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Feb 22, 2017 - ABSTRACT: Three sub-group lipases of Burkholderia cepacia lipase (BCL), Rhizomucor ... It reveals that the immobilized RML exhibited be...
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Various Types of Lipases Immobilized on Dendrimer-Functionalized Magnetic Nanocomposite and Application in Biodiesel Preparation Yanli Fan, Caixia Ke, Feng Su, Kai Li, and Yunjun Yan* Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, P. R. China

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S Supporting Information *

ABSTRACT: Three sub-group lipases of Burkholderia cepacia lipase (BCL), Rhizomucor miehei lipase (RML), and Candida rugosa lipase (CRL) were covalently immobilized on dendrimer functionalized magnetic carbon nanotube and used as catalysts to catalyze biodiesel production. The effects of imprinting molecule, organic solvent, water, methanol, temperature and time interval of methanol addition on the yield of biodiesel were optimized. The results showed that bioimprinting could greatly enhance catalytic performances of the three immobilized lipases. The obtained lipases were then employed to catalyze biodiesel production, and the achieved optimum conditions were: for BCL, water content 5 wt. %, reaction temperature 35°C, and with tbutanol as reaction medium, methanol : oil molar ratio 4:1, its highest biodiesel yield attained 96.4%; for RML, water content 10 wt. %, reaction temperature 50 °C, n-octane as the reaction medium, methanol : oil molar ratio of 5:1, the utmost biodiesel conversion rate was up to 96.2%; and for CRL, water content 7.5 wt. %, reaction temperature 40 °C, isooctane as the reaction medium, methanol:oil molar ratio of 4:1, the best yield reached 85.1%. It was borne out that the effect of time interval of methanol addition on the biodiesel conversion was more obvious for the immobilized RML and CRL than BCL. Furthermore, waste vegetable oil was also explored for biodiesel preparation vs soybean oil. It reveals that the immobilized RML exhibited best catalysis toward both feedstock in its corresponding solvent systems.

1. INTRODUCTION To substitute fossil fuels, biodiesel, i.e. fatty acid alkyl esters (FAAEs), has drawn increasing attention as a biodegradable and renewable fuel not only because of lower exhaust emissions, such as CO, SOx and HC,1 but also its being satisfactory and practical for both direct-injection and indirecttype diesel engines.2 It is usually prepared by transesterification of oils/fats or esterification of fatty acids with short chain alcohols. Enzymatic approaches (lipase catalysis) occupy the advantage relative to chemical methods due to the lower energy consumption, easy recovery of product, more environmentallybenign process, and compatibility with a wide variety of feedstock, especially with high free fatty acid content.3 Lipases (triacylglycerol hydrolases, EC 3.1.1.3) widely distribute in animals, plants and microbes, among which, those from microbes are the main resources.4 So far, over 65 microbial species are known to produce lipases,5 especially in the genera of Mucor, Rhizopus, Yarrowia, Candida, Bacillus, Pseudomonas, Burkholderia, etc. Based on substrate specificity, Pleiss et al.6 and Naik et al.7 subdivided lipases into three subgroups: (a) lipases with a crevice-like binding site located near the protein surface (such as lipases from Rhizomucor and Rhizopus); (b) lipases with a funnel-like binding site (for example, lipases from Candida antarctica, Pseudomonas, and Burkholderia); and (c) lipases with a tunnel-like binding site (for instance, lipase from Candida rugosa). Typical commercial lipases of the above three sub-groups, such as Rhizomucor miehei lipase, Burkholderia cepacia lipase, Candida rugosa lipase, were abundantly reported to be employed for biodiesel production.8−10 In particular, immobilized forms are more favored, mainly because high operational stability and © 2017 American Chemical Society

reusability of the immobilized enzymes make it possible to employ them in a batch reaction (easy recovery), or in a continuous procedure for a long time. This technique finally defrays biofuel production cost. Till now, various kinds of carriers are widely used for enzyme immobilization. Among them, several types of magnetic nanostructured materials have been proved to have great potential for immobilization of lipases, like magnetic nanoparticles, nanotubes, and grapheme, etc.11−13 Nanoscale materials possess many unique properties that enhance the efficiency of biocatalysts, including effective enzyme loading, higher surface area, and reducing mass transfer resistance.14 Furthermore, the conjugates endowed with magnetism can be easily separated from the reaction medium by using a magnetic field. The methods of enzymatic immobilization mainly include adsorption, entrapping cross-linking and covalent attachment.15 Therefore, covalent attachment has drawn increasing attention because it has the advantage of strong interactions between the support and the enzyme which makes enzyme leakage uncommon.16 However, covalent bonds formation usually adversely affect the conformation of the enzyme, leading to decreased catalytic activity. But adopted proper approach such as oriented-immobilization might eliminate this adverse effect.17 In addition, researches demonstrate that molecular bioimprinting and interfacial activation are the effective methods to improve enzymatic activity and stability in non-aqueous Received: January 5, 2017 Revised: February 22, 2017 Published: February 22, 2017 4372

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Energy & Fuels media.18,19 Meanwhile, proper organic solvents can also boost the activity of lipase in biodiesel synthesis.20 The main reasons for the use of organic solvent in transesterification reactions are to shape a homogeneous reaction system, which can decrease the viscosity of the reaction mixture, increase the diffusion rate and reduce mass transfer problems around the enzyme.21 A detailed comparison on enzymatic biodiesel preparation with different lipases has been elaborated by Babaki et al.,22 where Candida antarctica lipase B (CALB), Thermomyces lanuginosus lipase (TLL) and Rhizomucor miehei lipase (RML) were immobilized onto SBA-epoxy. In our previous work, a process of employing immobilized lipase on dendrimer-functionalized magnetic carbon nanotube (mMWCNTs-PAMAM) was successfully developed.23 The immobilized lipase exhibited easy recovery, high stability and excellent operational reusability for biodiesel production. Therefore, in this work, to generalize this new immobilization method, based on the substrate specificity of lipases and the merits of mMWCNTs-PAMAM matrix possessing magnetic properties and massive active sites to increase the loading of enzyme, the typical representatives of three sub-group lipases with different substrates specificities, B. cepacia lipase (BCL), R. miehei lipase (RML), and C. rugosa lipases (CRL), were selected as target proteins to be immobilized on mMWCNTs-PAMAM. To further achieve more satisfactory catalytic performance, the strategy based on bioimprinting−immobilization was also adopted to modify lipases. The effects of organic solvents, water, methanol content, reaction temperature and time interval of methanol addition into the reaction mixture on the methanolysis catalyzed by the three immobilized lipases were investigated. Additionally, a comparison of the catalytic efficiencies in transesterification for biodiesel synthesis from soybean oil and waste vegetable oil by the three immobilized lipases was carried out.

