Reversible Immobilization of Invertase on Sepabeads Coated withPolyethyleneimine: Optimization of the Biocatalyst’s Stability

Rodrigo Torres, Cesar Mateo, Manuel Fuentes, Jose M. Palomo, Claudia Ortiz,Roberto Fernandez-Lafuente,* and Jose M. Guisan*

Departamento de Biocatalisis, Instituto de Catalisis, CSIC, Campus Universidad Autonoma, Cantoblanco,28049 Madrid, Spain

Andrea Tam and Moreno Daminati

Resindion SRL, Mitsubishi Chemical Corporation, Milan, Italy

Invertase from S. cerevisiae has been immobilized by ionic adsorption on Sepabeadsfully coated with PEI. The enzyme was strongly adsorbed on the support (no desorptionof the invertase was found under conditions in which all of the enzyme was releasedfrom conventional anionic exchanger supports (e.g., DEAE-agarose)). Nevertheless,the enzyme could still be desorbed after its inactivation, and new fresh enzyme couldbe adsorbed on the supports without detrimental effects on enzyme loading. This is amultimeric enzyme, its minimal oligomerization active state being the dimer, but undercertain conditions of pH and concentration it may give larger multimers. Veryinterestingly, results suggested that the adsorption of the enzyme on this large andflexible polymeric bed was able to freeze some of the different oligomeric structures ofthe enzyme. Thus, we have found that the enzyme immobilized at certain pH values(pH 8.5) and high enzyme concentration, in which the main enzyme structure is thetetramer, was more stable than immobilized preparations produced in conditions underwhich oligomerization was not favorable (dimers at low enzyme concentration) or itwas too high (e.g., hexamers-octamers at low pH value). The optimal enzymepreparation remained fully active after a 15-day incubation at 50 °C and pH 4.5(conditions of standard industrial use) and presented an optimal temperatureapproximately 5 °C higher than that of soluble enzyme.

Introduction

The advantages of the use of immobilized enzymes aremany, and some of them may have a special relevancein the area of food technology. In this industrial area,the control of the expenses must be very strict becauseof the low added value of products. When covalentimmobilization does not provide a significant improve-ment of the enzyme properties (e.g., stabilization of theenzyme via multipoint covalent attachment) (Gianfredaand Scarfi, 1991; Blanco et al., 1988; Guisan et al., 1993;Fernandez-Lafuente et al. 1997; Mateo et al., 2000B), itwould be economically and technically inconvenient.

Reversible enzyme immobilization is a very powerfultool that may be considered to solve this cost problem.Such immobilization techniques may allow (i) a very easyprotocol for simple immobilization, and (ii) the reuse ofthe supports (e.g., expensive materials having excellentmechanical and hydrodynamic properties) and the sub-sequent reduction of industrial wastes. Reversible im-mobilization could provide the possibility of using suchenzymes in an immobilized form and, in this way, havingthe advantages of the use of immobilized enzymes, savingtime and costs (Rosevear, 1984; Royer, 1980; Klibanov,

1983; Kennedy et al., 1990; Katchalski-Katzir, 1993;Hartmeier, 1985; Chibata et al., 1986; Gupta, 1991).

There are some interesting protocols for reversibleimmobilization of proteins, e.g., thiol-disulfide exchangeof proteins containing surface thiol groups (Batista-Vieraet al., 1991, 1994, Brena et al. 1993), immobilized metalchelate supports (Brena et al., 1994), and interaction withphenyl boronates (Batista-Viera et al., 1988). However,the most general, easiest to perform, and oldest protocolfor reversible immobilization of enzymes (with an easyregeneration of the support) is the adsorption of enzymeson ionic exchange resins (mainly on anionic exchangesupports). In fact, the first immobilization and the firstindustrial process developed with immobilized enzymes(resolution of D,L-amino acids by amino acylase) wasperformed by using such enzyme preparations (Katch-alski-Katzir, 1993). However, immobilization of enzymeson conventional ionic exchanger resins is frequently notvery strong, and most proteins are fully desorbed fromsuch matrices at moderate-low ionic strength (e.g., 0.2M NaCl) or if a pH shift occurs during the reaction. Inthis way, high concentrations of ionizable substrates,changes of pH during the reaction, etc., can promoteundesirable leakage of immobilized enzyme from thesupport, resulting in an apparent inactivation of theenzyme preparation and a certain undesired contamina-tion of the product (Rosevear, 1984; Royer, 1980; Kli-

* To whom correspondence should be addressed. Tel: 34 91 58548 09. Fax: 34 91 585 47 60. E-mail: rfl@icp.csic.es/jmguisan@icp.csic.es.

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10.1021/bp020082q CCC: $22.00 © 2002 American Chemical Society and American Institute of Chemical EngineersPublished on Web 11/09/2002

banov, 1983; Kennedy et al., 1990). Particularly, such isthe case when using ionic supports that have beendesigned for enzyme purification, in which it is necessaryto desorb the enzyme under mild conditions.

