Improved Performance of Magnetic Cross-Linked Lipase Aggregates By

molecules

Article Improved Performance of Magnetic Cross-Linked Lipase Aggregates by Interfacial Activation: A Robust and Magnetically Recyclable Biocatalyst for Transesteriﬁcation of Jatropha Oil

Weiwei Zhang 1, Huixia Yang 1, Wanyi Liu 1, Na Wang 2,* and Xiaoqi Yu 2,* 1 School of Chemistry and Chemical Engineering, National Demonstration Center for Experimental Chemistry Education, Ningxia University, Yinchuan 750021, China; [email protected] (W.Z.); [email protected] (H.Y.); [email protected] (W.L.) 2 Key Laboratory of Green Chemistry Technology, Ministry of Education, College of Chemistry, Sichuan University, Chengdu 610064, China * Correspondence: [email protected] (N.W.); [email protected] (X.Y.); Tel.: +86-288-541-5886 (N.W. & X.Y.)

Received: 18 November 2017; Accepted: 2 December 2017; Published: 7 December 2017

Abstract: Lipases are the most widely employed enzymes in commercial industries. The catalytic mechanism of most lipases involves a step called “interfacial activation”. As interfacial activation can lead to a signiﬁcant increase in catalytic activity, it is of profound importance in developing lipase immobilization methods. To obtain a potential biocatalyst for industrial biodiesel production, an effective strategy for enhancement of catalytic activity and stability of immobilized lipase was developed. This was performed through the combination of interfacial activation with hybrid magnetic cross-linked lipase aggregates. This biocatalyst was investigated for the immobilization of lipase from Rhizomucor miehei (RML). Under the optimal conditions, the activity recovery of the surfactant-activated magnetic RML cross-linked enzyme aggregates (CLEAs) was as high as 2058%, with a 20-fold improvement over the free RML. Moreover, the immobilized RML showed excellent catalytic performance for the biodiesel reaction at a yield of 93%, and more importantly, could be easily separated from the reaction mixture by simple magnetic decantation, and retained more than 84% of its initial activities after ﬁve instances of reuse. This study provides a new and versatile approach for designing and fabricating immobilized lipase with high activation and stability.

Keywords: lipase; interfacial activation; surfactant; magnetic nanoparticles; cross-linked enzyme aggregates; enzyme immobilization; biodiesel

1. Introduction Lipase is one of the most widely used enzymes, and plays an important role in biotechnological and industrial processes including biodiesel, food, detergent, textile, environmental industries, oleochemical industries, as well as in pharmaceutical applications. This is due to their high activity, wide sources and broad range of substrates [1,2]. Under physiological conditions, lipases catalyze the hydrolysis of the ester bonds in the molecules of triglycerides. These substrates are practically insoluble in water, so the reaction is catalyzed at the water–lipid interface, at which most lipases express higher catalytic activity than in aqueous solution. This phenomenon of increased lipase activity at the interface is termed “interfacial activation” [3]. As is well known, the active site of the lipase is covered by a ﬂexible region called a “lid”, which is composed of either one or two amphiphilic a-helices, and blocks substrate access in the closed conformation. In lipases undergoing interfacial activation, the interaction with a drop of oil or a hydrophobic phase can cause opening of the lid to make the active site accessible [4]. As the interfacial activation can lead to a signiﬁcant increase in

Molecules 2017, 22, 2157; doi:10.3390/molecules22122157 www.mdpi.com/journal/molecules Molecules 2017, 22, 2157 2 of 16 catalytic activity, it is of profound importance for all applications of lipases, and should always be taken into account when developing lipase immobilization methods [5]. Previously, different activation methods have been used to immobilize enzymes and can improve their catalytic performance to some extent. These include increasing the hydrophobicity of the supporter for immobilization [6], ion-paired lipases [7], surfactant-coated lipases [8], microemulsions [9], and microemulsion-based organogels [10,11]. As mentioned above, surfactants can activate lipases in different immobilization procedures. An example of these procedures involves coating lipase with surfactant. This is a simple and efficient process demonstrating good stability in a wide range of organic solvents, and can be combined with other immobilization methods [12]. The physical modification of lipases with surfactants can mimic the lipid–water interface, causing opening of the lid to shift the lipase conformational equilibrium toward the open form. This is analogous to the interfacial activation-based molecular bioimprinting [13]. However, there are individual variations among the lipases in surfactant-coating activation. Thus, a concrete analysis should be made when different lipases are used. Moreover, to fully exploit the technical and economical advantages of lipases, it is recommended to use them in an immobilized form to reduce cost and increase stability of the free lipase. Various methodologies of enzyme immobilization have been established by previous studies including adsorption, ionic binding, covalent modification, entrapment, and encapsulation [14,15]. Efficient immobilization protocols are the result of perfect matching of factors depending on the enzyme, the process, and the support for immobilization [16,17]. In general, lipases can be immobilized using most of the methods developed for enzyme immobilization [18,19]. In the past two decades, cross-linked enzyme aggregates (CLEAs) have provided an innovative, versatile and industrially potent immobilization strategy, arousing extensive attention due to the simplicity of its preparation and robustness of the immobilized enzymes [20]. It has also been successfully applied in the preparation of lipases [21,22]. The CLEA technique appears to involve a superior immobilization method, with prominent advantages over other methods including high volumetric activity, functional stability, multiple recycling potential, and the fact that no purified enzymes are required [23–25]. Furthermore, the cross-linking step gives the aggregates a more stabilized structure by generating covalent linkages between enzyme molecules. This renders them permanently insolubilized [26] whilst maintaining their pre-organized superstructure and catalytic activity [27]. Despite the simplicity and low cost of preparing CLEAs, a common problem for CLEAs is their irregular shapes and sizes (in the range of 5–100 µm). These factors may lead to slow diffusion of substrates into the CLEAs, or their softness may hinder the process of recovery [28]. These CLEAs are not mechanically resistant, and may require physical support to increase their rigidity for some industrial applications [29]. Although various modifications have been made to further stabilize the CLEAs [30], magnetic bio-separation technology is a promising strategy for the preparation of immobilized enzymes. This technique can be easily performed using an external magnetic field, and provides enhanced stability over repeated uses in continuous bioseparation processes [31–33]. Regarding industrial use, attention should also be paid to magnetic carriers because of their low cost and excellent separability. In this work, (3-aminopropyl)triethoxysilane (APTES) was grafted onto the outer surfaces of the magnetic nanoparticles in order to attain the desired properties: high saturation magnetization and sufficient active sites to efficiently improve enzyme loading. Biodiesel, which consists of a mixture of fatty acid alkyl esters, offers a promising solution for the energy crisis due to its reputation as a sustainable and renewable alternative to fossil fuels. This reputation is based on biodiesels biodegradability, low emission of environmental pollutants and wide range of feedstocks [34]. In light of the current global environment, transesterification of oil feedstock catalyzed by immobilized lipase has been regarded as one of the most promising techniques for preparing biodiesel. This is due to their high activity and selectivity for alcoholysis of triglycerides under mild reaction conditions [35]. Therefore, potential industrially relevant biocatalysts are in high Molecules 2017, 22, 2157 3 of 16 demand.Molecules 2017 However,, 22, 2157 biocatalysts that are capable of both increased enzyme loading and improved3 of 15 enzymatic stability are rare. In this study, a strategy integrating surfactant-activated magnetic cross-linked enzyme In this study, a strategy integrating surfactant-activated magnetic cross-linked enzyme aggregates aggregates was developed and investigated for the immobilization of lipase from Rhizomucor miehei was developed and investigated for the immobilization of lipase from Rhizomucor miehei (RML). (RML). This was performed in order to combine advantageous properties such as high specific This was performed in order to combine advantageous properties such as high specific enzyme activity, enzyme activity, stable open-lid conformation of lipase, ease of separation and recovery, and stable open-lid conformation of lipase, ease of separation and recovery, and sufficient active sites, sufficient active sites, into a single system for lipase immobilization. The APTES functionalized into a single system for lipase immobilization. The APTES functionalized magnetic supports were magnetic supports were used as a rigid and multifunctional cross-linking reagent, able to reduce the used as a rigid and multifunctional cross-linking reagent, able to reduce the mobility of the lipase lid mobility of the lipase lid and produce immobilized lipases with a stabilized open form. Some specific and produce immobilized lipases with a stabilized open form. Some specific characteristics of the characteristics of the immobilized RML were examined further, including optimization of the immobilized RML were examined further, including optimization of the immobilization conditions immobilization conditions and the stability of the immobilized enzyme. Moreover, the prepared and the stability of the immobilized enzyme. Moreover, the prepared immobilized lipase was utilized immobilized lipase was utilized to catalyze transesterification for biodiesel production. to catalyze transesterification for biodiesel production.