on the surface of mMWCNTs-NH2 (Scheme S1). The detailed preparation procedures are provided in the Supporting Information. 2.4. Lipase Immobilization. mMWCNTs-PAMAM composites (100 mg) were dispersed in absolute ethanol and followed by adding an amount of glutaraldehyde (five different concentrations at 2.5, 5, 7.5, 10, 12.5 wt. %) and shaken in a thermostatic shaker at a stirring speed of 200 rpm at 30 °C for 10 h. The product was taken out by magnetic separation, washed several times with de-ionized water to remove excess glutaraldehyde. The obtained sample was defined as mMWCNTs-PAMAM-GA. Then, BCL (150, 200, 250, 300, 350 mg) and CRL (10, 20, 30, 40, 50 mg) powder respectively were dissolved in 5 mL phosphate buffer solution (0.05 M) and RML (0.4, 0.5, 0.6, 0.7, 0.8 mL) sample was added to 4.6, 4.5, 4.4, 4.3 and 4.2 mL phosphate buffer solution, respectively. The three enzymes solution were mixed with the carriers (mMWCNTs-PAMAM-GA, 100 mg) in a rotary shaker at a stirring speed of 200 rpm at various temperature (25, 30, 35, 40, 45, 50 °C) and time (1, 2, 2.5, 3, 3.5, 4, 4.5 h). The mixture was separated magnetically thoroughly and rinsed with fresh buffer to remove unbound and nonspecific absorption lipases. 2.5. Bioimprinting Procedure. Bioimprinting procedure as per the method of Lv et al.25 with slight modification was carried out prior to immobilization procedure to improve the activity of the immobilized lipases. BCL (250 mg), CRL (30 mg) powder were respectively dissolved in 4.9 mL 0.05 M phosphate buffer solution and RML (0.5 mL) sample in 4.4 mL. Bioimprinting molecules (lauric acid, oleic acid, linoleic acid, triolein, olive oil) were resolved in the mixture solvent of isopropanol (0.1 mL) and Tween 60 (100 mg) as surfactant. The mixture was added to the enzyme solution (the final concentration of the bioimprinting molecule was 0.075 mmol/mL) and incubated under the conditions of rotating speed 200 rpm at room temperature for 20 min. After incubation, the bioimprinted lipases were immobilized according to the procedures described in Section 2.4. Then, the bioimprinting molecules were removed with 5 mL isooctane, and then the immobilized−bioimprinted lipases were collected using a magnet and dried in a vacuum desiccator at room temperature. 2.6. Lipase Activity Assay. The activities of the immobilized and free lipases were analyzed using the method described previously.26 A certain amount of the immobilized and free lipases were added to 10 mL mixture containing 1-dodecanol (0.2 M) and lauric acid (0.2 M) in isooctane with addition 0.01 mL water, and the reactions were implemented at a certain temperature for 30 min with continuous shaking at 200 rpm. 1 mL sample was sampled and mixed with 15 mL of ethanol-acetone (1:1, v/v) to stop the reaction. The remaining acid in the sample was detected by titration with NaOH solution (0.05 M). Phenolphthalein solution (1%, w/v) was used as pH indicator. One unit of enzyme activity (U) was defined as the amount of lipase that consumed 1μmol of lauric acid per minute under the assay conditions. The amount of immobilized enzyme was detected as per the method from Bradford with bovine serum albumin (BSA) as the standard.27 The immobilization efficiency (%), activity recovery (%) and specific activities (U/g-protein) were calculated via Eqs. (1)−(3).28

2. MATERIALS AND METHODS 2.1. Materials. Burkholderia cepacia lipase (BCL, initial specific activity 4.51 × 103 U/g-protein) powder with protein content 0.8% was purchased from Amano Enzyme Inc. (Nagoya, Japan). Candida rugosa lipase (CRL, 7.94 × 103 U/g-protein) powder of 4.2% (mixtures of isozymes) and Rhizomucor miehei lipase (RML, 4.61 × 103 U/g-protein) of 3.7 mg/mL were bought from Sigma Aldrich (St. Louis, MO, USA). Multi-walled carbon nanotubes (MWCNTs, purity >95%) were commercially obtained from Nanotech Port Co. Ltd. (Shenzhen, China). Waste vegetable oil was obtained from ZTE Agrivalley Co. Ltd. (Hubei, China). The acid value was at 11.6 mg KOH/g. Soybean oil with 99% purity was purchased from local market. Other reagents such as methyl acrylate, ethylenediamine (EDA), 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde (GA, a commonly used non-toxic cross-linker) produced by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) were of analytical grade and used as received without any further purification. 2.2. Analysis of the 3D-Structure of BCL, CRL and RML. 3D structural models of BCL, RML and CRL (pdb identifiers: BCL, 3LIP; RML, 4TGL; CRL, 1CRL, all open configuration) obtained from the NCBI (http://www.ncbi.nlm.nih.gov/) were employed to analyze surface-exposed amino acid groups using PyMOL (2.7.6).24 2.3. Synthesis and Functionalization of mMWCNTs-PAMAM. mMWCNTs-PAMAM was synthesized and characterized according to the reported article.23 Briefly, the MWCNTs were oxidized by the nitro/sulfuric acid for 4 h to obtain the carboxyl grafted MWCNTs. Then, magnetic iron oxide nanoparticles were loaded onto the surfaces of MWCNTs (mMWCNTs) by the impregnation method, which was detailed in the Supporting Information. Amine-funtionalized mMWCNTs (mMWCNTs-NH2) were prepared with APTES via postsynthetic grafting. Subsequently, PAMAM dendrimer was grafted

