metal-organic framework and graphene have been effectively used for shielding of magnetic nanoparticles to construct the highly active and stable immobilized enzyme (Xie and Wang, 2014; Xie and Huang, 2018; Sargazi et al., 2018; Soozanipour et al., 2019). Because of high biocompatibility, great surface capacity, and rich oxygen-containing functionalities, graphene oxide (GO) can be used as a perfect support for high yield of enzyme immobilization and coating of magnetic nanoparticles. The enzymes can be immobilized on graphene oxide through non-covalent interactions such as π–π stacking, Vander Waals, and hydrophobic interactions and also covalent attachment using a cross-linking agent (Patel et al., 2017; Kashefi et al., 2019). Furthermore, an efficient and simple strategy to improve the enzyme efficiency and reusability has been established as Cross-Linked Enzyme Aggregates (CLEAs). The CLEAs nanohybrids exhibited some advantages such as high thermal stability, high enzyme efficiency, high organic solvents stability, and tolerance to autoproteolysis (Torabizadeh and Mikani, 2018; Lucena et al., 2019). Therefore, in the present study at first the screening of an efficient lipase (thermophilic, inducible, high methanol-tolerant) has been performed from Gehver hot spring. Then, CLEAs of this potent lipase was successfully prepared and was covalently immobilized onto the aminofunctionalized magnetic graphene oxide (maGO) and finally its application in the heterogeneous synthesis of biodiesel from non-edible oil feedstocks has been evaluated. The resulting nanobiocatalyst (maGOCLEAs-lip) magnetically recycled from reaction medium after applying a magnetic aside.
2.2. Characterization of immobilized lipase Scanning electron micrographs (SEMs) of magnetic graphene oxide (mGO), amino functionalized magnetic graphene oxide (maGO) nanocomposites and amino functionalized magnetic graphene oxide (maGO) CLEAs lipase (maGO-CLEAs-lip) were acquired on JEOL JSM6360 (Germany) scanning electron microscope (SEM) run at 5 kV. Elemental EDX analysis from selected part of SEM of maGO-CLEA-lip was also done for elemental mapping. FT-IR spectra were identified on a Shimadzu IR-Prestige-21 spectrometer in the spectrum of 400–4000 cm−1 subsequent the KBr pellet methods. 2.3. Biochemical characterization of lipase To exam the irreversible thermal inactivation of both forms of lipase, the lipase solution was incubated at optimal temperature for 3 h. At different time interval, aliquots were picked up and scanned for remaining activity. Kinetic factors of both free and maGO-CLEAs-lip were examined using several concentrations of p-nitrophenyl palmitate in 100 mM phosphate buffer (pH 7.0) at 45 °C. In both forms, 2 mg of lipase was used in assay reaction. The amounts of Vmax, Km factors for free and maGO-CLEAs-lip were considered from Lineweaver Burk plot of the initial reaction rates equivalent to different substrate concentrations. 2.4. Biodiesel construction Enzymatic transesterification reactions were carried out by free and maGO-CLEAs-lip nanocomposite and maintained for 48 h with a stirring speed of 160 rpm. The reaction consists of 0.4 g oil (oil from Ricinus communis), methanol (1:3 M ratio between R. communis oil and methanol) and 0.2% enzyme (free or correspond lipase on support) (w/w, based on the oil weight, g). At diverse time intervals (6, 12, 18 and 24 h), 100 µL of reaction blend was picked up and diluted with the same volume of n-hexane solvent. Afterward, the sample was gathered and the upper layer (10 µL) was performed to gas chromatography (GC) investigation for biodiesel measurement (Wang et al., 2017; Malekabadi et al., 2018).
