Effects of Additives on Lipase Immobilizationin Microemulsion-Based Organogels

Wei-Wei Zhang & Na Wang & Ling Zhang &

Wan-Xia Wu & Cheng-Li Hu & Xiao-Qi Yu

Received: 1 October 2013 /Accepted: 20 January 2014# Springer Science+Business Media New York 2014

Abstract An inexpensive, facile, and environmentally benign method was developed toimprove the activity and stability of Candida rugosa lipase (triacylglycerol acylhydrolase)immobilized on microemulsion-based organogels (CRL MBGs) via the addition of additivesduring immobilization. The additives used were polyethylene glycol (PEG) or polysaccha-rides. This study is the first report on the effect of additives in CRL MBGs. Among the testedadditives, PEG produced the most improvement in the immobilized CRL, enhancing itsstability in organic solvents (specifically polar solvents). The results of circular dichroismand fluorescence spectra experiments indicated that exposure of the acidic CRL to electroneg-ative additives in the buffer, such as polyethylenimine and the electropositive surfactantcetyltrimethylammonium bromide, may change the lipase secondary structure, ultimatelycausing enzyme inactivation. However, sodium bis(2-ethylhexyl)sulfosuccinate and PEG2000 had minimal effects on the secondary structure of CRL. The CRL MBGs containingPEG 2000 demonstrated remarkable retention of their catalytic activity during the recyclingtest. No significant changes in enzymatic activity were observed, even after nine runs, and90 % of the original yield was maintained after 15 cycles.

Keywords Lipase . Immobilization . Additive . Stability .MBGs

Introduction

Enzymes have been used extensively as biocatalysts in a number of biochemical processes forvarious industrial purposes, including pharmaceutical, food, flavor, and agrochemical appli-cations [1–4]. However, their lack of reusability and low stability has generally been consid-ered barriers to the development of continuous operations and large-scale applications.Enzyme immobilization is the most commonly used strategy to overcome these drawbacks,as it can efficiently improve the enzyme activity [5–8]. Various methodologies of enzyme

Appl Biochem BiotechnolDOI 10.1007/s12010-014-0746-0

W.<W. Zhang : N. Wang (*) : L. Zhang :W.<X. Wu : C.<L. Hu :X.<Q. Yu (*)Key Laboratory of Green Chemistry and Technology, Ministry of Education, College of Chemistry, SichuanUniversity, Chengdu 610064, People’s Republic of Chinae-mail: wnchem@scu.edu.cne-mail: xqyu@scu.edu.cn

immobilization, including adsorption, ionic binding, covalent modification, entrapment, andencapsulation, have been established by previous studies [9, 10].

Entrapment protects an enzyme by preventing direct contact with its substrates and thesurrounding environment, thereby preventing any negative influence on the enzyme structure.Of the several entrapment methods that have been developed, the sol–gel method is the mostprominent and widely used technique [11, 12]. The immobilization of enzymes inmicroemulsion-based organogels (MBGs) has become a more attractive approach [13], asthe relatively inert aqueous environment within the matrix causes little damage to the structureof the native enzyme [14]. In a previous study by Zhang et al. [15], sodium bis(2-ethylhexyl)sulfosuccinate (AOT)-coated Candida rugosa lipase (CRL; triacylglycerolacylhydrolase, E.C. 3.1.1.3, type VII) immobilized in microemulsion-based organogels(MBGs) was used for the synthesis of arylethyl acetate in organic solvents. To further improvethe activity and operational stability of this CRL MBGs for its potential use in continuousapplications, additives were introduced to the gelation process. These additives were intendedto protect the enzyme from external denaturing agents by forming a shield between the proteinand its reactive environment. The application of protective agents is a simple, fast, andeconomic method for improving the stability of an enzyme [16–18]. Similarly, biocatalystactivity can be significantly enhanced by immobilization in the presence of additives [19–21].Several substances have been used as additives, including surfactants [22], polyhydric alcohols[23], methyl esters [24], metal ions [25], and other chemical reagents [26]. These molecules arebelieved to function by providing additional sites for hydrogen bonding with the enzymesurface, decreasing dehydration, and providing thermodynamic barriers to unfolding [27].