Immobilization efficiency (%) =

immobilized protein × 100% total loading protein (1)

Activity recovery (%) specific activity of immobilized lipase = × 100% specific activity of adding free lipase

(2)

Specific activity (U/g‐protein) initial activity = protein content of immobilized lipase

(3)

2.7. General Procedures of Transesterification Reactions for Biodiesel Synthesis in Organic Solvents and GC Analysis. The reactions were conducted in a stoppered 50 mL shake flask in organic solvent system under a stirring rate of 200 rpm. The reaction mixture 4373

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RML

CRL

7.5 wt. % 0.5 mL 7.0 45 °C 4h

7.5 wt. % 30 mg 6.5 35 °C 2.5 h

a

BCL: Burkholderia cepacia lipase; RML: Rhizomucor miehei lipase; CRL: Candida rugosa lipase. The optimal conditions of five parameters of RML have been presented in ref 23. Note: ± indicates the standard deviation (SD). 4374

× × × × × 0.12) 0.17) 0.15) 0.15) 0.20)

CRL

± ± ± ± ± (3.34 (3.42 (3.59 (3.71 (3.92 105 105 105 105 105 × × × × × 0.012) 0.013) 0.012) 0.014) 0.015)

RML

± ± ± ± ± (1.276 (1.280 (1.283 (1.286 (1.291 0.25) 0.23) 0.17) 0.23) 0.20) ± ± ± ± ± 90.7 89.6 91.1 90.6 89.8 2.1 2.0 1.9 2.4 2.2

RML

± ± ± ± ± ± ± ± ± ± 86.4 85.1 87.2 87.2 86.7 1.7 2.1 1.9 1.9 2.4 ± ± ± ± ±

CRL

42.2 43.1 45.3 46.8 49.4 25.3 27.8 26.4 30.8 32.5

BCL

2.2 2.5 2.1 1.8 1.9

89.4 88.6 89.1 90.4 89.3

CRL

3.0 2.5 2.1 2.6 2.4

(8.38 (8.44 (8.59 (8.64 (8.73

± ± ± ± ±

BCL

× × × × ×

103 103 103 103 103

Specific activity (U/g)

BCL: Burkholderia cepacia lipase; RML: Rhizomucor miehei lipase; CRL: Candida rugosa lipase. The activity recovery and immobilization efficiency of RML have been presented in the published paper.23 Note: ± indicates the standard deviation (SD).

BCL 7.5 wt. % 250 mg 7.5 30 °C 2.5 h

a

Parameters Glutaraldehyde concentration Amounts of lipase pH value Reaction temperature Coupling time

± ± ± ± ±

Optimum value

RML

Table 1. Optimized Single Factor Conditions for the Three Lipases in the Immobilization Processa

2,769 2,775 2,781 2,789 2,799

3. RESULTS AND DISCUSSION 3.1. Synthesis of Immobilized Lipases. Three sub-group lipases, B. cepacia lipase (BCL), R. miehei lipase (RML) and C. rugosa lipases (CRL) were immobilized on mMWCNTsPAMAM. During the immobilization process, the immobilization conditions will change due to the difference in lipase sources. In our previous work, the effects of glutaraldehyde concentration, lipase loading, pH value, immobilization temperature and coupling time on the immobilization efficiency and activity recovery of RML were investigated.23 Herein, the effects of immobilization parameters on BCL and CRL were further examined. As shown in Tables 1 and 2, all the lipases immobilized on the dendrimer-functionalized magnetic carbon nanotube showed excellent activity recovery except CRL. When

5.2 4.7 3.9 5.0 4.6

where Asample: the peak area of the free fatty acids in the sample; f 0: the response factor; Ainternal: the peak area of the internal standard; Winternal: the mass (g) of the internal standard; and Wsample: the mass (g) of the sample. 2.8. Statistics Analysis. All trials were conducted in three parallel replicates, and the data were analyzed by the software Origin 8.0 (OriginLab Co., Northampton, MA, USA).

± ± ± ± ±

(5)

BCL

WinternalA sample

185 187 190 191 193

WsampleA internal

Parameters

(4)

Glutaraldehyde concentration Amounts of lipase pH value Reaction temperature Coupling time

A internal Winternal

Immobilization efficiency (%)

f0 =

A samplef0

Activity recovery (%)

Biodiesel yield (%) =

Table 2. Corresponding Activity Recovery, Immobilization Efficiency and Specific Activity of the Three Immobilized Lipases under Their Optimized Single Factor Conditions for the Five Key Parametersa

includes soybean oil (2.19 g), methanol, immobilized lipases (10 wt. %, the specific activities of BCL is 5.62 × 104 U/g-protein, RML 1.58 × 105 U/g-protein and CRL 5.71 × 103 U/g-protein), organic solvent (20 wt. %), and some water. All dosage percentages were based on the oil mass, unless otherwise stated. To avoid the inhibitory effect of methanol on the immobilized lipases, methanol was respectively added in three steps at the same interval. GC analysis: some samples were collected from the reaction mixture at specified time and centrifuged at 13800×g for 5 min to obtain the supernatant. 10 μL of supernatant, 290 μL hexane and 300 μL of 1.0 mg/mL heptadecanoic acid methyl ester (as internal standard, hexane as solvent) were mixed thoroughly for gas chromatographic analysis. The methyl ester content was analyzed using a GC-9790 gas chromatograph (Agilent HP-INNOWAX capillary column 30 m × 0.25 mm × 0.25 μm, J&W Scientific, Folsom, CA). The operating conditions: the mixed sample (1.0 μL) above was injected into the GC, and the column initial temperature was 180 °C and increased to 230 °C at a rate of 3°C min−1 and then maintained at 230 °C for 3 min. The injector and detector temperature were set at 230°C and 280°C, respectively. The biodiesel yield (%) was defined as the total FAAE content in the conversion oil sample. The biodiesel yield was calculated with Eqs. (4) and (5),28

103 103 103 103 103

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Table 3. Comparison the Reaction Components/Conditions and Biodiesel Yields Using the Immobilized Lipases in the Present Work and Previous Worksa