2. Results and Discussion

2.1. Characterization of the Immobilized Lipase In thisthis study,study, aa strategystrategy integratingintegrating interfacialinterfacial activation,activation, magneticmagnetic nanoparticles,nanoparticles, and lipaselipase CLEAs immobilization was developed in order to combine advantageous properties into a single system for for enzyme enzyme immobilization. immobilization. These These properties properties include include ease ease of separation of separation and recovery, and recovery, and andsufficient sufﬁcient enzyme enzyme loading. loading. As Asillustrated illustrated in inFigure Figure 11,, aminopropyl-functionalizedaminopropyl-functionalized magneticmagnetic nanoparticles werewere readily readily prepared prepared and and subsequently subsequent employedly employed for thefor immobilizationthe immobilization of lipase of lipase from Rhizomucorfrom Rhizomucor miehei miehei. .

Figure 1. Schematic illustration of the immobilization process to produce Rhizomucor miehei (RML) cross-linked enzyme aggregates (CLEAs), magnetic RML CLEAs,CLEAs, andand surfactant-activatedsurfactant-activated magnetic RML CLEAs.

As cancan bebe seenseen in in Figure Figure2, all2, all of theof the magnetic magnetic immobilized immobilized RMLs RMLs exhibited exhibited rapid rapid separation separation from thefrom reaction the reaction mixture mixture using a magnet.using a Oncemagnet. the Once magnetic the ﬁeldmagnetic has been field removed, has been the removed, immobilized the enzymeimmobilized can beenzyme easily can dispersed be easily by simpledispersed shaking. by simple This shaking. can provide This ancan easy provide and efﬁcientan easy wayand toefficient separate way immobilized to separate enzyme immobilized from a suspensionenzyme from system. a suspension The successful system. immobilization The successful of immobilization of lipase onto the APTES-Fe3O4 nanoparticles was identified by FT-IR spectroscopy and scanning electron microscopy.

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lipase onto the APTES-Fe3O4 nanoparticles was identiﬁed by FT-IR spectroscopy and scanning electronMolecules 2017 microscopy., 22, 2157 4 of 15

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Figure 2. 2. SchematicSchematic of the of simple the simple magnetic magnetic separation separation of surfactant-activated of surfactant-activated magnetic RML magnetic CLEAs: RML (A) CLEAs:immobilized (A) immobilizedlipase dispersed lipase in reaction dispersed mixture; in reaction (B) immobilized mixture; (B lipase) immobilized collected by lipase an external collected magnet. by an externalFigure 2. magnet. Schematic of the simple magnetic separation of surfactant-activated magnetic RML CLEAs: (A) Figureimmobilized 3 shows lipase the dispersed comparative in reaction FT-IR mixture; spectra (B) of immobilized the magnetic lipase core, collected APTES-functionalized by an external magnet. Fe 3O4 particles,Figure RML3 shows CLEAs, the comparativeand magnetic FT-IR RML spectra CLEAs. of theIn the magnetic spectrum core, of APTES-functionalized magnetic RML CLEAs, Fe new3O4 particles,characteristicFigure RML 3 peaksshows CLEAs, at the 584 comparative and cm magnetic−1 correspond FT-IR RML spectrato CLEAs.the Fe-O of the vibrations In magnetic the spectrum of core, the APTES-functionalizedmagnetite of magnetic core. RML This CLEAs, can Fe 3beO4 newascribedparticles, characteristic to RML the successfulCLEAs, peaks and binding at magnetic 584 cmof −Fe 1RML3Ocorrespond4 particles. CLEAs. toInAs the compared spectrum Fe-O vibrations with of magneticthe magnetic of the RML magnetite nanoparticles, CLEAs, core.new characteristic peaks at 584 cm−1 correspond to the Fe-O vibrations of the magnetite−1 core. This−1 can be Thisthe immobilized can be ascribed lipase to thedisplayed successful additional binding IR of bands Fe3O 4situatedparticles. at As1641 compared cm and with 1567 the cm magnetic, which nanoparticles,representascribed tothe the amide thesuccessful immobilized I (the stretchingbinding lipase of vibrations Fe displayed3O4 particles. of C=O additional As groups) compared IR and bands withamide situatedthe II magnetic(N-H at bending 1641 nanoparticles, cm and−1 C-Nand 1567stretching)the immobilized cm−1, whichbands lipase[36] represent of displayedRML, the respectively. amide additional I (the stretchingFurthermore, IR bands vibrations situated after the ofat lipase C=O1641 groups)cm loading,−1 and and it1567 was amide cm noted−1 II, which (N-H that bendingnewrepresent bands and the at C-N amide2938 stretching) cm I (the−1 and stretching 2859 bands cm [ 36vibrations−1 ]appeared of RML, of respectively.for C=O magnetic groups)Furthermore, RML and amide CLEAs, II after (N-Hwhich the bending was lipase reasonably and loading, C-N itattributedstretching) was noted to bands thatC-H new stretching[36] bands of RML, vibrations, at 2938 respectively. cm− which1 and Furthermore, 2859also indicated cm−1 appeared after the thesuccessful for lipase magnetic loading, loading RML itof wasCLEAs,lipase. noted which that wasnew reasonably bands at 2938 attributed cm−1 and to C-H2859stretching cm−1 appeared vibrations, for magnetic whichalso RML indicated CLEAs, thewhich successful was reasonably loading ofattributed lipase. to C-H stretching vibrations, which also indicated the successful loading of lipase.