Polyethylene glycol (PEG) is a key building block in research additives due to its biologicalproperties and unique physical attributes. As a neutral, water-soluble synthetic polymer, PEGis used in the biotechnology industry for its relatively nontoxic properties and high biocom-patibility. According to Soares et al. [21], PEG protects enzymes from denaturing effectswithout affecting their reaction rates. Therefore, in the present study, the effect of PEGs on thecatalytic activity of CRL MBGs was investigated. The effects of various reactiontemperatures, solvents, and storage durations on CRL MBG catalysis were analyzed.Selected critical properties of the additives, such as their category, additive loading,and addition sequence, were considered. CRL MBGs incorporating PEG 2000 weretested under a set of relevant process conditions in a batch system to further study themechanisms involved. This study is the first report on the effect of additives in CRLMBGs. Scanning electron microscopy (SEM) was used to study the MBG capsulesurface morphology. Circular dichroism and fluorescence spectra were used to evalu-ate the influence of additives on the advanced CRL structures, and changes in theobserved spectra of the additive-containing CRL formulations were noted.

Materials and Methods

Materials

AOT was obtained from Acros Organics (USA). Commercial CRL (type VII, 739 U/mg),gelatin (from porcine skin, type II), PEG, polyethylenimine (PEI), and sodium alginate werepurchased from Sigma-Aldrich (USA) and used without further purification. 2-Phenylethanoland β-cyclodextrin were purchased from Aladdin (China). All organic solvents and otherchemicals used were of analytical reagent grade. Double-distilled water was used throughoutthe experiments.

Appl Biochem Biotechnol

Preparation of CRL MBGs

The method for CRL MBG preparation is similar to the method described in a previous study[15]. Lipase (15 mg/mL), in the presence of 1 mM AOT, was incubated overnight at 4 °C inphosphate buffer (pH 7.2, 0.1 M). After 24 h, the lipase solution was added to a reversemicellar solutions of AOT/buffer/isooctane (Wo=60). The thermodynamically stable reversemicellar solution was prepared by mixing each component in a suitable ratio. Lipase-containing microemulsions were prepared by adding the previously prepared AOT-coatedCRL solution to the AOT reverse micellar solution with the appropriate water content in theisooctane. This solution was shaken briefly, immediately added to a second solution of 14 %(w/v) gelatin in a phosphate buffer (pH 7.2, 0.1 M) at 55 °C, stirred vigorously untilhomogeneous, and cooled to 25 °C. The gelatin was dissolved in a phosphate buffer,autoclaved, and cooled to 55 °C. The gel was then poured into plastic plates and kept overnightfor air drying. The dried gel was cut into smaller pieces on the following day and later used forthe transesterification reaction.

After sterilization, CRL MBGs with different additives were prepared by introducing theindividual additives at concentrations of 9.5 % (w/w) to the gelatin solution. The resultingsolutions were stirred until homogeneous. The lipase-containing microemulsion mixtures wereadded to their respective solutions. The lipase without additives served as the control setup.

The CRL MBGs with PEG 2000 were prepared in different sequences by mixing PEG2000 into the lipase solution (incubated overnight at 4 °C), the reverse micellar solution, andthe gelatin solution both before and after sterilization.

The CRL MBGs with different concentrations of PEG 2000 were prepared by mixing PEG2000 into the gelatin solution at concentrations of 1.7, 6.0, 9.5, 12.0, 14.3, 17.9, and 21.4 %(w/w).

Enzymatic Reaction Conditions

In addition to the experiments exploring the effects of solvents, other experiments wereperformed in solvent-free systems. CRL MBGs with and without additives were weighedand added to 2 mL of vinyl acetate containing 20 mg/mL 2-phenylethanol, unless otherwisenoted. The time course study was conducted at 50 °C for CRL, the as-produced CRL MBGs,and the CRL MBGs with different additives. This reaction was performed in 50 mL vinylacetate containing 2 mg/mL 2-phenylethanol. Each reaction mixture was incubated at 200 rpmin a temperature-controlled shaker at the desired temperature. Samples were withdrawn fromthe reaction medium at regular intervals and analyzed by high-performance liquid chromatog-raphy (HPLC). The transesterification reaction was conducted at a larger scale using 50 g 2-phenylethanol, as described in detail in “Results and Discussions”. All experiments wererepeated at least three times.

Tolerance to Solvents and Temperatures of the CRL MBGs

The CRL MBGs with and without different molecular weights of PEG were used fortransesterification in various organic solvents. Free CRL served as the control setup. Isopropylether, dioxane, toluene, tetrahydrofuran (THF), hexane, tert-butyl alcohol, acetone, and ace-tonitrile were selected as the solvents used in the different reaction systems.

