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Research ArticleReceived: 8 September 2008 Revised: 29 September 2008 Accepted: 29 September 2008 Published online in Wiley Interscience: 10 November 2008

(www.interscience.wiley.com) DOI 10.1002/jctb.2080

A simple method for biocatalyst immobilizationusing PVA-based hydrogel particlesPedro Fernandes,∗ Marco P.C. Marques, Filipe Carvalho andJoaquim M.S. Cabral

Abstract

BACKGROUND: The aim of this study was to evaluate the feasibility of enzyme immobilization in PVA particles through extrusionof LentiKatLiquid in polyethylene glycol. Inulinase, with invertase activity for sucrose hydrolysis, was used as model system.

RESULTS: Inulinase was effectively immobilized in PVA particles. The pH optimum of the enzyme activity was broadened forlower pH values. Mechanical instability of the PVA under prolonged incubation above 55 ◦C was observed. A 1.8-fold increase inthe apparent KM (Michaelis constant) suggests diffusion limitations as a result of immobilization. The immobilized biocatalystexhibited considerable operational stability, since a decrease of roughly 10% in the product yield after 24 h biotransformationruns was observed in trials performed at 50 ◦C, following 20 repeated, consecutive batches.

CONCLUSION: The results obtained highlight the potential of PVA-based particles obtained through extrusion into PEG for theproduction of suitable biocatalysts for application in large-scale processes.c© 2008 Society of Chemical Industry

Keywords: inulinase; sucrose hydrolysis; polyvinyl alcohol; LentiKatLiquid; PEG 600

INTRODUCTIONHydrogels are broadly acknowledged to be suitable materialsfor the immobilization of enzymes and whole cells, given theirbiocompatibility, low coefficient of friction, and high watercontent. The resulting biocatalysts are used in biotransformationsystems with application in several areas, among them food andfeed industries.1 The wider application of hydrogel-encapsulatedbiocatalysts is impaired by several features, namely the lowmechanical stability of most hydrogels, (particularly those fromnatural polymers) and enzyme leakage through the pores of the gellattice.2 Polyvinyl alcohol-based matrices have been shown to leadto particles with considerable chemical and mechanical stability,providing a suitable alternative to overcome the drawbacksof natural polymers while retaining the benefits inherent totheir use.3,4 Several methods have been developed to formhydrogels from polyvinyl alcohol (PVA), namely freeze-thawing,5,6

irradiation3 or chemical cross-linking.7 – 9 A different approach,which has reached commercial application, relies on the controlleddrying of a PVA-based hydrogel (LentiKatLiquid), and subsequentchemical stabilization, leading to lens-shaped particles.10,11 In thepresent work, a modified GeniaLab methodology is used forthe encapsulation of a commercial inulinase preparation, withinvertase activity, in PVA capsules. In the proposed methodology,the PVA capsules are obtained from LentiKat liquid uponextrusion into polyethylene glycol (PEG), where gelification occursinstantaneously, in the form of hemispheric-like capsules, thusavoiding the need for the partial drying process required to obtainlens-shaped particles, Lentikats . The methodology presented isreminiscent of the classic protocol for immobilization in calciumalginate gel particles. In the present work, the method is performed

with a relatively low molecular weight enzyme (inulinases fromAspergillus spp. are roughly under 80 kDa12), doing away withprevious enzyme processing (i.e. cross-linking) suggested for lowmolecular weight enzymes before immobilization in Lentikats .13

This has often been used for the immobilization of large molecularweight enzymes (above 100 kDa), such as glucoamylase, invertaseor dextransucrase, with application in aqueous media.14 – 16

The invert sugar mixture obtained through enzymatic sucrosehydrolysis has the advantage of being colorless, as opposed tothe colored products obtained through acid hydrolysis, mostlydue to the presence of hydroxyl methyl furfural.17,18 The use of animmobilized biocatalyst is favored in this particular application,to prevent product contamination with protein matter, reducingoperational costs, increasing the stability of the enzyme, andenabling repeated use or a continuous mode of operation.17 – 19

Although most research work aimed at the improvement of thisbioconversion process has focused on the use of immobilizedinvertases, a different approach, based on the use of inulinase withactivity for sucrose hydrolysis, has also been suggested.12,20

The kinetic parameters, pH and temperature profiles of free andimmobilized inulinase were matched and the operational stabilityof the immobilized enzyme was assessed, based on repeated,

∗ Correspondence to: Pedro Fernandes, IBB, Institute for Biotechnology andBioengineering, Centre for Biological and Chemical Engineering, InstitutoSuperior Tecnico, 1049-001 Lisboa, Portugal. E-mail: Pedro.fernandes@ist.utl.pt

IBB, Institute for Biotechnology and Bioengineering, Centre for Biological andChemical Engineering, Instituto Superior Tecnico, 1049-001 Lisboa, Portugal

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consecutive batch runs, with an initial sucrose concentration of10.0% (w/v).

