Characterization of Free and Immobilized Thermomyces lanuginosusLipase for Use in Transesterification Reactions

Carlos Eduardo Barao,1,2 Leandro Daniel de Paris,2

Joao Henrique Dantas,2 Cleide Mara Faria Soares,3

Heizir Ferreira de Castro,4 Gisella Maria Zanin,2

Flavio Faria de Moraes2

1Instituto Federal do Parana, Campus Paranavaı, Paranavaı, Brazil2Universidade Estadual de Maringa, Departamento de EngenhariaQuımica, Maringa, Brazil

3Instituto de Tecnologia e Pesquisa, Universidade Tiradentes,Aracaju, Brazil

4Escola de Engenharia de Lorena, Universidade de Sao Paulo,Lorena, Brazil

AbstractThermomyces lanuginosus lipase, in both its free form as wellas immobilized in sol-gel matrices produced using the pre-cursor tetraethoxysilane (TEOS) and dried using the xerogeltechnique, was used in transesterification reactions between 2-phenylethyl alcohol and vinyl acetate. The free lipase was firstcharacterized in terms of its transesterification activity at37�C, with a value of 1,233 U/g obtained and lower tempera-tures providing higher activity. The influence of humidity onthe enzyme immobilized with TEOS was evaluated using a 22

factorial design (varying the conditions of humidity and tem-perature). The results indicate that higher humidity (30%) andlower temperature (40�C) provided the best transesterificationactivity, and that these two factors and their interaction had asignificant influence on activity (at the 95% confidence level).Under these conditions, the biocatalyst presented a surfacearea of 502 m2/g, a pore volume of 0.643 cm3/g, and an averagepore diameter of 51.2 A. These results reflect its effectivenessin this type of reaction. Differences between the support aloneand the immobilized biocatalyst were observed using scanningelectron microscopy.

Key Words: Lipase, immobilization, sol-gel, humidity, trans-esterification

Introduction

There is considerable interest in the use of enzymes asbiocatalysts due to their potential for application inmany reactions used in scientific research, pharma-cology, and industry. Lipases are widely used in

diverse reactions involving triglycerides, including hydrolysis,esterification, transesterification, interesterification, and others.Lipases are also used in the manufacture of a variety of com-mercial products, in which they help to produce high yieldsselectively with high degrees of purity and low levels of con-tamination. However, the high costs associated with the useof lipases remain a challenge. Immobilization techniques thatenable reuse of the enzyme have been used to help bringdown costs and make lipase use economically viable at indus-trial scale.

Immobilization is a generic term employed to describe theretention of a biologically active catalyst within the interior of areactor or analytical system. A biocatalyst generally consists ofan enzyme or a mixture of enzymes found in cells that are re-tained in the interior of the pores or on the surface of a materialused as a support. The enzyme-support complex retains thephysical characteristics of the support while providing the bio-logical activity of the enzyme in a soluble form. This system canbe used in continuous reactors, with easy separation of the cat-alyst and product and greater process efficiency.1

The sol-gel immobilization process involves the synthesis ofan inorganic matrix through the formation of a sol and itstransformation into a humid gel. Removal of the liquid results inthe formation of a dry porous gel in the form of a dense amor-phous solid.2 Chemical agents are added to the sol in order tocontrol the drying and reduce the processing time, as well asto avoid the appearance of cracks in the membranes duringthe drying step. There are three main stages of the procedure:complexation (hydrolysis and polycondensation), solidifica-tion by cooling (gelation), and drying. In the first two stages,different components are uniformly incorporated together ina colloidal polymer contained in a solvent. In the final stage,a three-dimensional coagulate is produced from the two-dimensional polymer. In these polymerizations, the composi-tional homogeneity of the liquid state is maintained in the geland is ultimately reflected in the catalyst. The coagulate ob-tained has a small quantity of unreacted water, large quantitiesof diol/monoalcohols that are physically adsorbed and absorbedby the three-dimensional structure, and some unhydrolyzed or-ganic residues that remain unchanged after the drying process.3

The xerogel composites normally show high porosity as well ashigh concentrations of metal oxides. The nature and degree ofthe dispersion of oxide particles captured within the pores of thematrix are important factors influencing the characteristics ofthe xerogel.4

The enzyme chosen in this work was a Thermomyces lanugi-nosus lipase (lipolase) produced during submerged fermentation

DOI: 10.1089/ind.2014.0011 ª M A R Y A N N L I E B E R T , I N C . � VOL. 10 NO. 4 � AUGUST 2014 INDUSTRIAL BIOTECHNOLOGY 305

using a genetically modified strain of Aspergillus oryzae. Thecommercial enzyme is available in granular or liquid forms withdeclared activities of 100,000 U/g. This lipase is 1,3-specificand acts to break the 1,3 bonds of the triacylglycerol ester.