The adsorption of enzymes on composites based on thefull covalent coating of porous rigid supports with flexiblepolymers containing a very high density of ion exchangemoieties (e.g., polyethyleneimine) has been proposed asa very suitable method for reversible but very strongprotein immobilization (Mateo et al., 2000a). Desorptionof proteins from these supports was found to require theuse of denaturing conditions (very low pH, very high ionicstrength), but this desorption would be necessary onlywhen the enzyme had become already inactivated andwas ready to be discarded. Some authors even claim somestabilizing effects of PEI by diverse causes (Anderssonand Hatti-Kaul, 1999; Bryjak, 1995).

The invertase from Saccharomyces cerevisiase (EC3.2.1.26) is an enzyme with special relevance in foodtechnology and is extensively used in the production offructose syrup from sucrose. Although the dimer has beendescribed as the smaller active form, there are somereports that show a certain tendency to form largeroligomers (from dimer to octamers) depending on theconditions of the medium (pH, ionic strength, oligosac-charides, etc.) (Chu et al., 1983; Tammi et al., 1987; Kernet al., 1992; Trimble and Maley, 1977; Esmon et al., 1987;Reddy et al., 1990).

In the literature it is possible to find many examplesof immobilization of this enzyme by different techniques(Tumturk et al., 2000; Akgol et al., 2001; Arica et al.,2000; Ettalibi and Beratti, 2001; Tanriseven and Dogan,2001; Erginer at el, 2000; D’Souza and Godbole, 2002;Vrabel et al., 1997), although this complex enzyme is usedin free form in many cases, which generates someproblems in the downstream processing of the productarising from contamination of the syrup with the protein(which produces colored substances at high tempera-tures).

Materials and Methods

Materials. Invertase (â-D-fructofuranosidase; EC.3.2.1.26) from baker’s yeast was supplied by Novo Nor-disk and used without further purification. Activity was110 IU/mg powder at 25 °C and pH 4.5 (1 g of powderpresented 75 mg of protein). Sucrose was obtained fromSigma Chemical Co., polyethyleneimine (PEI) of 25 kDamolecular weight was purchased from Aldrich ChemicalCo., and Sepabeads EC-EP3 (an epoxy-acrylic resinhaving epoxy residues in its surface) was kindly donatedby Resindion SRL (Mitsubishi Chemical Co.). This sup-port has 30-35 µmol of epoxy groups per wet gram ofsupport. Sepabeads resins did not swell when passedfrom dry form to wet form, 1 mL of packed supportcorrespond to 0.7 g of wet support. DEAE-agarose wasobtained from Bio-Rad (Hercules, PA), and Sepharose CL-6B was obtained from Pharmacia. Protein concentrationwas determined by Bradford’s method (1976) usingbovine serum albumin (BSA) as standard. Other reagentswere of analytical grade.

Methods. Preparation of Sepabeads EC-EP3-PEISupport. To modify the epoxy-supports with polyethyl-eneimine (PEI), the epoxy support was incubated at pH11 with 25% PEI for 48 h, and then the remaining epoxygroups were blocked by incubation with 2 M ethylene-diamine at pH 10 for 72 h. This step was carried out toensure that no free epoxy groups remained in thesupport, to prevent any possibility of covalent attachment

between the enzyme and the support. Finally, all primaryamino groups were blocked by reaction with glycidol asdescribed elsewhere (Mateo et al., in preparation). Thistreatment prevented the formation of colored productsduring reaction with sugars at high temperatures.

Invertase Activity Determination. Standard inver-tase activity was determined by monitoring the hydroly-sis of 5 mL of 100 mM sucrose in 0.1 M acetate buffer atpH 4.5 and 25 °C. At different times, 100-µL samplesfrom the reaction solution were withdrawn. When usingsoluble enzymes, to stop the enzymatic reaction, 20 µLof 0.1 M NaOH was added to inactivate the invertase,followed by addition of 20 µL of 0.1 M HCl. Glucoseproduced by sucrose hydrolysis was measured spectro-photometrically at 505 nm using an enzymatic method(Glucose Trinder) (20 µL of reaction sample and 1 mL ofthe Glucose Trinder kit).

Gel Filtration Experiments. Gel permeation chro-matography experiments were performed at 4 °C usingSepharose CL-6B (1.0 cm × 40 cm; Fractionation range60-2000 kD; Pharmacia Co) and eluted at 0.25 mL/minwith 5 mM sodium phosphate. Protein was detected at280 nm, and invertase activity of the collected fractionswas determined as described above. Molecular weightmarkers (Dextran blue, tyroglobuline, bovine serumalbumin, bovine myoglobin, penicillin G acylase from E.coli) were used for estimation molecular weight of dif-ferent forms of invertase.