The tolerance to temperature changes was studied by conducting the same reaction at sixdifferent temperatures: 10, 20, 30, 37, 50, and 60 °C. A control setup was maintained at eachtemperature, with free lipase as the catalyst.

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Circular Dichroism and Fluorescence Spectroscopy

The circular dichroism (CD) spectra were obtained using a Chirascan CD spectropolarimeter.Scans of the samples with and without additives were performed within the UV range of 180 to300 nm in 1-nm increments. Scanning was performed at a rate of 30 nm/min and atemperature of 25 °C. The sample-cell path length was fixed at 1.0 cm. The spectralvalues of the control were subtracted from the observed results. The CRL concentra-tion was 1.0 mg/mL (in 50 mM PBS, pH 7.2) in the setups with and withoutadditives (surfactant, 2.5×10−3 mM; polymer, 0.5 mg/mL). The samples were testedafter storage at 4 °C for 24 h.

The fluorescence excitation and emission spectra were obtained using a FluoroMax-4spectrofluorophotometer (Horiba Jobin Yvon). The same sample concentrations used in thecircular dichroism experiment were used in these experiments. Scans of the samples wereperformed in the UV range of 300 to 550 nm (λex=286 nm; slits, 5 nm/5 nm).

Morphological Analysis of the Capsule Surface

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

Reusability and Storage Stability of CRL MBGs

To analyze the reusability of the CRLMBGs, the transesterification reaction was performed asdescribed above. Upon completion of one cycle, the immobilized enzyme was then recoveredby filtration. The recovered CRL MBGs were washed three times with vinyl acetate to ensurethe complete removal of the product and substrates. The residual solvent was subsequentlyremoved using N2, and fresh solvent was reintroduced into the system. This procedure wasrepeated for several cycles.

Both the free and immobilized enzymes were stored at 4 °C and at room temperature. Thestorage stability of the enzymes was determined by comparing the sample activity at regulartime intervals.

Preparative-Scale Reaction

The preparative-scale experiments were performed in a 250-mL flask that was incubated withshaking at 200 rpm in a temperature-controlled shaker. 2-Phenylethanol (50 g) was added to100 mL of vinyl acetate and heated to 50 °C. PEG 2000-containing MBGs with 200 mg ofCRL were then added to the mixture.

Analytical Procedures

The quantitative analysis of the samples was performed by HPLC through a reverse-phase column (Welchrom-C18, 5 μm, 4.6 mm×150 mm; Welchrom) using aShimadzu LC-2010A HT apparatus equipped with a 254-nm UV detector. Methanolwith 30 % (v/v) water was used as the mobile phase at a split flow rate of 0.8 mL/min. The ester content was identified and quantified by comparing the retention timeand peak area of the reaction sample with the standard sample. Pure phenylethylacetate was used as the external standard.

Appl Biochem Biotechnol

Results and Discussions

Additives and Their Stabilizing Effect

The gel was doped with additives during gelation to preserve the CRL catalytic activity duringthe sol–gel process. With the exception of PEI, all other additives significantly enhanced thereaction yield, as shown in Fig. 1. In particular, the immobilized lipase containing PEGs withdifferent molecular weights demonstrated higher activity, with approximately 70 % yield,whereas the CRL MBGs without additives only produced 34 % yield. The presence of PEGduring the CRL immobilization process most likely affected the hydration level of CRL bymodifying the hydrophilicity of the lipase microenvironment. A similar mechanism wasproposed by Rocha et al. [28], and Guncheva and Zhiryakova [29] likewise demonstrated thatPEG 2000 had a beneficial effect on both the stability of CRL biocatalytic preparation andCRL activity. Moreover, the molecular weight of PEG affected the activity of the CRLMBGs.PEG 8000 most likely reduced the enzyme/substrate contact via diffusional limitation, whereasthe short chain of PEG 800 could not efficiently protect the CRL molecules.

Polyethyleneimine is a positively charged, branched polyelectrolyte (with a similar back-bone to that of PEG, a nonionic polymer) that can cause enzyme inactivation, although it hasvarious biotechnological applications. Previous studies have shown that PEI has a stabilizingeffect on several enzymes in solution [30, 31]. However, in the case of our experiment, PEIaddition led to lipase inactivation, and flocculates visible to the naked eye formed after CRLwas mixed with PEI. CRL is an acidic protein with an isoelectric point of 5.6. Consequentlythe electrostatic interactions between oppositely charged polymer/protein pairs affected theexisting protein-solvent and intramolecular protein interactions maintaining the CRL confor-mation, thereby decreasing the observed lipase activity. Mazzaferro et al. [32] reported that PEIfunctioned as a deactivator of acidic porcine heart lactate dehydrogenase (LDH), whichsupports the result of this study.