Support

Immobilization method

Enzyme binding efficiency (%)

Magnetic nanoparticles

Covalent bonding

84%

Magnetic microsphere

Covalent bonding

83%

Magnetic silica nanocomposite

Physical adsorption

97%

Dendrimer-coated magnetic carbon nanotubes

Covalent bonding

87% (BCL) 89% (RML) 90% (CRL)

a

Reaction condition Methanolysis of soybean oil (12 h); oil : alcohol molar ratio 1:3; 50 °C; No water; solvent-free system; 40% immobilized lipase Thermomyces lanuginosa (w/w oil) Methanolysis of soybean oil (24 h) ; oil:alcohol molar ratio 1:5; 35 °C; 0.1% water; solvent-free system; 50% immobilized lipase Candida rugosa (w/w oil) Methanolysis of olive oil (30 h); oil:alcohol molar ratio 1:4; 40 °C; 10% water; solvent-free system; 11% immobilized lipase Burkholderia sp. C20 (w/w oil) Methanolysis of soybean oil (24 h); oil:alcohol molar ratio 1:4; 35 °C; 5% water; 20% t-butanol; 10% immobilized BCL (w/w oil). Methanolysis of soybean oil (36 h); oil:alcohol molar ratio 1:5; 50 °C; 10% water; 20% n-octane; 10% immobilized RML (w/w oil). Methanolysis of soybean oil (40 h); oil:alcohol molar ratio 1:4; 40 °C; 7.5% water; 20% isooctane; 10% immobilized CRL (w/w oil).

Biodiesel yield (%)

Reuse (reaction cycle)

ref.

87%

5 (50% conversion)

11

86%

5 (47% conversion)

33

92%

10 (60% conversion)

32

96% (BCL) 96% (RML) 85% (CRL)

10 (89% conversion) 10 (81% conversion) 10 (58% conversion)

This study

Note: BCL: Burkholderia cepacia lipase; RML: Rhizomucor miehei lipase; CRL: Candida rugosa lipase.

for CRL. In addition, the activity of enzyme in water environment will decrease with prolonging of coupling time. In general, the activity recovery increases with the extension of the coupling time and then decreases as the coupling time extension. Nevertheless, the rate of enzyme activity decreased and the time required to maintain enzyme activity for different enzymes are also different. As the results show, the optimum coupling times for BCL, RML and CRL were 2.5 h, 4 h and 2.5 h, respectively. Under the optimal conditions, the activity recovery of the immobilized BCL and RML were dramatically improved compared with the immobilized CRL. The reason is not only related to the unique characteristics of the lipase structure, but also to the immobilization method. As is well known, the mechanism for improving the activity and stability of the immobilized lipase is extremely complicated. It is mainly attributable to a combination of the following factors. First, the active centers of most lipases are covered by a so-called “lid” structure, which controls access of the substrate(s) to the active site. The secondary structure of the lipase would probably change during immobilization, and the “lid” might be opened to some extent for the substrate(s), which would provide an easier access, leading to an increase in lipase activity.31 The second factor is related to the immobilization method. Here, the three lipases, BCL, RML and CRL, were respectively immobilized on a uniform matrix by covalent bond according to the distribution of amino groups on enzyme molecules, because most amino groups are located far from the catalytic active center in comparison to other functional groups (carboxyl, hydroxyl and sulfhydryl groups). As shown in Figure S1, the distributions of ε-NH2 residues of BCL and RML are both non-uniform, mainly far from the catalytic center (Fig. S1b and d). However, its distribution in CRL (Fig. S1e and f) is nearly uniform with some amino residues distributed near the catalytic center, which may be the main cause resulting in loss of activity after immobilization.17 Other active groups of CRL, such as carboxyl, hydroxyl and sulfhydryl groups, are also unsuitable. Therefore, physical immobilization methods such as adsorption, entrapment, and encapsulation might be a better choice for the immobilization of CRL. There are some reports about the influences of the support on the loading and the enzymatic activity of the immobilized lipases on magnetic nanocomposites.11,32,33 Table 3 presents a

the glutaraldehyde concentration was at 7.5 wt. %, the activity recovery of the three immobilized lipases all arrived at their highest values (BCL: 185%, RML: 2,769%, CRL: 42.2%). The probable reason is because the same amount of glutaraldehyde was required to completely activate the amino group of the same carrier. At this concentration (7.5 wt. %) of glutaraldehyde, the immobilization efficiencies of the three lipases were 86.4% (BCL), 89.4% (RML) and 90.7% (CRL), and the corresponding specific activities were 8.38 × 103 U/g (BCL), 1.276 × 105 U/g (RML) and 3.34 × 103 U/g (CRL), respectively. However, the effects of other parameters on the immobilization of the three enzymes varied to some extent. The optimum dosage of enzymes used differed owing to the different protein contents (BCL: 0.8%, RML: 3.7 mg/mL, CRL: 4.2%) and amino groups contents on the surface of the enzyme molecules (BCL: 2.2%, RML: 2.6%, CRL: 3.7%). In fact, the higher amount of the enzyme loading, the easier the aggregation of enzyme would occur, which could decline the activity recovery. Actually, the highest activity recovery was not obtained at the highest loading of the enzymes. While the dosage of enzymes were respectively 250 mg (BCL), 0.5 mL (RML) and 30 mg (CRL), the activity recovery of the three immobilized lipases reached the highest points (Table 2). Meanwhile, pH value is a critical factor in enzyme immobilization. Panzavolta et al.29 have reported that pH value had little effect on immobilization efficiency while great influence on the esterification activity. It is because enzymes are differently charged in various pH values, which is conducive to the active conformation. As per the theory of “pH memory”, enzymes maintain the ionization as they turn towards the organic phase from aqueous. So, various conformation was preserved,30 and the highest activity recoveries were obtained with the pH value severally in 7.5 (BCL), 7 (RML) and 6.5 (CRL). For the immobilization temperature, enzyme activity generally increases with the elevation of temperature to a certain level, and thereafter too much high temperature will lead to protein denaturation and thus decline the activity recovery. The denaturation and inactivation temperatures of the three lipases are different, so the most appropriate immobilization temperatures are also different. The optimum temperatures were 30 °C for BCL, 45 °C for RML and 35 °C 4375