The enzyme, either free or immobilized in a sol-gel matrixproduced using a silica precursor (tetraethoxysilane, TEOS),was employed in the transesterification reaction between 2-phenylethyl alcohol and vinyl acetate, producing 2-phenylethylacetate. The effects of biocatalyst humidity and reaction tem-perature were evaluated, and morphological analysis of thebiocatalyst was performed using the Brunauer-Emmett-Teller(BET) technique and scanning electron microscopy (SEM).

Materials and MethodsMATERIALS

T. lanuginosus lipase (Lipolase) and TEOS were obtainedfrom Sigma-Aldrich (St. Louis, MO). The following analytical-grade reagents were used: olive oil, anhydrous ethanol, 2-phenylethylalcohol, vinyl acetate, ethanolamine, ethanol, hydrochloric acid,ammonium hydroxide, heptane, acetone, powdered gum arabic,and polyethylene glycol (PEG-1500; Synth, Campinas, Brazil).

DETERMINATION OF PROTEIN CONTENTThe protein content of the free lipase enzyme was determined

by the colorimetric method employing Coomassie Brilliant Bluereagent and standard bovine serum albumin, as described byBradford.5

DETERMINATION OF TRANSESTERIFICATION ACTIVITYBY PRODUCTION OF 2-PHENYLETHYL ACETATE

The transesterification activity of the enzyme in its freeand immobilized forms was measured using the reaction of 2-phenylethyl alcohol with vinyl acetate, forming 2-phenylethylacetate. The reaction product was measured using high-performanceliquid chromatography (HPLC), with an octadecylsilane col-umn and an ultraviolet detector operated at 254 nm. The mobilephase was a mixture of acetonitrile and water, at a flow rate of1.0 mL/min. The reaction mixture was prepared using 0.6 mLof 2-phenylethyl alcohol, 2.4 mL of vinyl acetate, and 20 mg offree or immobilized enzyme, followed by agitation for 20 min atthe desired temperature. Subsequently, 0.1 mL of the mixturewas removed using a pipette and added to 0.6 mL of isopropylether, followed by 1:10 dilution in the acetonitrile/water mobilephase. The products were then quantified by HPLC. One unit ofenzymatic activity was defined as the quantity of enzyme thatproduced 1 lmol of 2-phenylethyl acetate per min, at a giventemperature. The activities of free and immobilized enzymewere measured in triplicate and expressed in U/g (activity unitsper gram of enzyme).

IMMOBILIZATION PROCEDURE USING TEOSThe sol-gel solution was prepared according to the method-

ology described in Patent PI0306829-3.6 TEOS was diluted inabsolute ethanol under a nitrogen atmosphere. A pre-hydrolysissolution of 0.22 mL of HCl dissolved in 5.0 mL of ultrapurewater was then added, at 35�C, and the mixture was maintainedunder agitation for 90 min. The enzyme (2.7 g) dissolved in

deionized water was added, together with an additive (16 mL ofa solution prepared with 0.8 g of PEG-1450 in 20 mL of deio-nized water). Gelation of the silica was achieved by addingammonium hydroxide (concentration 28–30%) dissolved inethanol (hydrolysis solution). After 24 h, the material was fil-tered under vacuum, using hexane and acetone to remove theexcess water. For preparation of the support alone (control),the enzyme solution was substituted by water. After preparation,the biocatalysts were dried by evaporation in a desiccator, withhumidity controlled by the Karl Fischer technique.

PHYSICAL CHARACTERIZATION (SURFACE AREA,PORE DIAMETER, AND PORE VOLUME)

Sample analysis was performed using a NOVA 1200 instru-ment (Quantachrome; Boynton Beach, FL). The surface area,average pore size, and average pore volume were determined viathe BET method using N2 adsorption at 77 K. Before analysis,samples were submitted to a thermal treatment at 60�C, undervacuum, to eliminate any water existing within the pores of thesolids.

SEMSEM analyses were performed using a Leica Model LEO 440i

(Leica Microsystems; Wetzlar, Germany). Portions of the en-zymatic preparations were attached to the sample holders usingcarbon tape and coated with a layer of carbon under vacuum.

Table 1. Transesterification Activity of the Free Enzymeat Different Temperatures

TEMPERATURE (�C)TRANSESTERIFICATION

ACTIVITY (U/g)a

37 1,230 – 20.0

45 1,091 – 10.0

53 1,068 – 15.0

aspecific activity.