Figure 3. The FT-IR spectrum of (A) Fe3O4 nanoparticles; (B) (3-aminopropyl)triethoxysilane (APTES)-

functionalized Fe3O4 nanoparticles; (C) RML CLEAs; (D) magnetic RML CLEAs; (E) AOT-activated

magneticFigure 3. RMLThe FT-IR CLEAs. spectrum (Sodium of (bis-2-(ethylhexyl)A) Fe3O4 nanoparticles; sulfosuccinate, (B) (3-aminopropyl)triethoxysilane AOT). (APTES)- Figure 3. The FT-IR spectrum of (A) Fe3O4 nanoparticles; (B) (3-aminopropyl)triethoxysilane 3 4 (APTES)-functionalizedfunctionalized Fe O nanoparticles; Fe3O4 nanoparticles; (C) RML CLEAs; (C) RML(D) magnetic CLEAs; RML (D) CLEAs; magnetic (E) AOT-activated RML CLEAs; (SEMmagneticE) AOT-activated studies RML monitored CLEAs. magnetic (Sodium the RML morphological bis-2-(ethylhexyl) CLEAs. (Sodium changes sulfosuccinate, bis-2-(ethylhexyl) of the magnetic AOT). sulfosuccinate, nanoparticles AOT). and different immobilized RML preparations, as illustrated in Figure 4. The typical SEM image of the bare Fe3O4 particlesSEM in studies Figure monitored 4A indicates the thatmorphological the aggregates changes of Fe of3O the4 particles magnetic exhibit nanoparticles an obvious and spherical different SEM studies monitored the morphological changes of the magnetic nanoparticles and different shape,immobilized and present RML preparations, a clearly tight as illustratedstructure. inThis Figure is due 4. The to typicalthe strong SEM magnetic image of dipole–dipolethe bare Fe3O4 immobilized RML preparations, as illustrated in Figure4. The typical SEM image of the bare Fe 3O4 interactionsparticles in Figurebetween 4A Fe indicates3O4 nanoparticles. that the aggregates Figure 4B of showsFe3O4 particles representative exhibit SEMan obvious images spherical of the particles in Figure4A indicates that the aggregates of Fe 3O4 particles exhibit an obvious spherical aminopropyl-functionalizedshape, and present a clearly Fe 3tightO4 particles. structure. As displayedThis is due in thisto the figure, strong APTES-Fe magnetic3O4 nanoparticlesdipole–dipole shape, and present a clearly tight structure. This is due to the strong magnetic dipole–dipole showedinteractions a rough between surface Fe 3morphology,O4 nanoparticles. with Figurean approximate 4B shows size representative of 30–50 nm, SEM implying images that of thethe modificationaminopropyl-functionalized of ATPES on the Fe surface3O4 particles. of the Fe As3O displayed4 nanoparticles in this could figure, weaken APTES-Fe the aggregation3O4 nanoparticles of the magneticshowed ananoparticles. rough surface As morphology, indicated in Figurewith an 4C, approximate RML CLEAs size display of 30–50 a smooth nm, implyingsurface, and that their the lengthsmodification ranged of from ATPES 300 on to the1000 surface nm. This of the indicate Fe3O4s nanoparticles that the surface could of CLEAs weaken became the aggregation more compact, of the perhapsmagnetic improving nanoparticles. enzyme As indicatedrigidity for in furtherFigure 4C,applications. RML CLEAs However, display this a smooth also limits surface, the diffusionand their lengths ranged from 300 to 1000 nm. This indicates that the surface of CLEAs became more compact, perhaps improving enzyme rigidity for further applications. However, this also limits the diffusion

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interactions between Fe3O4 nanoparticles. Figure4B shows representative SEM images of the aminopropyl-functionalized Fe3O4 particles. As displayed in this figure, APTES-Fe3O4 nanoparticles showed a rough surface morphology, with an approximate size of 30–50 nm, implying that the modification of ATPES on the surface of the Fe3O4 nanoparticles could weaken the aggregation of the magnetic nanoparticles. As indicated in Figure4C, RML CLEAs display a smooth surface, andMolecules their 2017 lengths, 22, 2157 ranged from 300 to 1000 nm. This indicates that the surface of CLEAs became5 of 15 more compact, perhaps improving enzyme rigidity for further applications. However, this also of substrate to access lipase. When modified Fe3O4 particles were added before lipase precipitation, the limits the diffusion of substrate to access lipase. When modified Fe3O4 particles were added before lipasemagnetic precipitation, particles could the magneticact as cores particles during could lipase act precipitation, as cores during whilst lipase also precipitation,cross-linking with whilst lipase. also cross-linkingTherefore, this with procedure lipase. Therefore,could not only this procedureprevent th coulde formation not only of preventsecondary the particles formation (Figure of secondary 4D) but particlesalso improve (Figure the4 stabilityD) but alsoof the improve immobilized the stability enzymes. of theIt is immobilizednoteworthy that enzymes. the formed It is APTES-Fe noteworthy3O4 nanoparticles not only display magnetic behaviours, but also have large active surface available for that the formed APTES-Fe3O4 nanoparticles not only display magnetic behaviours, but also have largelipase active immobilization. surface available After forcoating lipase with immobilization. AOT, RML AfterCLEAs coating were withuniformly AOT, RMLdispersed CLEAs on were the uniformlymagnetic particles dispersed (Figure on the 4E). magnetic These particlesobservations (Figure suggested4E). These that observations the coating suggestedlayer of surfactants that the coatingcould prevent layer of the surfactants formation could of large prevent aggregates. the formation Therefore, of large the aggregates. specific surface Therefore, area of the the specific RML surfaceCLEAs areacould of be the effectively RML CLEAs improved. could be effectively improved.

FigureFigure 4.4. SEMSEM imagesimages ofof (A(A)) FeFe3O3O44 nanoparticles;nanoparticles; ((BB)) APTES-functionalizedAPTES-functionalized FeFe33OO44 nanoparticles; ((CC)) RMLRML CLEAs;CLEAs; ((DD)) magneticmagnetic RMLRML CLEAs;CLEAs; ((EE)) AOT-activatedAOT-activated magneticmagnetic RML RML CLEAs. CLEAs.

InIn addition,addition, the the magnetic magnetic measurements measurements of the magneticof the magnetic nanoparticles nanoparticles and different and immobilized different RMLimmobilized preparations RMLwere preparations displayed were in Figure displayed5. As illustrated in Figure in 5. Figure As 5illustrated, the magnetization in Figure curves 5, the exhibitmagnetization no hysteresis, curves indicating exhibit no the hy superparamagneticsteresis, indicating characterthe superparamagnetic of all these samples. character The of saturation all these magnetizationsamples. The ofsaturation immobilized magnetization RML preparations of immobilized is lower thanRML that preparations of magnetic is nanoparticles, lower than that owing of tomagnetic the presence nanoparticles, of lipase-loading. owing to Generally, the presence these of properties lipase-loading. allow for Generally, easy and these rapid separationproperties ofallow the AOT-activatedfor easy and rapid magnetic separation RML of CLEAs the AOT-activate from the reactiond magnetic mixture RML using CLEAs a magnet. from the reaction mixture using a magnet.

Figure 5. Magnetic hysteresis loops of Fe3O4 nanoparticles, APTES-functionalized Fe3O4 nanoparticles, magnetic RML CLEAs and AOT-activated magnetic RML CLEAs.

Molecules 2017, 22, 2157 5 of 15 of substrate to access lipase. When modified Fe3O4 particles were added before lipase precipitation, the magnetic particles could act as cores during lipase precipitation, whilst also cross-linking with lipase. Therefore, this procedure could not only prevent the formation of secondary particles (Figure 4D) but also improve the stability of the immobilized enzymes. It is noteworthy that the formed APTES-Fe3O4 nanoparticles not only display magnetic behaviours, but also have large active surface available for lipase immobilization. After coating with AOT, RML CLEAs were uniformly dispersed on the magnetic particles (Figure 4E). These observations suggested that the coating layer of surfactants could prevent the formation of large aggregates. Therefore, the specific surface area of the RML CLEAs could be effectively improved.

Figure 4. SEM images of (A) Fe3O4 nanoparticles; (B) APTES-functionalized Fe3O4 nanoparticles; (C) RML CLEAs; (D) magnetic RML CLEAs; (E) AOT-activated magnetic RML CLEAs.