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enhancement of near 70% with respect to non-bioimprinted CRL. Similarly, the activity recovery of Yarrowia lipolytica lipase LIP2 were also significantly improved after imprinting with and imprinting molecule compared with non-treated YlLIP2.36 These results manifest that different lipases all can be enhanced to some degree via imprinting technology. 3.3. The Effects of the Organic Solvents and Water Content on the Biodiesel Production. It is widely recognized that organic solvent features not only affect the mass transfer in the reaction system, but also greatly influence on the enzyme structure and activity. Enzyme shows different catalytic activity, substrate selectivity, operating stability and kinetic characteristics in different solvents. Lu et al.37 studied the performances of Candida sp. 99-125 catalyzing methanolysis of glycerol trioleate in twelve different solvents and found that the fundamental influence of organic solvents on enzymatic catalysis was hydrophobicity (log P, logarithm of the partition coefficient (P) of the solvent in 1-octanol and water two-phase system). An optimal organic solvent could improve the mutual solubility of hydrophilic alcohols and hydrophobic triglycerides and thus protect the enzymes from denaturation at high alcohol concentrations. Table 4 enucleates the effect of different

comparison between mMWCNTs-PAMAM in this investigation and previously reported immobilization on other magnetic micro/nanocomposite supports. As mentioned above, the transesterification performance of the three proposed lipases with different substrate specificity immobilized on mMWCNTs-PAMAM were comparable or better than that obtained from other immobilized lipases. Moreover, the three immobilized lipases have higher water tolerance and reusability than the other immobilized lipases (Table 3). Thus, the immobilized lipases developed in this study seem to be more effective and have the potential for practical applications in enzymatic biodiesel synthesis processes, especially for nonedible oil with high content of water as substrate. 3.2. Bioimprinting. The strategy of molecular bioimprinting is to cause a ligand-induced beneficial conformational change in the enzyme in aqueous solution, and later employ it in non-aqueous media where the enzyme is supposed to maintain the imprinted conformation and keeps high catalysis activity. Generally, fatty acid substrate analogues are utilized as templates for the bioimprinting of lipases.34 Herein, the effects of five imprinting molecules on the biocatalytic activity of the three lipases were examined (Fig. 1). Lauric acid was used as

Table 4. Effect of Solvents with Different log P Values on the Enzymatic Biodiesel Productiona Yield (%)

Figure 1. Illustration of bioimprinting affecting on the biocatalytic capability of three lipases. The reaction conditions: immobilized lipases loading 0.1 g, stirring speed 200 rpm, room temperature, incubated time 1 h, bioimprinting molecular amount 0.075 mmol/ mL).

Organic solvents

log P

Solvent free n-Nonane Isooctane n-Octane n-Heptane n-Hexane n-Pentane Toluene Benzene Phenol Dihydroxybenzene t-Butanol N,NDimethylformamide

5.1 4.7 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.8 −1.0

BCL 42.5 40.4 69.4 52.9 62.1 45.8 39.2 43.5 40.3 38.6 40.8 80.6 37.6

± ± ± ± ± ± ± ± ± ± ± ± ±

RML 3.6 4.1 2.9 4.8 3.1 2.9 2.5 2.8 4.0 2.3 2.6 4.7 1.7

30.5 32.1 44.6 49.6 36.7 31.4 29.9 38.5 30.6 28.4 26.6 42.2 20.5

± ± ± ± ± ± ± ± ± ± ± ± ±

2.8 4.6 3.4 2.6 1.9 2.5 3.1 1.7 0.9 1.8 2.3 4.1 2.6

CRL 21.4 23.8 35.3 29.5 26.3 25.8 26.8 27.9 29.6 20.7 25.4 30.9 26.7

± ± ± ± ± ± ± ± ± ± ± ± ±

1.7 0.9 0.7 3.2 4.3 2.5 0.8 0.8 1.5 3.8 2.0 3.4 0.7

a

BCL: Burkholderia cepacia lipase; RML: Rhizomucor miehei lipase; CRL: Candida rugosa lipase. The reactions were performed at 40 °C, 200 rpm for 12 h. 10 wt. % immobilized lipases were added to 2.19 g of soybean oil containing 20 wt. % solvent (from log P = 5.1 to log P = −1.0) and 5 wt. % water (all dosage percentages were based on the oil mass); methanol was added only once by the molar ratio of methanol : oil molar ratio 4:1. The values are averages, and ± indicates the standard deviation (SD).

template, the activity recovery of the immobilized BCL was enhanced from 192.0% to 1,244%. Oleic acid chosen as template, the maximal activity recovery of CRL was 72.6%, and that of RML was 3,437%, which were 1.47-fold and 1.25-fold enhancement over the non-bioimprinted immobilized enzymes, respectively. Moreover, the immobilization efficiency of the three lipases had no obvious change after imprinting with different imprinting molecules. The reasonable explanation for this dramatic enhancement of activity imprinted with lauric acid to BCL and oleic acid to CRL and RML was probably that the resemblance of these molecules to the natural substrates of the enzymes contributes to forming an enzyme-support complex with a very suitable open conformation favorable for the access of substrates.35 Foresti et al.18 reported immobilizing Candida rugosa lipase on polypropylene by physical adsorption. The activity of the immobilized lipase was greatly enhanced via bioimprinting-immobilization technique, with specific activity

solvents on the three lipases with methanol added only once, and Table 5 lists biodiesel yield of methanolysis reaction in varied solvents systems and water amounts. It can be clearly seen that the yield of the reaction is remarkably dependent on the type of solvents. The lipase in various solvents also required different optimal amount of water to retain its maximum activity. As shown in Table 4, biodiesel yield catalyzed by the immobilized BCL did not exceed 70% except t-butanol, which showed a yield of 80.6%. Similarly, when the immobilized RML was used as a catalyst, the highest biodiesel yield was obtained in n-octane, which was close to 50%. While isooctane was the optimal organic solvent for the immobilized CRL (35.3%). In the more 4376