MATERIAL AND METHODSFructozyme L, a commercial preparation of inulinase fromAspergillus niger, was provided by Sigma (St Louis, MO, USA),polyethylene glycol (PEG) 600 was from Fluka (Deisenhofen,Germany), LentiKat liquid, a PVA-based material was fromGeniaLab (Braunschweig, Germany), and sucrose was from Tate &Lyle - Acucares de Portugal, SA (Santa Iria de Azoia, Portugal). Allother chemicals were of analytical grade from various suppliers.

Enzyme immobilizationIn order to entrap inulinase in PVA, the LentiKat liquid washeated at 95 ◦C and then cooled to 40 ◦C. The enzyme suspension(0.5 mL of a 10-fold, 100 mmol L−1 acetate buffer, pH 4.5, dilutedcommercial preparation) was added to 10 mL of the LentiKat

liquid and mixed under magnetic stirring. The enzyme-richsolution was extruded through a needle (Therumo, 20 G × 2′′,Therumo Medical Corp., Somerset, NJ, USA) into 150 mL of PEG600, under mild magnetic stirring, where capsules were formed.After a 2 h period, the 3 mm sized hemispheric capsules wereharvested, and thoroughly washed with acetate buffer, weighedafter removal of excess buffer with qualitative filter paper, soakedin acetate buffer for 2 h, harvested and weighed again afterremoval of excess buffer with qualitative filter paper, and eitherimmediately used for bioconversion runs or stored at 4 ◦C until use.

Determination of the immobilization yield, of temperatureand pH profiles and of kinetic parametersExperiments were performed at the intended pH (3.5–6.0) andtemperature (40–65 ◦C), in 1.0 mL of a 10.0% (w/v) sucrose solutionin acetate buffer (100 mmol L−1), containing 50 mg of immobilizedinulinase or 20 µL of a 40-fold diluted preparation of free inulinase.Runs were performed in 1.5 mL screw-capped vessels and agitation(500 rpm) was promoted by magnetic stirring. Immobilizationyield was determined as the ratio between the specific activityof immobilized inulinase and that of the soluble inulinase, in a30 min run performed at 50 ◦C, using a 10.0% (w/v) of sucrose inacetate buffer (pH 4.5). Samples (10 µL) were taken after 30 minand immediately assayed for reducing sugars. For kinetic runs,samples (10 µL) were taken after 5 min (free enzyme) and 10 min(immobilized enzyme). All runs were performed in triplicate, atleast. Kinetic parameters were determined using the commercialsoftware Leonora.21

03 4 5 6 7

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pH

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tivity

(%

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Figure 1. Influence of pH on the activity of free (dots) and immobilized(triangles) forms of the enzyme preparation. Bioconversion runs werecarried out at 50 ◦C, at different pH values (3.5–6.0).

Repeated batch hydrolysisReactions were performed in 1.5 mL screw-capped magneticallystirred (500 rpm) reactors at 50 ◦C or 55 ◦C, in 1.0 mL of 100 mmolL−1 acetate buffer (pH 4.5), containing 10.0% (w/v) of sucrose and50 mg of immobilized inulinase. Throughout each cycle (24 h run)10 µL samples were collected and assayed for reducing sugars.After each cycle, the immobilized biocatalyst was harvested,thoroughly washed with acetate buffer and used for the nextrun. All runs were performed in triplicate, at least.

Analytical methodsQuantification of reducing sugars was performed by the DNSmethod.22 Quantification of protein was performed by the BCAmethod23 using a commercial kit from Pierce Biotechnology (Rock-ford, IL, USA). Particles of immobilized biocatalyst were dissolvedin distilled water heated at 70 ◦C, before protein quantification.Liquid supernatants resulting from the immobilization proce-dures were also assayed for protein levels. The standard deviationfrom these determinations did not exceed 8%, except if statedotherwise.

RESULTS AND DISCUSSIONImmobilization yieldThe feasibility of the proposed methodology for enzyme immobi-lization was assessed primarily by evaluating the immobilizationyield (Table 1). No traces of protein were observed in the super-natants resulting from the immobilization method. Given the mildmethodology used for immobilization, the immobilization yield ismost probably suggestive of diffusion limitations.

Effect of pH on enzyme activityThe effect of pH on the initial activity of free and immobilizedinulinase was assessed at 50 ◦C, in the pH range 3.5–6.0 (Fig. 1).The optimum pH for sucrose hydrolysis remained unchanged uponimmobilization at 4.5. Interestingly the immobilized biocatalystdisplayed higher activity than the free form at lower pH. Thisfeature, which can be useful since it may reduce the risk of microbial

Table 1. Immobilization of inulinase in LentikatLiquid basedparticles

Immobilized biocatalystEnzyme loading(mg g−1

support)Immobilizaton

yield (%)

Hemispheric particles 0.13 ± 0.02 48 ± 5

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ty (

%)

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30 35 40 45 50 55 60 65 70

Temperature (°C)

Figure 2. Influence of temperature on the activity of free (dots) andimmobilized (triangles) forms of the enzyme preparation. Bioconversionruns were carried out at pH 4.5, at temperatures in the range 35–65 ◦C.