In addition, the magnetic measurements of the magnetic nanoparticles and different immobilized RML preparations were displayed in Figure 5. As illustrated in Figure 5, the magnetization curves exhibit no hysteresis, indicating the superparamagnetic character of all these samples. The saturation magnetization of immobilized RML preparations is lower than that of magnetic nanoparticles, owing to the presence of lipase-loading. Generally, these properties allow for easy and rapid separation of the AOT-activated magnetic RML CLEAs from the reaction mixture Moleculesusing a 2017magnet., 22, 2157 6 of 16

Figure 5. Magnetic hysteresis loopsloops ofof FeFe33O44 nanoparticles, APTES-functionalized FeFe33O4 nanoparticles, Moleculesmagnetic 2017, 22 RML, 2157 CLEAs andand AOT-activatedAOT-activated magneticmagnetic RMLRML CLEAs.CLEAs. 6 of 15

2.2. Optimization of Magnetic RML CLEAs Preparation Parameters

2.2.1. Effect of Precipitant The foremost step in preparation of CLEAs is prec precipitation.ipitation. The characteristics of the precipitant that allow it to achieve the maximum activity reco recoveryvery in aggregation can differ from one lipase to another. This This is is due due to to the the diversity of of biochemical biochemical and and structural structural properties properties of of the the enzymes enzymes [37]. [37]. Lipases from different microbial sources display differentdifferent glycosylated surfaces. This may result in different aggregation, packing and configuring configuring of the protein molecules with different precipitating reagents [26[26].]. To To limit limit maximum maximum enzyme enzyme activity activity in magnetic in magnetic RML CLEA RML preparation, CLEA preparation, five commonly five commonlyused protein used precipitating protein precipitating agents were agents investigated, were investigated, including including organic solvents organic solvents (acetone, (acetone, ethanol, ethanol,isopropanol), isopropanol), polyethylene polyethylene glycol (PEG glycol 800) (PEG and 800) ammonium and ammonium sulfate. As sulfate. shown As in shown Figure in6, differentFigure 6, differenttypes of precipitanttypes of exhibitedprecipitant varying exhibited degrees varying of activity degrees for CLEAs.of activity In addition, for CLEAs. significantly In addition, higher significantlyactivity of RML higher CLEAs activity was retainedof RML inCLEAs saturated was ammoniumretained in sulfate,saturated and ammonium can therefore sulfate, be used and as can the thereforeprecipitant be of used choice as the for furtherprecipitant studies. of choice for further studies.

Figure 6. EffectEffect of precipitant type on the activities of magnetic RML CLEAs.

2.2.2. Effect of Glutaraldehyde Concentration Cross-linking of the precipitated RML and amino functionalized magnetic nanoparticles is the second stepstep in in the the preparation preparation of magnetic of magnetic RML CLEAs.RML CLEAs. In this step, In this lipase step, aggregates lipase areaggregates permanently are permanently packed into an active and insoluble form. Glutaraldehyde is a powerful cross-linker that is widely used in the design of biocatalysts because it is cost effective and readily available in commercial quantities [38]. Since the enzyme activity, stability and particle size of the resulting magnetic CLEAs are deeply affected by the ratio of cross-linker to enzyme [39], magnetic RML CLEAs were prepared using different concentrations of glutaraldehyde. As shown in Figure 7, the transesterification yield of the magnetic RML CLEAs was enhanced using increasing glutaraldehyde concentrations up to 1.6% (v/v). However, the transesterification yield decreased with the addition of excessive amounts of glutaraldehyde. This result implies that excessive glutaraldehyde might result in increasing enzyme rigidity due to a more intensive cross-linking. This restricts enzyme flexibility and influences the active site availability, thus decreasing the activity of magnetic RML CLEAs.

2.2.3. Effect of Lipase-to-Nanoparticle Ratio To achieve effective immobilization, the transesterification yield was determined with the lipase-to-nanoparticle weight ratio varying from 1:2 to 2.5:1 during magnetic CLEA preparation. Accordingly, as shown in Figure 8, the transesterification yields decreased as the weight ratio of lipase and nanoparticle increased. A higher concentration of enzyme naturally leads to a higher enzyme loading amount. However, the loading efficiency decreases with increasing enzyme concentration. In order to maximize efficient utilization of magnetic nanoparticles, the optimal lipase-

Molecules 2017, 22, 2157 7 of 16 packed into an active and insoluble form. Glutaraldehyde is a powerful cross-linker that is widely used in the design of biocatalysts because it is cost effective and readily available in commercial quantities [38]. Since the enzyme activity, stability and particle size of the resulting magnetic CLEAs are deeply affected by the ratio of cross-linker to enzyme [39], magnetic RML CLEAs were prepared using different concentrations of glutaraldehyde. As shown in Figure7, the transesterification yield of the magnetic RML CLEAs was enhanced using increasing glutaraldehyde concentrations up to 1.6% (v/v). However, the transesterification yield decreased with the addition of excessive amounts of Molecules 2017, 22, 2157 7 of 15 glutaraldehyde. This result implies that excessive glutaraldehyde might result in increasing enzyme rigidityto-nanoparticle due to a weight more intensive ratio of cross-linking.1:1 was selected This as restricts the most enzyme appropriate flexibility option and influences for magnetic the activeRML siteCLEA availability, preparation. thus decreasing the activity of magnetic RML CLEAs.

Molecules 2017, 22, 2157 7 of 15 to-nanoparticle weight ratio of 1:1 was selected as the most appropriate option for magnetic RML CLEA preparation.

Figure 7. Effect of glutaraldehyde concentration on the on the activities of magnetic RML CLEAs. Figure 7. Effect of glutaraldehyde concentration on the on the activities of magnetic RML CLEAs.

2.2.3. Effect of Lipase-to-Nanoparticle Ratio To achieve effective immobilization, the transesterification yield was determined with the lipase-to-nanoparticle weight ratio varying from 1:2 to 2.5:1 during magnetic CLEA preparation. Accordingly, as shown in Figure8, the transesterification yields decreased as the weight ratio of lipase and nanoparticle increased. A higher concentration of enzyme naturally leads to a higher enzyme loading amount. However, the loading efficiency decreases with increasing enzyme concentration. In order to maximize efficient utilization of magnetic nanoparticles, the optimal lipase-to-nanoparticle weightFigure ratio of7. Effect 1:1 was of glutaraldehyde selected as the concentration most appropriate on the optionon the activities for magnetic of magnetic RML CLEARML CLEAs. preparation.

Figure 8. Effect of the lipase-to-nanoparticle ratio on the activity of magnetic RML CLEAs.