Table 2. Transesterification Activities Obtained Usinga 22 Experimental Design

TEMPERATURE(�C) HUMIDITY (%)

TRANSESTERIFICATIONACTIVITY (U/g)

40 ( - 1) 10 ( - 1) 1,382

60 ( + 1) 10 ( - 1) 1,403

40 ( - 1) 30 ( + 1) 1,814

60 ( + 1) 30 ( + 1) 1,327

50 (0) 20 (0) 1,473

50 (0) 20 (0) 1,560

50 (0) 20 (0) 1,480

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Results and DiscussionCHARACTERIZATION OF THE FREET. LANUGINOSUS LIPASE

The average protein concentration ofthe commercial T. lanuginosus lipasepreparation, in units of mass of proteinper volume of solution, was 27.3 mg/mL.

Measurements of the transesterifica-tion activity of the free enzyme wereperformed at different temperatures(Table 1) and showed that the highestactivity was achieved at the lowest tem-perature. Temperature has a major influ-ence on the activity, selectivity, andstability of a biocatalyst, as well as onthe reaction equilibrium; this reactionwas used to characterize the enzymebecause it is employed during the pro-duction of biodiesel.

The T. lanuginosus lipase is highlystable in aqueous media and maintainsits activity between pH 7 and 11, whichis a broad range for an enzyme. The li-pase also remains reasonably active attemperatures of 55–60�C, even thoughrecommended temperatures are between 30–40�C, as observedpreviously.7

INFLUENCE OF THE HUMIDITY OF THE IMMOBILIZEDENZYME ON THE TRANSESTERIFICATION REACTION

An evaluation of the influence of the humidity of the im-mobilized enzyme on the transesterification reaction involving2-phenylethyl alcohol and vinyl acetatewas then performed. The conditions oftemperature and humidity were variedusing a 22 experimental design (Table 2)in order to identify the conditions thatprovided the greatest transesterificationactivity.

The results showed that the enzymewas most active under conditions oflower temperature (as also found for freelipase) and higher humidity, achieving1,814 U/g at 40�C and 30% humidity. Intransesterification reactions, the pres-ence of small quantities of water gener-ally helps to improve reaction yield, asthe presence of water within the poressustains the activity of the lipase andfacilitates catalyst reuse. According toAhn et al., adding 1.0 mL of water per0.33 g of biocatalyst resulted in high se-lectivity towards unsaturated methylfatty acids produced during methanolysisof soya oil using lipase immobilized onmesoporous silica and increased the sta-bility of the immobilized lipase consid-

erably.8 Other studies have also demonstrated the need to addwater in processes involving lipases, such as the transesterifica-tion of palm oil with ethanol and methanol, where Candida ru-gosa lipase immobilized on activated carbon was used at a ratio of0.5 mg of enzyme to 1.0 mL of water.9

The Pareto estimated effects diagram (Fig. 1) revealed thatall the parameters influenced transesterification activity, withthe greatest effect being due to the interaction, followed by

Fig. 1. Pareto diagram of estimated standardized effects on the transesterification activityof immobilized T. lanuginosus lipase.

Fig. 2. Normality graph of observed values as a function of the values predicted by theregression model for the transesterification activity of immobilized T. lanuginosus lipase.

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temperature and humidity. These findings were in agreementwith the analysis of variance, with p-values <0.05 for the interac-tion. An R2 value of 0.9643 was obtained for the regression model.

The regression model describing the transesterification activityof T. lanuginosus lipase immobilized on TEOS, as a function ofreaction temperature and humidity, could be described by:

Trans: act: = 1491:7 - (116:5 · H)

+ (88:86 · T) - (126:58 · H · T)

A normality graph (Fig. 2) was constructed in order to con-firm that the conditions of normality required by the model weresatisfied, with the errors obtained in the analysis of variancebeing independent and normally distributed. Fig. 2 shows thatone point is slightly removed from the regression line for theexperimental data. However, despite this feature, there was nosignificant departure from normality.

The results are summarized in the response surface graphshown in Fig. 3, from which it is clear that the greatest trans-esterification activity was obtained at lower temperature andhigher humidity.