Carbohydrates are known to stabilize various proteins in naturally dehydrated environ-ments. During lyophilization, carbohydrates can preserve the crucial water layer at the surfaceof proteins and protect their secondary structure. In this study, polysaccharides such as

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Fig. 1 Time course of CRL MBGs with and without various additives. Superscript a indicates thattransesterification was carried out at 50 °C and 200 rpm for 12 h in vinyl acetate. 2-Phenylethanol concentrationwas 2 mg/mL

Appl Biochem Biotechnol

cellulose, oligosaccharides such as β-cyclodextrin, chitosan with different molecular weights,and sodium alginate were used as additives. These additives improved the activity of CRLMBGs to various degrees.

The immobilized enzymes with different amounts of PEG 2000 added were selected as thetemplates to explore the mechanisms by which additives influence enzyme activity. The effectof additive loading was studied. The activity of CRL MBGs varied with increasing PEG 2000content, presenting a bell-shaped curve (Fig. 2, inner). The best enzyme activity was obtainedwith a PEG 2000 concentration of 9.5 %. Decreasing activity was eventually observed withincreasing amounts of PEG. This trend was interpreted as a result of the strong influence of thehigh PEG concentration on the enzyme microenvironment. High concentrations of PEGproduced a more hydrophilic microenvironment, which could reduce the partition betweenthe hydrophobic substrate in the vicinity of the active site of the enzyme. Moreover, theaccumulation of alcohol molecules on the enzyme in more hydrophilic microenvironmentscould inhibit the reaction, obeying the Ping Pong Bi-Bi mechanism [33, 34].

Because the different addition sequences disparately affected the different components ofthe immobilization system, the effect of different additive mixing sequences on the lipaseactivity was investigated. The PEG 2000 addition sequence had no significant effect on theactivity of the CRL MBGs (data not shown). Thus, PEG most likely did not have a significantfunction in the individual components of the immobilization system, despite its effect on theentire structure of the immobilized enzyme.

The suspension of enzymes in organic solvents can cause conformational changes in theenzymes and thus change their substrate specificities [35]. These enzymes become easilydenatured, and their catalytic activities are lost in the presence of organic solvents. Variouslow-polarity, water-immiscible solvents (high log P) were tested in this study, including

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Fig. 2 Effect of PEG 2000 concentrations on activity of CRL MBGs. Superscript a indicates thattransesterification was carried out at 50 °C and 200 rpm for 12 h in vinyl acetate. 2-Phenylethanol concentrationwas 2 mg/mL

Appl Biochem Biotechnol

toluene, isopropyl ether, and n-hexane. Furthermore, high-polarity, water-miscible solventswith low log P values (acetone, acetonitrile, 1,4-dioxide, t-butanol, and THF) were investi-gated at 37 °C. The catalytic activity of the CRL MBGs was clearly improved by PEG in mostsolvents, except for isopropyl ether (Fig. 3a), and the activity mainly followed the log P trends.Higher activity was obtained when solvents with higher Log P values were used. However, theprotective effect of the additives was better in the more polar solvents (e.g., 1,4-dioxane withLog P of −1.1, acetonitrile with Log P of −0.33). Lower log P values correspond to lesshydrophobic solvents. Compared to the hydrophobic solvents, the hydrophilic ones couldmore easily rob the “essential water” bound to the lipase, which was necessary to preserve theflexibility of the enzyme conformation, thereby deactivating the lipase [36]. The enzyme thusbecame less stable, and its conformation may have changed. The results demonstrate that PEGcan prevent the removal of the essential water from the enzyme molecules by polar solvents,though the effect of the PEG molecular weight on solvent tolerance did not follow a regularpattern.