2.3. Surfactants Activated Lipase Magnetic CLEAs RML is a stable and widely used lipase with a molecular size of 31,600 Da and an isoelectric point (pI) of 3.8. The structure of RML revealed a Ser-His-Asp trypsin-like catalytic triad with an active serine buried under a 15 amino acid long “lid” [40]. In the closed conformation, the lid covers the active site, thereby blocking the access of substrate molecules [3]. Interaction with a hydrophobic phase can cause opening of the lid, making the active site accessible. Moreover, considering that RML tends to form bimolecular aggregates with reduced activity, and the presence of detergents may break these dimers, the use of surfactant-coated procedure may be a good option to create interfacially activated and monomeric RML molecules. Figure 8. Effect of the lipase-to-nanoparticle ratio on the activity of magnetic RML CLEAs. To fix the lipase in an “open conformation”, an integrated strategy of interfacial activation and 2.3.covalent Surfactants cross-linking Activated immobilization Lipase Magnetic was CLEAs developed, and surfactant-coated RML was sequentially used for magnetic CLEAs preparation. As shown in Figure 9, the presence of AOT or Tween 80 during RML is a stable and widely used lipase with a molecular size of 31,600 Da and an isoelectric the magnetic CLEAs preparation promotes the activity of magnetic RML CLEAs to varying degrees point (pI) of 3.8. The structure of RML revealed a Ser-His-Asp trypsin-like catalytic triad with an compared to the free RML and RML CLEAs. When Tween 80 was used, magnetic RML CLEAs were active serine buried under a 15 amino acid long “lid” [40]. In the closed conformation, the lid covers more active at lower concentrations (0.5 to 1.5 mM). This decreased with increasing concentrations, the active site, thereby blocking the access of substrate molecules [3]. Interaction with a hydrophobic suggesting that Tween 80 could not provide effective activation of RML. Meanwhile, the activity of phase can cause opening of the lid, making the active site accessible. Moreover, considering that RML magnetic RML CLEAs significantly increased with the addition of AOT, indicating that AOT was the tends to form bimolecular aggregates with reduced activity, and the presence of detergents may optimal amphiphile for the activation of RML, and that the optimum concentration was 4.0 mM. break these dimers, the use of surfactant-coated procedure may be a good option to create Under the optimal conditions, the activity recovery of the AOT-activated magnetic RML CLEAs interfacially activated and monomeric RML molecules. was as high as 2058%, with a 20-fold improvement over the free RML. As is well known, the To fix the lipase in an “open conformation”, an integrated strategy of interfacial activation and mechanism for improving the activity and stability of the immobilized lipase is extremely covalent cross-linking immobilization was developed, and surfactant-coated RML was sequentially complicated. The significant increase in enzyme activity is not only related to the immobilization used for magnetic CLEAs preparation. As shown in Figure 9, the presence of AOT or Tween 80 during method, but also to the unique characteristics of lipase, as well as the carrier properties. The active the magnetic CLEAs preparation promotes the activity of magnetic RML CLEAs to varying degrees compared to the free RML and RML CLEAs. When Tween 80 was used, magnetic RML CLEAs were more active at lower concentrations (0.5 to 1.5 mM). This decreased with increasing concentrations, suggesting that Tween 80 could not provide effective activation of RML. Meanwhile, the activity of magnetic RML CLEAs significantly increased with the addition of AOT, indicating that AOT was the optimal amphiphile for the activation of RML, and that the optimum concentration was 4.0 mM. Under the optimal conditions, the activity recovery of the AOT-activated magnetic RML CLEAs was as high as 2058%, with a 20-fold improvement over the free RML. As is well known, the mechanism for improving the activity and stability of the immobilized lipase is extremely complicated. The significant increase in enzyme activity is not only related to the immobilization method, but also to the unique characteristics of lipase, as well as the carrier properties. The active

Molecules 2017, 22, 2157 8 of 16 Molecules 2017, 22, 2157 8 of 15

2.3.centers Surfactants of most Activated lipases Lipaseare covered Magnetic by CLEAs a so-called “lid” structure, which controls access of the substratesRML isto a stablethe active and widely site. The used secondary lipase with ast molecularructure of size the of lipase 31,600 probably Da and an changes isoelectric during point (pI)immobilization, of 3.8. The structure and the lid of RMLmight revealed be opened a Ser-His-Asp to some extent trypsin-like for the substrates. catalytic triadThis would with an provide active serineeasier buriedaccess underby the a 15substrates, amino acid leading long “lid”to an [ 40increase]. In the in closed lipase conformation, activity. Molecular the lid coversdynamics the activesimulations site, thereby of the blockingRML lid thepostulat accessed of that, substrate among molecules other interactions, [3]. Interaction Arg86 with within a hydrophobic the lid stabilized phase canthe open-lid cause opening conformation of the lid, of makingthe protein the through active site multiple accessible. hydrogen Moreover, bonds considering to the protein that surface RML tends [41]. toMoreover, form bimolecular the electrostatic aggregates interactions with reduced of Arg86 activity, play andan important the presence role of in detergents terms of both may the break intrinsic these dimers,stability the and use displacement of surfactant-coated of the lid. procedure This action may occurs be a through good option enhancement to create interfaciallyof hinge mobility activated in a andhigh monomeric dielectric medium RML molecules. [42]. Therefore, the interaction between the anionic head group in the AOT moleculeTo ﬁx and the the lipase positively in an “open charged conformation”, Arg86 residue an integratedmight be able strategy to shift of the interfacial lipase conformational activation and covalentequilibrium cross-linking toward the immobilization open form, and was stabilize developed, the open and forms surfactant-coated of RML. Furthermore, RML was the sequentially coating of usedanionic for AOT magnetic could CLEAs provide preparation. a better micro-aqueous As shown in Figure environment9, the presence for lipase of AOT molecules. or Tween It 80 may during also thecause magnetic greater CLEAs dispersion preparation and decrease promotes agglomeration the activity ofby magnetic exerting RML a smaller CLEAs effect to varying on the degrees three- compareddimensional to thestructure free RML of negatively and RML CLEAs.charged Whenlipase. Tween These 80coated was used,molecules magnetic tend RMLto offer CLEAs improved were moreenzyme active activity. at lower Furthermore, concentrations APTES (0.5 functionalized to 1.5 mM). This ma decreasedgnetic nanoparticles with increasing can provide concentrations, a larger suggestingsurface area that and Tween sufficient 80 could active not sites, provide resultin effectiveg in activationhigher immobilization of RML. Meanwhile, efficiency the and activity larger of magneticcontact area RML between CLEAs signiﬁcantlythe substrate increased and the withenzyme the. addition This can of effectively AOT, indicating decrease that mass AOT wastransfer the optimalresistance, amphiphile leading to for higher the activation enzyme activity. of RML, and that the optimum concentration was 4.0 mM.

Figure 9. Effect of activation of different surfactantssurfactants onon activityactivity ofof magneticmagnetic RMLRML CLEAs.CLEAs.

2.4. Application in Biodiesel Production Under the optimal conditions, the activity recovery of the AOT-activated magnetic RML CLEAs was as high as 2058%, with a 20-fold improvement over the free RML. As is well known, 2.4.1. Effect of Organic Solvents on Biodiesel Production the mechanism for improving the activity and stability of the immobilized lipase is extremely complicated.To estimate The the signiﬁcant practical application increase in of enzyme immobilized activity RML, is not AOT-activated only related tomagnetic the immobilization RML CLEAs method,were also but investigated also to the as unique efficient characteristics nano-biocatalysts of lipase, for asenzymatic well as the transesterification carrier properties. in production The active centersof biodiesel of most from lipases jatropha are covered oil. A byseries a so-called of experiments “lid” structure, was performed which controls to determine access of the the substrates optimal toconditions the active for site. fatty The acid secondary methyl ester structure (FAME) of theprod lipaseuction. probably This result changes suggested during that immobilization, activation of andsurfactants the lid mightbefore be immobilization opened to some could extent anchor for the the substrates. lipase in Thisan activated would provide state, thus easier keeping access it by in the an substrates,active form leading for subsequent to an increase application. in lipase activity. Molecular dynamics simulations of the RML lid postulatedThe reaction that, among medium other plays interactions, a significant Arg86 role within in ma theintaining lid stabilized the catalytic the open-lid activity conformation and stability ofof thean enzyme. protein through In order multiple to select hydrogenthe suitable bonds medium to the for protein the lipase surface reaction, [41]. Moreover, the production the electrostatic of FAMEs interactionswas performed of Arg86 in various play an conventional important role organic in terms solvents of both at the 40 intrinsic °C and stabilitythe results and compared displacement to the of theresults lid. obtained This action from occurs free RML. through Interestingly, enhancement AOT-activated of hinge mobility magnetic in a RML high CLEAs dielectric produced medium yields [42]. Therefore,greater than the 20% interaction in all of betweenthe anhydrous the anionic solvents head (except group for in thetoxic AOT 1,4-dioxane molecule and and THF), the positively whereas chargedlittle product Arg86 was residue observed might when be able free to RML shift was the used lipase (Figure conformational 10). The solubility equilibrium of jatropha toward oil the is much open higher in tert-butanol, t-BME and isopropyl ether, resulting in a decrease in mass transfer resistance of the substrates and substantial enhancement of FAME yields. Meanwhile, the hydrophilic THF and 1,4-dioxane could more easily strip away the essential water bound to the lipase by participating in