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contamination during long-term operation under incubation atsuch pH values, was also reported by Rebros and co-workers,16

upon immobilization of invertase in lenticular-shaped particles,LentiKats. Overall broadening of the pH optimum for sucrosehydrolysis following immobilization of invertase and inulinase inhydrogels, compared with the free form, has been reported.12,16,24

In the present case, however, the enhancement of activityfollowing immobilization was observed only for lower pH values.Partial enhancement of the pH optimum for invertase followingimmobilization in agarose-guam gum was also reported by Bagaland Karve,25 but this effect was observed only for higher pH values,in the range 4.0–6.0, whereas in the range 3.0–4.0 free invertaseoutperformed the immobilized enzyme. These effects have beenascribed to secondary interactions between the enzyme and theimmobilization support.16

Effect of temperature on enzyme activityFree inulinase displayed the highest activity at 60 ◦C (Fig. 2).Immobilized inulinase was also tested in the temperature range35–65 ◦C and biocatalyst activity consistently increased withtemperature. At 60 ◦C, however, capsules displayed an evidentdecay in physical stability after a 30 min incubation period, andat 65 ◦C actually melted; thus the highest activity for immobilizedbiocatalyst was obtained at 55 ◦C. Lack of mechanical stabilityat temperatures above 55 ◦C was also reported by Rebros andco-workers16 for invertase immobilized in lens-shaped particles,obtained by the traditional partial drying process.

Effect of immobilization on kineticsThe kinetic parameters of the free and immobilized enzyme weredetermined at pH 4.5 and 50 ◦C (Table 2). An increase in theapparent KM (Michaelis constant) was observed following immo-bilization, the decreased affinity of the enzyme towards substratemost likely resulting from hindered substrate diffusion inside thematrix. This is a feature commonly reported for invertase/inulinase

Table 2. Kinetics constants obtained for sucrose hydrolysis with freeand immobilized inulinase

Biocatalyst KM (g L−1) Vmax (greducing sugars L−1 min−1)

Free enzyme 43 ± 5 1.5 ± 0.1

Immobilized enzyme 76 ± 9 1.2 ± 0.1

entrapped in hydrogels for sucrose hydrolysis.12,16,18,25,26 Theroughly 1.8-fold increase in KM following immobilization com-pares favorably with the KM increases previously reported (1.9to 3.60) except for the data reported by Bagal and Karve.25 Cu-riously, the greatest increase in KM was observed for invertaseentrapped in lens-shaped particles, despite their thinness, in therange 200–400 µm, which is suggestive of reduced diffusionlimitations.16

Biocatalyst reuseImmobilized biocatalyst was used repeatedly in several consecu-tive 24 h batch hydrolysis runs at 50 ◦C, with an initial substrateconcentration of 10% (w/v), which was almost fully converted inthe first run (Fig. 3). A decrease of roughly 10% in final productyield was observed after 20 runs, suggesting high operationalstability of the immobilized biocatalyst.

Storage stabilityDecay of enzyme activity over time upon storage in solution isa well-known feature, although immobilization usually tends todecrease such an effect.16,19 In the present case, after a 30 dayperiod of storage at 4 ◦C in acetate buffer (pH 4.5, 100 mmol L−1),the immobilized inulinase retained about 90% of the initial activity,a promising result.

CONCLUSIONSInulinase with invertase activity was effectively immobilized inPVA based particles obtained through extrusion of Lentikat

liquid into PEG 600. Immobilization broadened the pH optimumof the enzyme for lower pH values. The highest activity of the freeenzyme was observed at 60 ◦C, however PVA particles proved to bemechanically unstable under prolonged incubation above 55 ◦C,thus this temperature was set as the optimum for the immobilizedenzyme. Diffusion limitations were observed as an outcome ofimmobilization. Immobilized inulinase displayed high operationalstability, since it was used in 20 consecutive batch runs at 50 ◦C,with no significant loss of activity. The immobilized biocatalystalso proved stable upon storage. Overall, and considering alsothe simplicity of the immobilization methodology and the easyhandling of the particles, this work indicates the potential ofenzyme immobilization in PVA-based particles obtained throughextrusion into PEG, for the production of biocatalysts withapplication in relevant biotransformation processes.

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140

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Batch runs

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oduc

t yie

ld (

%)

Figure 3. Effect on the final product yield of the repeated use of immobilized inulinase for the hydrolysis of a 10.0% (w/v) sucrose solution. Batch runs(24 h) were performed at 50 ◦C and pH 4.5. In the first cycle a 94 ± 8 g L−1 product titer was observed.

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ACKNOWLEDGEMENTSP. Fernandes thanks Fundacao para a Ciencia e Tecnologia (FCT) forsupport under Programme Ciencia 2007. M.P.C. Marques thanksFundacao para a Ciencia e a Tecnologia (Portugal) for financialsupport in the form of PhD grant SFRH/BD/24433/2005.

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