To further study solvent tolerance at higher temperatures, isopropyl ether was chosen as thesolvent. As can be seen in Fig. 3a, free lipase showed higher activity than CRL MBGs at37 °C. When the temperature rose to 50 °C, the CRL MBGs containing PEG demonstratedsuperior performance, with reaction conversion greater than 90 % after 12 h (Fig. 3b). Thisimprovement was due to substrate diffusion in the immobilized enzyme, which was limited bymass transport phenomena and decreased with increasing temperature. At high temperatures,better substrate molecule diffusion can be achieved because of the reduced viscosity of thereaction medium. However, the improvement in the activity of CRL MBGs with PEG 8000 at50 °C may demonstrate the strong diffusion limitation caused by PEG 8000 in CRL MBGs.Moreover, the CRL MBGs with PEG demonstrated higher enzymatic activity compared to thesimple immobilized enzyme, thereby indicating that PEG can efficiently reduce mass transportphenomena in the immobilized structure.

The thermal stability of the CRL MBGs with PEG 2000 was also investigated and wascompared to that of the control MBGs without additives, as well as that of free CRL, in thetemperature range from 10 to 60 °C. The results are shown in Fig. 4. Compared to free lipase, adistinct enhancement in the thermal stability was observed with the immobilized enzyme. Theactivity of free CRL clearly decreased when the temperature was above 40 °C, whereas nearlyno loss of activity was observed for CRL MBGs with or without PEG 2000. Therefore, theMBG structure could protect the encapsulated enzyme molecules remarkably from thermaldenaturation, and the PEG molecules provided no further improvement to the system.

Circular Dichroism and Fluorescence Spectra

Circular dichroism spectroscopy in the far UV range was used to investigate the influence ofsurfactants and polymers on the secondary structure of CRL at pH 7.2 and 25 °C. The spectraof the CRL samples with AOT and PEG 2000 coincided well with the original enzymesolution with no significant differences (Fig. 5a). Thus, AOT and PEG 2000 had no significanteffect on the secondary structure of CRL. In the case of CTAB and PEI, the ellipticity wasdiminished, and visible flocculates formed immediately after the additive was mixed in. CRL(PI=5.6) is an acetic protein that is negatively charged in a buffer of pH 7.2. The additive PEIis a weak polybase with a branched structure, whereas CTAB is a cationic surfactant. Thestrong interaction between the cationic head group in the additive molecules and the negativelycharged lipase could have induced changes in the secondary structure of CRL, which mayhave reduced the observed enzyme activity. Because the complexation of oppositely chargedmacromolecules has been shown to cause the formation of coacervates or precipitates that

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undergo phase separation [32], the decreased signal was mainly attributed to the phaseseparation of the PEI-protein complexes. The CD spectra of CRL in the presence of CTABand PEI exhibited a decrease in α-helix content, which can be attributed to the partial

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unfolding of the protein. This mechanism may account for the CRL inactivation observed inthe immobilization process.

The fluorescence spectra of the samples verified the enzyme activity and were in agreementwith the CD spectra. The spectra of the CRL samples containing AOT and PEG 2000concurred with those of the original CRL solution, as shown in Fig. 5b. However, thefluorescence intensity of CRL in the presence of CTAB and PEI was significantly increasedwith a slight redshift, which reflected the changes in the advanced structure of the lipase.

Morphological Analysis

A comparison of vacuum freeze-dried CRL MBG capsules with and without additivesis presented in Fig. 6a, f, showing the formation of a three-dimensional network inthe capsule. The network dimensions became more uniform when PEG was added,with a slight increase in diameter. When the PEG 2000 concentration was relativelylow (Fig. 6c, d, g, h), the gel was unable to form a complete and uniform network.With increasing PEG 2000 content in the system, the network structure of theimmobilized enzyme gradually became complete, smooth, and uniform. However,the system was delaminated when PEI was added, and a homogeneous gel phasecould not form. The diameter of the network with PEI increased distinctly, as shownin Fig. 6i, j. PEI addition also produced a microenvironment of pores with greaterdiameters, which may have improved the diffusion of substrate molecules. Further-more, the catalytic activity of the enzyme was drastically reduced. These phenomenaindirectly support the strong inactivation of enzymes caused by electrostaticinteractions.