Molecules 2017, 22, 2157 9 of 16 form, and stabilize the open forms of RML. Furthermore, the coating of anionic AOT could provide a better micro-aqueous environment for lipase molecules. It may also cause greater dispersion and decrease agglomeration by exerting a smaller effect on the three-dimensional structure of negatively charged lipase. These coated molecules tend to offer improved enzyme activity. Furthermore, APTES functionalized magnetic nanoparticles can provide a larger surface area and sufﬁcient active sites, resulting in higher immobilization efﬁciency and larger contact area between the substrate and the enzyme. This can effectively decrease mass transfer resistance, leading to higher enzyme activity.

2.4. Application in Biodiesel Production

2.4.1. Effect of Organic Solvents on Biodiesel Production To estimate the practical application of immobilized RML, AOT-activated magnetic RML CLEAs were also investigated as efficient nano-biocatalysts for enzymatic transesterification in production of biodiesel from jatropha oil. A series of experiments was performed to determine the optimal conditions for fatty acid methyl ester (FAME) production. This result suggested that activation of surfactants before immobilization could anchor the lipase in an activated state, thus keeping it in an active form for subsequent application. The reaction medium plays a significant role in maintaining the catalytic activity and stability of an enzyme. In order to select the suitable medium for the lipase reaction, the production of FAMEs was performed in various conventional organic solvents at 40 ◦C and the results compared to the results obtained from free RML. Interestingly, AOT-activated magnetic RML CLEAs produced yields greater than 20% in all of the anhydrous solvents (except for toxic 1,4-dioxane and THF), whereas little product was observed when free RML was used (Figure 10). The solubility of jatropha oil is much higher in tert-butanol, t-BME and isopropyl ether, resulting in a decrease in mass transfer resistance of theMolecules substrates 2017, 22, and2157 substantial enhancement of FAME yields. Meanwhile, the hydrophilic THF9 andof 15 1,4-dioxane could more easily strip away the essential water bound to the lipase by participating in non-covalent solvent protein interactions, resulting inin distortion of their active conformation and thus loss ofof their their activity. activity. This This substantial substantial enhancement enhancemen suggestedt suggested that surfactant that surfactant activation activation can be combined can be withcombined covalent with cross-linking covalent cross-linking to anchor the to lipaseanchor in th ane openlipase active in an form,open thusactive enhancing form, thus their enhancing stability intheir polar stability solvents. in polar As depictedsolvents. inAs Figure depicted9, the in highestFigure 9, yield the highest of FAMEs yield was of obtainedFAMEs was in tertobtained-butanol in fortert-butanol both free for (47%) both and free immobilized (47%) and RMLimmobilized (93%). ItRML has been(93%). reported It has been that biodieselreported productionthat biodiesel in theproduction presence in of thetert -butanolpresence improvesof tert-butanol the solubility improves of methanolthe solubility and reducesof methanol the inhibitory and reduces effect the of glycerol,inhibitory therefore effect of improvingglycerol, therefor the yielde improving of reaction the [43 yield]. of reaction [43].

Figure 10. Effect of different solvents on AOT-activated magnetic RML CLEAs catalyzed biodiesel production from jatrophajatropha oil.oil.

2.4.2. Effect of Temperatures on Biodiesel Production As temperature is an important factor to be considered in the enzymatic preparation of biodiesel, biodiesel production was carrying out in isopropyl ether and tert-butanol at different temperatures (30–60 °C). According to Figure 11, AOT-activated magnetic RML CLEAs were more active in the range of 30–60 °C than free RML, either in isopropyl ether or tert-butanol. Increasing lipase stability after immobilization can be attributed to rigidification of the lipase structure, which prevents unfolding of the protein. As the temperature gradually increases from 30 to 60 °C, there is a rise in the biodiesel yield catalyzed by immobilized RML in isopropyl ether. However, the optimum temperature of the free form was found to be 50 °C in the same medium. Meanwhile, when tert- butanol is used as the solvent, the optimum temperature for AOT-activated magnetic RML CLEAs was found to be 40 °C, with biodiesel yields of about 93%. Based on the results, 40 °C was selected for further studies.

Figure 11. Effect of temperatures on AOT-activated magnetic RML CLEAs-catalyzed biodiesel production from jatropha oil in isopropyl ether and tert-butanol.

Molecules 2017, 22, 2157 9 of 15

non-covalent solvent protein interactions, resulting in distortion of their active conformation and thus loss of their activity. This substantial enhancement suggested that surfactant activation can be combined with covalent cross-linking to anchor the lipase in an open active form, thus enhancing their stability in polar solvents. As depicted in Figure 9, the highest yield of FAMEs was obtained in tert-butanol for both free (47%) and immobilized RML (93%). It has been reported that biodiesel production in the presence of tert-butanol improves the solubility of methanol and reduces the inhibitory effect of glycerol, therefore improving the yield of reaction [43].

Figure 10. Effect of different solvents on AOT-activated magnetic RML CLEAs catalyzed biodiesel production from jatropha oil. Molecules 2017, 22, 2157 10 of 16 2.4.2. Effect of Temperatures on Biodiesel Production 2.4.2.As Effect temperature of Temperatures is an important on Biodiesel factor Production to be considered in the enzymatic preparation of biodiesel, biodieselAs temperature production iswas an importantcarrying out factor in isopropyl to be considered ether and in thetert enzymatic-butanol at preparation different temperatures of biodiesel, biodiesel(30–60 °C). production According was to Figure carrying 11, out AOT-activate in isopropyld ethermagnetic and RMLtert-butanol CLEAs at were different more temperatures active in the (30–60range of◦C). 30–60 According °C than to free Figure RML, 11, either AOT-activated in isopropyl magnetic ether RMLor tert CLEAs-butanol. were Increasing more active lipase in the stability range ofafter 30–60 immobilization◦C than free RML,can be either attributed in isopropyl to rigidifi ethercation or tert of-butanol. the lipase Increasing structure, lipase which stability prevents after immobilizationunfolding of the can protein. be attributed As the temperature to rigidiﬁcation graduall of they lipase increases structure, from 30 which to 60 prevents °C, there unfolding is a rise ofin the protein.biodiesel As yield the temperaturecatalyzed by gradually immobilized increases RML from in isopropyl 30 to 60 ◦ C,ether. there However, is a rise in the the optimum biodiesel yieldtemperature catalyzed of bythe immobilized free form was RML found in isopropyl to be 50 ether.°C in However,the same medium. the optimum Meanwhile, temperature when of tert the- freebutanol form is wasused found as the to solvent, be 50 ◦ Cthe in optimum the same medium.temperature Meanwhile, for AOT-activated when tert -butanolmagnetic isRML used CLEAs as the solvent,was found the to optimum be 40 °C, temperature with biodiesel for yields AOT-activated of about magnetic93%. Based RML on the CLEAs results, was 40 found °C was to beselected 40 ◦C, withfor further biodiesel studies. yields of about 93%. Based on the results, 40 ◦C was selected for further studies.