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Table 5. Biodiesel Yield of Methanolysis Catalyzed by Three Immobilized Lipases in Different Solvents Systems and Water Amountsa Yield (%) Control

2.5 wt. % water

5 wt. % water

Solvents

BCL

RML

CRL

BCL

RML

CRL

BCL

RML

CRL

Isooctane n-Octane t-Butanol

36.6 ± 2.6 29.6 ± 3.4 25.6 ± 2.9

18.8 ± 3.1 20.4 ± 4.3 15.4 ± 3.5

19.5 ± 2.8 14.6 ± 2.1 6.1 ± 1.9

65.4 ± 2.8 41.7 ± 2.9 68.9 ± 0.7

26.4 ± 3.0 30.3 ± 3.2 28.8 ± 4.5 Yield (%)

28.6 ± 3.6 20.9 ± 2.2 7.8 ± 3.7

47.2 ± 2.6 38.2 ± 3.7 50.3 ± 2.6

39.8 ± 3.4 44.9 ± 1.8 41.4 ± 2.0

40.4 ± 1.5 32.6 ± 2.3 25.5 ± 2.7

7.5 wt. % water

10 wt. % water

12.5 wt. % water

Solvents

BCL

RML

CRL

BCL

RML

CRL

BCL

RML

CRL

Isooctane n-Octane t-Butanol

47.2 ± 2.6 38.2 ± 3.7 50.3 ± 2.6

39.8 ± 3.4 44.9 ± 1.8 41.4 ± 2.0

40.4 ± 1.5 32.6 ± 2.3 25.5 ± 2.7

38.6 ± 3.7 24.4 ± 1.5 29.1 ± 1.6

46.9 ± 2.6 55.7 ± 2.1 45.7 ± 0.9

30.7 ± 1.4 26.1 ± 2.7 31.7 ± 1.8

30.6 ± 1.6 21.7 ± 2.4 28.7 ± 2.4

43.7 ± 2.9 48.4 ± 2.3 42.5 ± 1.7

23.4 ± 1.6 23.6 ± 1.6 20.8 ± 2.8

BCL: Burkholderia cepacia lipase; RML: Rhizomucor miehei lipase; CRL: Candida rugosa lipase. The reactions were performed at 40 °C, 200 rpm for 12 h. 10 wt. % immobilized lipases were added to 2.19 g of soybean oil containing 20 wt. % solvent (all dosage percentages were based on the oil mass); methanol was respectively added to the system in three steps at 0 h, 4 h, and 8 h by the molar ratio of methanol : oil molar ratio 4:1. The values are averages, and ± indicates the standard deviation (SD).

a

was found that a certain amount of water was required in the reaction mixtures for the three lipases. The biodiesel yields catalyzed by the three immobilized lipases increased to different degree with the increase of water amount in isooctane, n-octane and t-butanol systems. And the highest conversion rate of 84.5% was obtained with 5 wt. % water for the immobilized BCL in the t-butanol system. Meanwhile, the biodiesel yield slightly increased to a high yield at 10 wt. % of water content in n-octane system for the immobilized RML, and 7.5 wt. % for CRL in isooctane system. 3.4. The Effects of Methanol Concentration on Biodiesel Production. Methanol serves as a reaction substrate of transesterification and excessive methanol tends to push the reaction process to the synthesis direction, but is harmful to the lipases. Addition of appropriate amounts of alcohols to the reaction mixture can increase reaction rate and degree of transesterification reaction. So, the optimal amount of methanol added to the reaction for each lipase was tested. As shown in Figure 2, the biodiesel yields grew with the increase of methanol amount, while higher methanol content would significantly lower the enzyme activities of the three immobilized lipases. Consequently, the minimal stoichiometric methanol to oil ratio of 5:1 for the immobilized RML, 4:1 for BCL and CRL were chosen in further experiments. 3.5. The Effects of Reaction Temperature on Biodiesel Production. In order to prevent the lipases from thermal inactivation, the enzymatic transesterification is generally conducted at lower temperature compared with chemical reactions. Meanwhile, increasing the reaction temperature tends to push the reaction process to endothermic direction, which is propitious to the biodiesel production.42 Therefore, temperature effects on methanolysis catalyzed by the immobilized lipases (BCL, RML and CRL) in organic solvents were examined. The optimum operational temperatures for the three immobilized lipases varied greatly. The biodiesel yield of the immobilized BCL, RML and CRL showed their maximum values at 35, 50 and 40 °C, respectively, further increase of temperature will result in decrease of biodiesel yield (Fig. 3). 3.6. The Effects of Time Interval of Methanol Addition on Biodiesel Production. It is known that over 1/2 M equivalent of alcohols added to the reaction mixture at the beginning will inactivate the activity of enzyme. Stepwise

hydrophilic solvents system, such as phenol, dihydroxybenzene and N,N-dimethylformamide, the yields were all not very high. Some former studies have elucidated that enzymes showed higher activity in relatively hydrophobic organic solvents (log P > 2) which have been tried as reaction medium for biodiesel production.38 However, some research showed that there was not a linear correlation between the solvents and the biodiesel yield, but an approximate S-shaped curve.37 In this study, we also found that there was not a linear correlation between solvents and the biodiesel yields for the three lipases in transesterification reaction. The explanation might due to the fact that different lipase sources with specific immobilized method would have unique characteristics in organic solvents. In addition, it can be found that t-butanol was a relatively excellent medium for these three immobilized lipases, which was perhaps that because methanol and glycerol have good solubility in t-butanol solvent. So, the negative effects on lipases activity and stability caused by methanol and glycerol could be weakened and lipases still exhibited good stability in such reaction medium. Meanwhile, the yields catalyzed by the immobilized RML and CRL compared with BCL were very low in most cases, revealing that the activities of RML and CRL might be more severely inhibited by the excess methanol. When three-step methanol addition strategy was employed, all biodiesel yields were markedly enhanced in their corresponding optimal organic solvents (Table 5). Furthermore, RML and CRL are known to exhibit much higher tolerance to water,39,40 so several hydrophobic solvents such as isooctane and n-octane were beneficial to RML and CRL due to the fact that they do not partially replace the residual protein-surface bound water. Accordingly, t-butanol, n-octane and isooctane were employed as the reaction media for further transesterification catalyzed by the immobilized BCL, RML and CRL, and three-step methanol addition procedure was selected in the following experiments. Water content of the reaction mixture has significant influence on the catalytic activity and stability of lipases, especially in different solvent systems, which is not only related to the unique characteristics of lipases acting at oil-water interface, but also correlated with the reaction equilibrium.41 Moreover, the optimum water content generally depends on the type of lipases. So, water amount varied from 0 to 12.5 wt. % was determined for each immobilized lipase. From Table 5, it 4377