2. Methods 2.1. Lipase production and immobilization Soil and water samples were collected from Gehver hot spring (63 °C and pH 5.7) located in Jiroft (Iran). The best lipase producing bacteria was screened and identified based on our previous reports (Malekabadi et al., 2018). Protein purification was also performed by 85% (W/V) ammonium sulphate and Q-Sepharose column based on our previous reports (Azadian et al., 2016; Ramezani-Pour et al., 2015; Azadian et al., 2017). Graphene oxide (GO) was produced by oxidizing graphite substrate according to the Hummers procedure with slight modification (Hummers and Offeman, 1958). Fe3O4 nanoparticles were also produced and activated with amino functional groups (Reza et al., 2010). Then, these nanoparticles were functionalized with 3-aminopropyl triethoxysilane (APTES) to acquire amino activated magnetic nanoparticles (Talekar et al., 2012; Xie and Huang, 2018). For synthesis of cross linked enzyme aggregates of lipase (CLEAs-lip), lipase MG10 (3.0 mg/mL) was dissolved in 100 mM of phosphate buffer (pH 7.5). After that, saturated ammonium sulphate (5.0 ml) was supplemented with the stirring for 1 h at 4 °C. Glutaraldehyde solution (40 mM) was supplemented and stirred for 4 h at 30 °C to obtain enzyme cross
3. Results and discussion 3.1. Lipase production and immobilization Lipase producing bacteria were screened in enrichment culture medium supplemented with olive oil as a sole source of carbon. Furthermore, methanol (30%, v/v) was also used to acquire the methanol tolerant lipase. The 16S rDNA gene of MG10 isolate was amplified and sequenced (Genbank Accession No. MF927590.1) and compared by BLAST investigation to other bacteria in the NCBI database. The results proposed a near relationship between MG10 isolate and the other members of the Enterobacter genus with an extreme sequence homology (99%) to Enterobacter cloacae. The phylogenetic tree (Fig. 1) designated that the strain MG10 was associated with Enterobacter species and used for the following study. 474
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Fig. 1. Phylogenetic tree of Entrobacter sp. MG10 based on 16S rDNA gene.
functionalized graphene oxide, in which aldehyde groups of glutaraldehyde making linkage between amine of lipase and amino functionalized magnetite nanomaterials (Xie and Huang, 2018). The SEM analysis of graphene oxide shown an irregular circular structure which was similar to the earlier reports, given that a bulky specific surface zone of the nanomaterials. Results of SEM image shown that lipase immobilization seem to diminish the construction of stacked GO structures. Elemental EDX investigation from particular part of SEM image of maGO-CLEAs-lip for elemental plotting obviously specifies the existence of associated atoms of support containing C, N, O, Si, P, S and Fe which displays the effective functionalization of APTES, particularly by noticing Si atom. Furthermore, the remarkable attendance of phosphorous atom can intensely endorse the effective lipase immobilization. Presence of functional groups on graphene oxide and lipase immobilization onto these nanoparticles were investigated by FTIR spectroscopy. The peak around 532–614 cm−1 could be evaluated to the stretching vibration of Fe–O in Fe3O4 nanoparticles, representing the presence of Fe3O4 in the graphene oxide which focused that the preparation of Fe3O4-graphene oxide nanoparticles was effective (Xie and Huang, 2018). Moreover, peaks at 1635 and 1636 cm−1 resemble C]O vibrations of the present carboxyl and carbonyl functional groups on the mGO and presence of amide linkage between glutaraldehyde with Fe3O4 nanoparticles and CLEAs (Xie and Huang, 2018). Additionally, a characteristic adsorption band achieved at 3447 cm−1 equivalent to the adsorbed H2O and OH group on the surface of mGO, which shown excessive absorbance in all of these nanoparticles and the amino functionalized magnetic graphene oxide-CLEA. FTIR spectrum of amino functionalized magnetic graphene oxide shows the presence of a peak in 2922 cm−1 spreads to aliphatic chain of coated APTES. After lipase immobilization on the maGO, the 614 cm−1 band owing to the stretching vibration of Fe–O in Fe3O4 nanoparticle was practically vanished, which signifying the covering of Fe3O4 by lipase. Moreover, FTIR spectrum of maGO-CLEAs-lip also shown two
Table 1 Kinetic parameters of free and immobilized enzymes.
Free enzyme maGO-CLEAs-lip
Km (mM)
Vmax (µmol/min)
0.148 0.0658
0.235 0.342
In each forms of enzyme, 2 mg of lipase was used. Table 2 Determination of thermal stability, recyclability, and protein leaching of maGOCLEAs-lip nanocomposite.