Figure 11.11. EffectEffect of of temperatures on AOT-activated magnetic RML CLEAs-catalyzedCLEAs-catalyzed biodieselbiodiesel production fromfrom jatrophajatropha oiloil inin isopropylisopropyl etherether andand tert-butanol.tert-butanol.

2.4.3. Reusability One of objectives of using an immobilized enzyme is to design a more efficient biocatalyst that can easily be recovered and reused. Magnetic immobilization offers the benefits of easy separation of enzyme from the reaction system, allowing for consequent reuse of enzyme, as well as the potential to run a continuous process. In this work, the reusability of AOT-activated magnetic RML CLEAs was evaluated in consecutive batches of the biodiesel reaction in isopropyl ether and tert-butanol, respectively. After completion of each batch, the immobilized enzyme was recovered by magnetic separation and washed with solvent to prepare it for the subsequent batch. The initial activity prior to the first recovery was taken as 100%, and the activity in the subsequent reactions was calculated accordingly. As presented in Figure 12, AOT-activated magnetic RML CLEAs performed well with repetition up to 5 cycles, with 84% and 81% of activity retained in isopropyl ether and tert-butanol respectively. This implies that the immobilized RML possesses excellent long-term operational stability. It is noteworthy that the AOT-activated magnetic RML CLEAs displayed a fast response (20 s) to an external magnetic field, which indicated that this level of saturation magnetization would be adequate for the separation of the immobilized enzyme. Molecules 2017, 22, 2157 10 of 15

2.4.3. Reusability One of objectives of using an immobilized enzyme is to design a more efficient biocatalyst that can easily be recovered and reused. Magnetic immobilization offers the benefits of easy separation of enzyme from the reaction system, allowing for consequent reuse of enzyme, as well as the potential to run a continuous process. In this work, the reusability of AOT-activated magnetic RML CLEAs was evaluated in consecutive batches of the biodiesel reaction in isopropyl ether and tert-butanol, respectively. After completion of each batch, the immobilized enzyme was recovered by magnetic separation and washed with solvent to prepare it for the subsequent batch. The initial activity prior to the first recovery was taken as 100%, and the activity in the subsequent reactions was calculated accordingly. As presented in Figure 12, AOT-activated magnetic RML CLEAs performed well with repetition up to 5 cycles, with 84% and 81% of activity retained in isopropyl ether and tert-butanol respectively. This implies that the immobilized RML possesses excellent long-term operational stability. It is noteworthy that the AOT-activated magnetic RML CLEAs displayed a fast response (20 s) to an Moleculesexternal2017 magnetic, 22, 2157 field, which indicated that this level of saturation magnetization would11 of be 16 adequate for the separation of the immobilized enzyme.

Figure 12. Reuse of AOT-activated magnetic RML CLEAs for biodiesel production from jatropha oil Figure 12. Reuse of AOT-activated magnetic RML CLEAs for biodiesel production from jatropha oil in in isopropyl ether and tert-butanol. isopropyl ether and tert-butanol.

3. Materials and Methods 3. Materials and Methods

3.1. Materials Lipase from Rhizomucor miehei (solution) and methyl ester standards (methyl palmitate, methyl stearate, methylmethyl oleate, oleate, methyl methyl linoleate, linoleate, and and methyl methyl tridecanoate) tridecanoate) were purchasedwere purchased from Sigma-Aldrich from Sigma- (St.Aldrich Louis, (St. MO, Louis, USA). MO, Substrates USA). containingSubstrates 3-aminopropyl containing 3-aminopropyl triethoxysilane triethoxysilane (APTES), glutaraldehyde (APTES), (25%,glutaraldehydev/v) and 2-phenyl (25%, v/v ethanol) and 2-phenyl (>98%, CP)ethanol were (>98%, purchased CP) were from purchased Aladdin (shanghai, from Aladdin China). (shanghai, Sodium bis-2-(ethylhexyl)China). Sodium bis-2-(ethylhexyl) sulfosuccinate (AOT) sulfosuccinate were obtained (AOT) from were Acros obtained (Pittsburgh, from Acros PA, USA). (Pittsburgh,Jatropha PA,oil wasUSA). obtained Jatropha from oil was Yanyuan obtained County, from Sichuan Yanyuan Province, County, China. Sichuan All Province, other chemicals China. wereAll other of analytical chemicals or chromatographicalwere of analytical gradesor chromatographical and used without grades further puriﬁcation.and used without Double furthe distilledr purification. water was employed Double throughoutdistilled water the was experiments. employed throughout the experiments.

3.2. Functionalization of Magnetic Nanoparticles

3 4 Magnetic Fe3OO4 nanoparticlesnanoparticles were were prepared prepared by the conventional co-precipitation method. In a 2 2 3 2 typical procedure, 1.20 gg (6.0(6.0 mmol)mmol) ofof FeClFeCl2··4H4H2O and 3.25 gg (12.0(12.0 mmol)mmol) ofof FeClFeCl3··6H6H2O were dissolved in 50 mL deionized water under nitrogen at room temperature. Then, 10 ml 25% ammonia solution was added dropwise under a nitrogen atmosphereatmosphere with vigorous stirring, resulting in the immediate formation of black precipitates. After After agin agingg in in the mother solution for 3 h, the obtained magnetic particles particles were were washed washed several several times times with with deionized deionized water water until untilneutral, neutral, and dried and at dried 100 °C at 100for 2◦ Ch. forTo obtain 2 h. To amino obtain functionalized amino functionalized magnetic particles, magnetic magnetic particles, nanoparticles magnetic nanoparticles were suspended were suspendedin a solution in composed a solution composedof 2.5 mL methanol, of 2.5 mL methanol,100 μL of 1003-aminopropylµL of 3-aminopropyl trimetoxysilane trimetoxysilane and 25 μL and of 25 µL of deionozed water. The resulting mixture was homogenized by ultrasonication for 30 min.

Then glycerol (1.5 mL) was introduced into the solution and allowed to reﬂux at 90 ◦C for 6 h with maximum mechanical agitation. Finally, the APTES-Fe3O4 nanoparticles were washed several times with methanol and deionized water, magnetically separated, and subsequently lyophilized prior to use.

3.3. Immobilization of Lipase

3.3.1. Preparation of RML CLEAs RML CLEAs were prepared according to the procedure reported by Jia et al. [33]. Firstly, 5 mL of saturated ammonium sulphate solution was added into 1 mL of RML solution (10 mg/mL, 0.1 M phosphate buffer, pH 7.0), and stirred for 30 min at 4 ◦C. After precipitation of RML, glutaraldehyde was added slowly to the ﬁnal concentration of 1.6% v/v, and stirred for 3 h at 30 ◦C. After cross-linking, 4 mL of phosphate buffer (0.1 M, pH 7.0) was used to dilute the suspension, and the mixture was then Molecules 2017, 22, 2157 12 of 16 centrifuged at 10,000 rpm for 5 min. The resultant precipitates were washed thrice with phosphate buffer and deionized water, lyophilized and ﬁnally stored at 4 ◦C.