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addition for the immobilized BCL. However, the biodiesel yields catalyzed by the immobilized RML and CRL were remarkably improved by lengthening the time interval (Figure 4b, c). The results indicated that bacterial lipase (BCL is from B. cepacia) was more resistant to organic solvents than fungal lipases (RML and CRL are from R. miehei and C. rugose, respectively).44 3.7. A Comparison of Biodiesel Production from Soybean Oil and Waste Vegetable Oil. The above researches have enunciated that the soybean oil can be effectively converted into biodiesel with the three immobilized lipases as catalysts under their corresponding optimal conditions. However, the cost of feedstock accounts for a large portion in biodiesel production cost. So, a kind of cheaper feedstock, waste vegetable oil was further investigated. Some specifications of waste vegetable oil have been tested and listed in Table S1, and it can be seen that there are obvious distinctions between soybean oil and waste vegetable oil, especially in water contents, free fatty acids (FFAs), stearic acid, oleinic acid and linoleic acid. The temporal kinetics of methanolysis for soybean oil and waste vegetable oil by the three immobilized lipases were presented in Figure 5a-c. For the immobilized BCL, the initial reaction rate of waste vegetable oil was higher than that of soybean oil, owing to the higher FFAs content; nevertheless, the final biodiesel yield of waste vegetable oil was much lower than that of the soybean oil, which might result from the negative effect of water existing in the waste vegetable oil (2.01%) and continuously produced by esterification of FFAs (Fig. 5a).20 In comparison, the yield of waste vegetable oil was similar with that of soybean oil for the immobilized RML, probably due to its higher tolerance to water up to 20% (w/ w).39 While for the immobilized CRL, the final yield of waste vegetable oil was much higher than that of soybean oil, which is perhaps because CRL is a versatile lipolytic enzyme with five individual isoforms45 and different isoforms exhibit remarkable variation in their catalytic efficiencies.46 Actually, Kuo et al.46 compared the transesterification activities of CRL isozyme (CRL1-CRL4) for three non-edible oils and found that CRL2 and CRL4 exhibited superior catalytic efficiencies for producing biodiesel from Jatropha curcas seed oil. A comparison of the transesterification catalyzed by the immobilized BCL, CRL and RML showed that biodiesel yields of BCL and RML were both up to 96% when soybean oil was used as raw material. However, the reaction time when the highest yield was achieved by the immobilized BCL was 10 h much shorter than that of the immobilized RML. As waste vegetable oil was used as raw material, the final biodiesel yield was 80.8%, much lower than that of the soybean oil for the immobilized BCL; while, for the immobilized RML, it was up to 92.1%, much close to that of the soybean oil for the immobilized CRL, less than 90%, though higher than that of soybean oil. The results are probably due to the following reasons: (i) the characteristics (such as trans-/esterification ability and substrate specificity) of the enzymes are much different; (ii) the compositions of the two feedstock differ from each other; (iii) it is closely related to the nature of the carrier and the immobilization method. Reasons (i) and (ii) have been respectively elaborated in Section 3.2 and 3.7. Moreover, previous report also suggested that the immobilized CRL could play a better role in the preparation of biodiesel by replacing organic solvents with ionic liquids.47 Additionally, the immobilized BCL could work better for biodiesel production

Figure 2. Effects of methanol concentration on biodiesel production. The reactions were performed at 40 °C, 200 rpm for 12 h; 10 wt. % immobilized lipases were added to 2.19 g of soybean oil containing 20 wt. % solvent; methanol was respectively added in three steps at 0 h, 4 h, and 8 h by the molar ratio of methanol : oil between 2:1 and 6:1; the water content 5 wt. % for immobilized BCL (Burkholderia cepacia lipase) in a t-butanol system, 7.5 wt. % for CRL (Candida rugosa lipase) in a isooctane system, and 10 wt. % for RML (Rhizomucor miehei lipase) in a n-octane system.

Figure 3. Effects of temperature on biodiesel production. The reactions were performed at temperatures from 30 to 55 °C, 200 rpm for 12 h; 10 wt. % immobilized lipases were added to 2.19 g of soybean oil containing 20 wt. % solvent; methanol was respectively added in three steps at 0 h, 4 h, and 8 h by a methanol : oil molar ratio of 5 : 1 for immobilized RML (Rhizomucor miehei lipase) and 4 : 1 for BCL (Burkholderia cepacia lipase) and CRL (Candida rugosa lipase); the water content was 5 wt. % for immobilized BCL in a t-butanol system, 7.5 wt. % for CRL in a isooctane system and 10 wt. % for RML in a noctane system.

addition of alcohols to the system is a widely adapted strategy to prevent enzyme from inactivation caused by alcohols.43 Moreover, the length of time interval of alcohol addition also has great influence on the enzyme activity. Fan et al.23 used the three-step method and obtained 93.1% yield with the time interval of 10 h, which was higher than that of 6 h time interval. Thus, the effects of time interval of methanol addition on biodiesel production for the three lipases were investigated. As can be seen from Fig.4a, there was no significant increase in conversion rates with extension of time interval of methanol 4378

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Figure 4. Effects of time interval of methanol addition on biodiesel production. The reaction condition: 10 wt. % immobilized lipases, 2.19 g of soybean oil containing 20 wt. % solvent, 200 rpm, (a) methanol : oil molar ratio of 4 : 1, the water content 5 wt. %, 35 °C for immobilized BCL (Burkholderia cepacia lipase) in a t-butanol system; (b) methanol : oil molar ratio of 5 : 1, the water content 10 wt. %, 50 °C, for immobilized RML (Rhizomucor miehei lipase) in a n-octane system; (c) methanol : oil molar ratio of 4 : 1, the water content 7.5 wt. %, 40 °C, for immobilized CRL (Candida rugosa lipase) in a isooctane system.