Free enzyme maGO-CLEAs-lip
Thermal stabilitya (%)
Recyclabilityb (%)
Leachingc (%)
50 85
– 70
– 11
In each forms of enzyme, 2 mg of lipase was used. a For thermal stability consideration, remaining activities (%) were measured after 3 h incubation in 50 °C. b For recyclability, remaining activity (%) was measured after 8 cycles. c For leaching consideration, the protein concentration was measured in the supernatant after 8 cycles.
Results of protein loading on this nanocomposite shown that, immobilization efficiency was achieved about 73%. After a magnet was positioned sidewise, maGO-CLEAs-lip showed fast response (60 s) to the peripheral magnetic field. It incomes that the maGO-CLEAs-lip particles were shown suitable magnetic concern even though layers of CLEAs-Lip were covered on their surfaces, wherein it is significant in term of lipase immobilization. 3.2. Characterization of the immobilized lipase Lipase MG10 was immobilized on the surface of magnetic 475
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absorption peaks at 2840 and 2922 cm−1 mentioning CeH stretching in eCH3 and eCH2e, which demonstrate the immobilization of enzyme on the support. In addition, the appearance two new FTIR absorption bands at 1404 and 1514 cm−1 owing to the lipase immobilization were discovered, which specified that the enzyme was covalently bounded to the maGO nanocomposites via amide linkage.
Appendix A. Supplementary data
3.3. Activity of free and maGO-CLEAs lipase
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Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2019.01.050. References
Results showed that, the maximum activity of both forms of enzyme was obtained at pH 8.0 and 9.0, respectively. Moreover, relative lipase activity of maGO-CLEAs-lip was faintly lower than free enzyme in acidic pH, but marginally greater than in basic pH. Results of thermal stability shown that, the lasting activity of the free lipase is 50% while the maGO-CLEAs-lip reserved 85% of its initial activity after 3 h of incubation at 50 °C. These results evidently designate that the immobilization of lipases onto maGO can avoid their conformation transition at high temperature, and improving their thermal tolerance. As shown in Table 1, Vmax of maGO-CLEAs-lip was slightly upper than free enzyme about 0.1 µmol/min, which directed the rate of pNPP hydrolysis was not significantly changed after maGO-CLEAs-lip preparation. In the case of maGO-CLEAs-lip, the detected lower Km value state a better lipase affinity for the pNPP substrate, about 2.25 folds. It approves that conformational changes by the reason of enzyme immobilization assistance the protein to appropriately turn its active site concerning the substrate (Talekar et al., 2012). Reusability results of maGO-CLEAs-lip shown that enzyme activity of maGO-CLEAs-lip was the highest up to 5 cycles, but it continuously decreased over 5 cycles. In addition, results showed that the cumulative leaching of 11% of the protein concentration in the supernatant after 8 cycles (Table 2). These results recommend that suitable cross-linking of enzyme and mGO nanomaterials produced stable maGO-CLEAs-lip (Talekar et al., 2012). Results of storage stability displayed maGOCLEAs-lip reserved about 75% of its original activity after 30 days of incubation, wherein free enzyme missed its preliminary activity at the similar time. These results verified that maGO-CLEAs-lip had chief protection on the storage stability of lipase (Yong et al., 2008). 3.4. Biodiesel production from Ricinus communis oil The highest biodiesel synthesis (26%) from R. communis oil was gained at room temperature after 24 h of incubation by Entrobacter Lipase MG10 (10 mg). Remarkably, maGO-CLEAs-lip formed the highest biodiesel construction (78%) from R. communis oil after 24 h. Besides, the immobilized MG10 lipase enriched biodiesel construction from R. communis oil about 3.0 folds at diverse time of incubation, as compared to free lipase. 4. Conclusion Lipase MG10 is a high potent lipase which was isolated from Gehver hot spring. The CLEAs of this lipase was immobilized on the magnetic amino functionalized graphene oxide. The observed lower Km value state 2.25 folds affinity for the pNPP substrate, as compared to free enzyme. The biodiesel yield of this nanocomposite was 3.0 folds higher than free enzyme, creation it capable as an outstanding nanobiocatalyst for effective production of biodiesel. Evaluation of this nanobiocatalyst for efficient biodiesel production from the other non-edible oil feedstocks and waste cocking oils is also in progress. Acknowledgements The authors express their gratitude to the Research Council of the Shahid Bahonar University of Kerman (Iran) and Research, Technology Institute of Plant Production (RTIPP).
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