3.3.2. Preparation of Magnetic RML CLEAs

Magnetic RML CLEAs were produced by mixing with 10 mg of APTES-Fe3O4 nanoparticles and 1 mL of RML solution (10 mg/mL, 0.1 M phosphate buffer, pH 7.0) and shaken for 15 min at 30 ◦C. Then 5 mL of precipitant was added with stirring at 4 ◦C for 30 min. After precipitation, glutaraldehyde was added dropwise into the suspension, and stirred for 3 h at 30 ◦C. All subsequent procedures were the same as those for the RML CLEAs preparation.

3.3.3. Preparation of Surfactants-Activated Magnetic RML CLEAs The surfactant-activated RML was prepared using anionic Aerosol OT (Sodium bis-2-(ethylhexyl) sulfosuccinate, AOT) and nonionic Tween 80 at various concentrations. Firstly, 1 mL of RML solution and an appropriate amount of surfactant were mixed and stirred at 4 ◦C for 30 min. After incubation for 24 h at 4 ◦C, the suspended solution was sequentially used for magnetic RML CLEAs preparation. Free RML, RML CLEAs and magnetic RML CLEAs served as controls.

3.4. Assay of Immobilized RML

3.4.1. Activity Assay The activities of free lipase and different immobilized preparations were determined by the transesteriﬁcation reaction of 2-phenyl ethanol and vinyl acetate. Firstly, 10 mg of 2-phenylethanol was mixed with 1 mL of vinyl acetate. To start the reaction, 10 mg of RML (the initial amount of RML CLEAs and magnetic RML CLEAs was 10 mg) was added, and the mixture was reacted in a temperature-controlled shaker at 30 ◦C, 220 rpm for 24 h. The reaction was terminated by the isolation of immobilized lipase using a magnet. The samples were withdrawn from the reaction medium at regular intervals and analyzed by high-performance liquid chromatography (HPLC). All experiments were repeated at least three times.

3.4.2. Biodiesel Production To evaluate the effectiveness of immobilized lipase catalyzed jatropha oil bioconversion, the transesteriﬁcation experiments were conducted as follows. In a typical experiment, the reaction was performed in a 10 mL screw-capped vessel containing 2.0 mL solvent, 0.5 g of jatropha oil and anhydrous methanol, at oil-to-methanol molar ratio of 1:3. The reaction was initiated by the addition of 20 mg RML (the initial amount of RML CLEAs and magnetic RML CLEAs was 20 mg). Then the mixture was incubated in a temperature-controlled shaker at 40 ◦C, 220 rpm for 48 h. All biodiesel reactions were performed in dried solvents without any water added. Aliquots (20 µL) of the reaction mixture were withdrawn at various time intervals throughout the reaction time, and then diluted with n-hexane for GC analysis. Effects of solvents and reaction temperatures on the biodiesel production were studied by single-factor experiment design.

3.4.3. Reusability The enzyme reuse was evaluated in the transesteriﬁcation reaction of jatropha oil with methanol. Upon completion of one cycle, the immobilized derivative was separated by a permanent magnet, washed thrice with solvent, and then re-suspended in a fresh reaction mixture for the next catalytic cycle. The biodiesel yield of the ﬁrst reaction was set as 100% and the ester yield in the subsequent reactions was calculated accordingly. Molecules 2017, 22, 2157 13 of 16

3.4.4. HPLC Analysis HPLC was conducted with Waters Associates equipment (Waters 2695 with 2998 Photodiode Array Detector). A C18 column was used in the HPLC experiments with MeOH/water = 80:20 (v/v). The wavelength of the UV detector was set at 254 nm, the column temperature was maintained at 30 ◦C during the assays, and the ﬂow rate was 0.8 mL/min.

3.4.5. GC Analysis The fatty acid methyl ester (FAMEs) content of the reaction mixture was quantiﬁed using a Fuli9790 gas chromatography (Fuli, China) equipped with an AT.SE-54 column (30 m × 0.25 mm × 0.33 µm). Nitrogen was used as the carrier gas at a constant ﬂow of 1.5 mL/min. The column temperature was held at 160 ◦C for 2 min, and raised at 10 ◦C/min to 240 ◦C, where it was then maintained for 10 min. The temperatures of the injector and the detector were set at 280 and 270 ◦C, respectively. By comparing the retention times and peak areas of standard fatty acid methyl ester peaks, the total quantities of biodiesel in the reaction mixtures were calculated.

3.5. Characterization

3.5.1. FT-IR The Fourier transform infrared (FT-IR) analysis was carried out using Shimadzu FTIR-4200 spectrometer in the range of 400 to 4000 cm−1. A standard KBr pellet technique was applied for the sample preparation.

3.5.2. SEM The morphology of the particle surface was observed using a scanning electron microscope (SEM, JSM 7500F; JEOL, Tokyo, Japan). The capsule was freeze-dried and coated with gold before it was analyzed.

3.5.3. Magnetization Measurements The magnetic properties of the magnetic nanoparticles and different immobilized RML preparations were detected at room temperature using a vibrating sample magnetometer (VSM, MicroSense EZ9, Lowell, MA, USA).

4. Conclusions In the present study, lipase from Rhizomucor miehei was activated with surfactant and used in preparation of magnetic CLEAs in order to obtain active hybrid catalysts for the transesterification of jatropha oil with methanol to produce fatty acid methyl esters (FAMEs). Since the coating of surfactant fixes RML in its open form, the activity of the final derivatives increased remarkably compared to the free RML. The nature of the precipitant and surfactant, as well as the concentration of glutaraldehyde, are the key factors affecting the immobilization of RML. Under the optimal conditions, the AOT-activated magnetic RML CLEAs displayed 20-fold improvement in transesterification activity compared with the free RML. This increased activity is attributed to the enhanced interfacial activation of lipase due to the interaction between lipase and appropriate surfactant coating. Moreover, the immobilized RML showed excellent catalytic performance for the biodiesel reaction and, more importantly, could be easily separated from the reaction mixture by simple magnetic decantation. It also retained more than 84% of its initial activity after five instances of reuse. The interfacial activation favors the open conformation of lipase, and consequently the substrate availability for the immobilized enzyme. Therefore, the combination of interfacial activation, magnetic nanoparticles, and cross-linked enzyme aggregates in lipase immobilization has high potential for industrial application, and presents an attractive potential process for other lipase immobilization in the future. Molecules 2017, 22, 2157 14 of 16

Acknowledgments: We gratefully acknowledge financial support of the Natural Science Foundation of Ningxia (No. NZ1645), the Major Innovation Projects for Building First-class Universities in China’s Western Region (No. ZKZD2017003), the Scientific Research Foundation of the Higher Education Institutions of Ningxia (No. NGY2017045), and the Scientific Research Start Funds of Ningxia University Talent Introduction (No. BQD2015012). We thank the Comprehensive Training Platform of the Specialized Laboratory of College of Chemistry of SCU for sample analysis. The Key Laboratory of Green Chemistry and Technology of the Ministry of Education of College of Chemistry of SCU are greatly appreciated for the biomass offer and analysis. We thank Ma Zhi, from School of Physics & Electronic-Electrical Engineering, Ningxia University for magnetization measurements. Author Contributions: Weiwei Zhang designed the experimental scheme and did most of the sample preparation and characterizations. Huixia Yang helped with the experiment. Weiwei Zhang, Wanyi Liu, Na Wang and Xiaoqi Yu contributed in the discussion, revision, and editing of the manuscript. All authors reviewed the main manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds are not available from the authors.

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