TEXTURAL CHARACTERIZATIONOF THE IMMOBILIZED ENZYME

The BET technique was used to determine the surface area,pore size, and pore volume of the biocatalyst at 30% humidity,and images were obtained using SEM. The values obtained were502 – 0.36 m2/g (surface area), 0.643 – 0.006 cm3/g (pore vol-ume), and 5.1 – 0.03 nm (average pore diameter). Character-ization of the porosity of biocatalysts can aid interpretation ofthe enzyme activity results. The values obtained here showedthat the biocatalyst possessed a high surface area that providedsubstantial contact with the substrate. The immobilization pro-cess was assisted by the presence of mesopores; release of theenzyme from pores of this size is much slower than from mac-ropores, while contact with the substrate is not restricted to theextent found for micropores. These physical characteristics in-dicate that the biocatalyst could be used in a wide range ofapplications. Similar findings have been reported for lipaseimmobilized on mesoporous silica (obtained using sodium sil-icate and a biodegradable gelatin) in the hydrolysis reaction oftriacetin, where the values obtained were 518.8 m2/g (surfacearea), 3.6 nm (pore size), and 1.34 cm3/g(pore volume). The preparation showedgood thermal stability and retained 45%of its initial activity after six consecutiveusages.10

The SEM micrographs of the support,either pure or containing the enzyme(Fig. 4), revealed the roughness of thematerials, especially in the case ofthe biocatalyst, where the presence oflarge quantities of material deposited onthe surface acted to increase the mea-sured porosity.

The images revealed that the enzy-matic preparations possessed irregu-lar surface morphology, suggesting that

there was incomplete homogenization during the biocatalystpreparation process, as expected for the sol-gel technique based onprevious work.11

ConclusionsThe lipase derived from T. lanuginosus was shown to be ef-

fective in transesterification reactions. Despite the fact that theenzyme was derived from a thermoresistant microorganism, itsactivity in the transesterification reaction was greater at lowertemperature. When the lipase was immobilized in a silica matrix(xerogel) produced using TEOS as a precursor, factorial designexperiments demonstrated that the transesterification activ-ity was significantly influenced by temperature, humidity, andthe interaction between these two parameters. Higher enzyme

Fig. 3. Response surface for the transesterification activity of im-mobilized T. lanuginosus lipase as a function of temperature andhumidity.

Fig. 4. Micrographs of the pure support (left) and the support with enzyme (right).

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308 INDUSTRIAL BIOTECHNOLOGY AUGUST 2014

activity was achieved at higher humidity and lower temperature(within the ranges tested). The structure of the biocatalyst, in-vestigated using BET and SEM analyses, revealed the existenceof a mesoporous structure with a high surface area, whichcontributed to the effectiveness of the biocatalyst.

AcknowledgmentsThe authors thank the Chemical Engineering Department of

State University of Maringa for the support and CAPES for fund-ing the Pro-engenharias project (process 23038-028317/2008-44).

Author Disclosure StatementNo competing financial interests exist.

R E F E R E N C E S

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2. Silva RF, Vasconcelos WL. Influence of processing variables on the porestructure of silica gels obtained with tetraethylorthosilicate. Materials Res1999;2:197–200.

3. Santos OAA. Hidrogenacao seletiva do acido oleico em catalisadores de rutenio.Tese de Doutorado. Faculdade de Engenharia Quımica, UNICAMP, Campinas,1999:181.

4. Avnir D, Braun Lev O, Ottolenghi M. Enzymes and other proteins entrapped insol–gel materials Chem Materials 1994;20:1605–1614.

5. Bradford MM. A rapid and sensitive method for the quantification ofmicrogram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem 1976;72:5.

6. INPI Patent submission No. PI0306829-3, September 11, 2003.

7. Fernandes MLM, Krieger N, Baron AM, et al. Hydrolysis and synthesis reactionscatalysed by Thermomyces lanuginosa lipase in the AOT/Isooctane reversedmicellar system. J Mol Cat B Enzym 2004;30(1):43–49.

8. Ahn KW, Ye SH, Chun WH, et al. Yield and component distribution of biodieselby methanolysis of soybean oil with lipase-immobilized mesoporous silica.Microporous Mesoporous Materials 2011;142:37–44.

9. Pirajan JC, Giraldo L. Study of immobilized candida rugosa lipase for biodieselfuel production from palm oil by flow microcalorimetry. Arab J Chem2011;4:55–62.

10. Wang X, Zhou G, Zhang H, et al. Immobilization and catalytic activity of lipaseon mesoporous silica prepared from biocompatible gelatin organic template.J Non-Crystal Solids 2011;357:3027–3032.

11. Barao CE, Paris LD, Dantas JH, et al. Characterization of biocatalysts preparedwith Thermomyces lanuginosus lipase and different silica precursors, driedusing aerogel and xerogel techniques. Appl Biochem Biotechnol 2013;172:263–274.

Address correspondence to:Carlos Eduardo Barao, PhDInstituto Federal do Parana

Campus Paranavaı�Rua Jose Felipe Tequinha 1400

Paranavaı�, PR, BrazilPhone: + 55 44 84069137

Fax: + 55 44 34820109

E-mail: carlos.barao@ifpr.edu.br

T. LANUGINOSUS LIPASE IN TRANSESTERIFICATION

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