Reusability, Storage Stability, and Preparative-Scale Reaction

Reusability is another crucial feature during the practical application of biocatalystsand is essential for the cost-effective use of the immobilized lipase, either in repeated

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Fig. 4 The temperature tolerance of the CRL MBGs with and without PEG 2000. Superscript a indicates thattransesterification was carried out at various temperatures and 200 rpm for 12 h in vinyl acetate. 2-Phenylethanolconcentration was 20 mg/mL

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baths or in continuous processes. Inactivation and enzyme leaching are the mostprominent drawbacks of the large-scale use of immobilized enzymes. The repeateduse of CRL MBGs with and without PEG 2000 was investigated in several batches oftransesterification runs at 50 °C (Fig. 7). No evident decrease in the catalyticefficiency of CRL MBGs with PEG 2000 was observed after nine runs of biotrans-formation. The system maintained 90 % yield, even after 15 cycles of repeated use.By contrast, the activity of the CRL MBGs without additives gradually decreased to75 % yield after 15 runs. The experimental data highlight the mechanical stability ofthe additives in the immobilization matrix, despite prolonged exposure to organicsolvents.

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The comparison of the storage stability of free and encapsulated CRL is shown inFig. 8. The activity of free CRL decreased by 10 % after 6 weeks of storage at roomtemperature and continued to decrease thereafter. It was found to have only 88 % ofits initial activity by the tenth week of storage. By contrast, the storage stability ofCRL was significantly improved after encapsulation, particularly with PEG 2000 asthe additive. No activity loss was observed during the first 6 weeks, and theremaining activity was as high as 97 % of the initial value even after storage atroom temperature for 10 weeks. Thus, the CRL MBGs have a distinct advantage overthe free enzyme after long-term storage.

To verify the feasibility of the proposed process at a larger scale, CRL MBGs with200 mg of CRL were added to a mixture of 50 g 2-phenylethanol in 100 mL of vinylacetate and then shaken at 50 °C. The isolated transesterification yield, as measuredby column chromatography, reached 90 % after 4 days. This result indicates thepossibility of applying the CRL MBGs in industrial chemical synthesis and otherbulk applications.

Fig. 6 SEM (before analyzing, the capsule was freeze-dried and gold coated) image of CRLMBG capsules withand without additives (a, b CRL MBGs; c, d CRL MBGs with PEG 2000 9.5 %; e, f CRL MBGs with PEG4000; g CRL MBGs with PEG 2000 1.7 %; h CRL MBGs with PEG 2000 6.0 %; i CRL MBGs with PEI 600; jCRL MBGs with PEI 25,000). The scale of every single SEM represents 100 um

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Fig. 7 Reusability of CRL MBGs with and without PEG 2000. Superscript a indicates that transesterificationwas carried out at 50 °C and 200 rpm for 24 h in vinyl acetate, and the MBGs was recycled 15 times. 2-Phenylethanol concentration was 20 mg/mL

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Conclusion

In summary, this study presents an inexpensive, simple, and rapid method to improvethe activity and stability of CRL MBGs by the introduction of additives (PEGs orpolysaccharides) during immobilization. The results indicate that PEG is a betteradditive with regard to transesterification activity, as higher enzyme activities wereobtained after immobilization for the same enzyme concentration. The addition of PEIdecreased the activity of CRL MBGs, and we have demonstrated that PEI causes theinactivation of CRL in buffer, as suggested by the circular dichroism and fluorescencespectra. This phenomenon was most likely due to the conformational changes in theimmobilized enzyme. While the addition of PEG provided no further improvements tothe thermal stability of CRL, the improved catalytic activity of the CRL MBGscaused by the additives was evident in many organic solvents, particularly in themore polar solvents. No significant changes to the CRL MBG activity were observedafter nine runs because of the presence of PEG 2000. Unlike the immobilizedenzymes without additives, the CRL MBGs with PEG 2000 could maintain a 90 %yield even after 15 cycles of repeated use.

The results of this study could serve as a guideline for the optimization of the immobili-zation conditions for immobilized lipases with noncovalently modified PEG. One of thelimitations to the use of enzymes is their initial cost. However, this study demonstrated thatthe immobilized enzymes can be reused numerous times without loss of activity, therebymaking their synthesis economical. Further economic advantages of large-scale biotechnolog-ical applications are highlighted, and the proposed method may be applied for a wide variety ofother lipases and esterases.

Acknowledgments This work was financially supported by the National Program on Key Basic ResearchProject of China (973 Program, 2013CB328900) and the National Natural Science Foundation of China (nos.21001077, 21321061, J1310008, and J1103315). We also thank the Sichuan University Analytical & TestingCenter for SEM analysis.

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Fig. 8 Storage stability of CRL MBGs with and without PEG 2000 at 4 °C and room temperature (RT).Superscript a indicates that transesterification was carried out at 37 °C and 200 rpm for 24 h in vinyl acetate. 2-Phenylethanol concentration was 20 mg/mL

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