Figure 5. Biodiesel production from soybean oil and waste vegetable oil. The reactions condition: 10 wt.% immobilized lipases, 2.19 g soybean oil (molecular weight: 877, 1.98 g waste vegetable oil (molecular weight: 792) containing 20 wt.% solvent, 200 rpm, (a) methanol : oil molar ratio of 4 : 1, the water content 5 wt.%, 35°C for immobilized BCL (Burkholderia cepacia lipase) in a t-butanol system; (b) methanol : oil molar ratio of 5 : 1, the water content 10 wt.%, 50°C, for immobilized RML (Rhizomucor miehei lipase) in a n-octane system; (c) methanol : oil molar ratio of 4 : 1, the water content 7.5 wt.%, 40°C, for immobilized CRL (Candida rugosa lipase) in a isooctane system.

Table 6. Reusability of the Three Immobilized Lipasesa

from raw oils having lower water content and FFAs, meaning it has higher ability to catalyze transesterification than esterification and less tolerance to the negative effect of moisture. In contrast, higher water content and FFAs such as waste vegetable oil would be suitable for RML, which showed best performance among the three lipase for biodiesel synthesis, indicating its high catalysis abilities for transesterification and esterification. 3.8. Operational Stability of Biocatalysts. One of the objectives of the immobilized enzyme is to design more efficient biocatalysts which can easily be recovered and reused. To investigate the reusability of the three biocatalysts for biodiesel production, the immobilized enzymes was recovered by magnetic separation after each batch and washed with their corresponding solvents for the subsequent batch and the next batch was carried out with fresh substrates under the same reaction conditions as described previously. The reusability of the immobilized enzymes were presented in Table 6. As can be seen, the biodiesel yields after 10 cycles for the immobilized BCL, RML and CRL were 89.4%, 80.5% and 58.3%, respectively. The results indicate that the immobilized lipases show good capability to be repeatedly used. The yield of biodiesel, catalyzed by immobilized Candida antarctica lipase on SBA-15 via physical adsorption, decreased by 8.9% after eight recycles.48 The gradual reduction in biodiesel yield was ascribed to both the leaching of the enzyme and loss of activity of the

Biodiesel yield (%) Cycles 1 2 4 6 8 10

BCL (24 h) 95.4 93.8 94.3 93.1 90.7 89.4

± ± ± ± ± ±

1.9 2.6 1.5 2.1 1.8 2.3

a

RML (36 h)b 96.3 92.9 89.2 86.9 84.1 80.5

± ± ± ± ± ±

1.3 2.0 1.7 1.4 2.5 1.6

CRL (40 h)c 84.6 80.2 76.5 69.9 65.7 58.3

± ± ± ± ± ±

1.5 2.1 2.4 1.2 1.6 2.0

a

BCL: Burkholderia cepacia lipase; RML: Rhizomucor miehei lipase; CRL: Candida rugosa lipase. The reaction conditions of the three immobilized lipases were the same as described previously. a, b, c refer to the reaction time of the immobilized BCL, RML and CRL for each batch (24 h, 36 h and 40 h, respectively). Note: ± indicates the standard deviation (SD).

immobilized lipase. Compared to physical adsorption, covalently linkage could strongly diminish the leaching of the enzyme. In addition, the stability and reusability of BCL was better than that of RML and CRL, which attributed to the tolerance of the enzyme and the system solvent. Isooctane and n-octane (log P > 4) are high hydrophobic solvent, which is beneficial for CRL and RML in single batch reaction. However, with the increase of the recycling number, methanol and byproduct glycerol will adsorb onto the surface of the immobilized lipase due to their poor solubility in isooctane 4379

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and n-octane solvents, enlarging mass transfer resistance and causing gradual inactivation of the enzyme, resulting in a reduction in the biodiesel yield.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.energyfuels.7b00036. Experimental Procedures: 1.1 Preparation of mMWCNTs; 1.2 Synthesis of mMWCNTs-NH2 by Aminosilane; 1.3 Surface Modification with PAMAM Dendrimer Table S1, Scheme S1, and Figure S1 (PDF)

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4. CONCLUSION Typical representatives, BCL, RML and CRL, of three subgroup lipases with different substrate specificities were respectively immobilized on a uniform magnetic nanocomposites material by covalent binding, and their characteristics for transesterification reaction were further investigated. Bioimprinting could improve catalytic performance of the three lipases. In the optimized conditions, all immobilized lipases could achieve high biodiesel yields, showing advantage of using CNTs as carriers for catalytic aim over other less durable nanomaterials. The immobilized BCL, RML and CRL exhibited well operational stability and reusability. However, the biodiesel yields achieved by the immobilized BCL and RML were higher than that of CRL. It was also demonstrated that waste vegetable oil could be effectively converted into biodiesel by the immobilized RML. This study implies that the immobilization strategy employed in this study can significantly enhance the catalytic activity and stability of lipases, but the enhancement is different to some extent for different enzymes. Specific conditions are still required for each lipase to catalyze transesterification and the immobilized RML may offer a promising solution to industrial scale biodiesel production for low-cost inedible oil. Furthermore, this work provides a new choice for immobilization of other enzymes with similar surface amino distribution and substrate specificity.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86-27-87792213; E-mail: [email protected]. ORCID

Yunjun Yan: 0000-0002-4225-6261 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was financially supported by the National Natural Science Foundation of China (No. 31170078), the National High Technology Research and Development Program of China (Nos. 2011AA02A204 and 2013AA065805), the Natural Science Foundation of Hubei Province (No. 2015CFA085), and the Fundamental Research Funds for HUST (Nos. 2014NY007 and 2014QN119). The authors thank Ms. Chen Hong, from the Centre of Analysis and Test, Huazhong University of Science and Technology for biodiesel analysis and Xiaotao Han from the National High Magnetic Field Center, HUST. 4380

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