Immobilization of Invertase. Immobilization wascarried out by adding the supports (both, DEAE-agaroseor PEI-Sepabeads) to a suspension of invertase in 5 mMsodium phosphate at the desired pH value and 4 °C(standard relation of volumes was 1 g of support to 10mL of enzyme suspension). After immobilization, theenzyme preparation was washed with distilled water andwith 25 mM sodium acetate pH 4.5 or 25 mM sodiumphosphate at pH 7, to equilibrate the enzyme preparationon the conditions desired (which depend on the nextexperiment). In this way, all immobilized preparationswere kept under exactly the same conditions for at least16 h before the experiments were performed.

Adsorption Strength Determination: Reuse ofSupport. The immobilized invertase was incubated atincreasing concentrations of NaCl at 25 °C and pH 7, andthe invertase activity of the suspension and supernatantwas assayed.

Enzyme Inactivation Experiments. Enzyme prepa-rations were incubated at different temperatures, pHvalues, and enzyme concentrations. Periodically, samplesof these suspensions were withdrawn and the remainingactivity was assayed as described above. Invertase re-sidual activity was expressed as a percentage of initialactivity at the given incubation time.

Results

Comparison of PEI-Coated Supports versus Con-ventional Cationic Supports. The enzyme was im-mobilized in 5 mM sodium phosphate at pH 7 on DEAE-agarose and PEI-Sepabeads, with an immobilization yieldhigher than 90% in both cases, after only 5 min ofenzyme-support contact. The activity was fully preservedduring the immobilization protocol in both cases.

The salt concentration at which the enzyme began tobe desorbed from the support was much higher usingPEI-Sepabeads than DEAE-agarose (Figure 1). While allthe enzyme was desorbed from agarose DEAE at only100 mM of NaCl, invertase began to be partially desorbedfrom the PEI support at more than 200 mM NaCl.

1222 Biotechnol. Prog., 2002, Vol. 18, No. 6

Moreover, Figure 2 shows that the invertase im-mobilized on PEI-Sepabeads was more stable both thanthe enzyme immobilized on conventional supports (DEAE-agarose) and the soluble enzyme.

The maximum load of this support was approximately30 mg of enzyme/gram of support with an activity ofaround 36,000 IU/g (around 30,000 per milliliter ofpacked immobilized preparation) of biocatalyst at 25 °Cand pH 4.5.

In this way, these new preparations could be used ina very wide range of conditions considering both thermalstability and adsorption strength of the enzyme to thesupport and giving very high volumetric activities.

Effect of pH of Immobilization on Stability ofImmobilized Enzyme. The enzyme remained fullyactive at pH 5.5-8.5 and 4 °C for 24 h; therefore theimmobilization could be performed in this wide range ofpH values.

The immobilization rate was very high in all cases, andthe maximum load of the support was similar (around30-35 mg of enzyme per gram of support), with activitiesretentions higher than 95% in all cases.

The stability of the free or immobilized (Figure 3)enzyme at acidic pH values was higher than the stabilityat alkaline pH values. Curiously, the stability of theenzyme immobilized at pH 8.5 was higher than that ofthe enzyme immobilized at pH 5.5 when both prepara-tions were inactivated at pH 4.5 and 50, 55 (Figure 4),or 60 °C.

This result suggests that the different immobilizedenzymes could present some differences promoted by thepH value of the immobilization. It has been described

that invertase presents different composition of multi-meric structures when changing the pH value (Tammiet al., 1987; Chu et al., 1983; Reddy et al., 1990). Also, ithas been reported that PEI-coated supports may permitthe stabilization of multimeric enzymes by implicatingseveral subunits in the immobilization (Mateo et al.,2000a). Thus, if the enzyme presented a different ag-gregation form during the immobilization, this structureseems to be preserved after the immobilization.

For this reason, we have tried to determine if theinvertase could be found in different multimeric statesunder the different immobilization conditions using GFC.Table 1 shows that while at pH 5.5 invertase seems tobe mainly in very high aggregation forms (mainly hexa-mers and octamers), at pH 8.5 there was a certaindistribution of oligomers, the tetramer being the pre-dominant form. This result seems to suggest that thequaternary structure of the invertase octamers could notbe fully stabilized by the relatively small PEI used in thiswork, enabling the releasing of some subunits at high Tthat could have a stability near to that of the free enzyme.

Figure 1. Desorption of invertase from S. cerevisae adsorbedon different anion exchanging supports at growing ionicstrength: (2) activity released from DEAE-agarose; (b) activityreleased from Sepabeads EC-EP3-PEI by incubation withincreasing NaCl concentrations (see Methods). Enzyme wasimmobilized at pH 7 as described in Methods, with a proteinloading of 25 mg/g of support. Desorption was performed at pH7.

Figure 2. Effect of the support on the stability of immobilizedinvertase: (2) soluble invertase; (9) DEAE-agarose preparation;(b) Sepabeads EC-EP--PEI preparation. Inactivations wereperformed at pH 4.5 and 55 °C. Enzyme was immobilized atpH 7 as described in Methods, with a protein load of 25 mgprotein/g of support.

Figure 3. Effect of pH on the stability of invertase SepabeadsEC-EP3-PEI immobilized preparations: (b) pH 5.5; (9) pH 7.0;(2) pH 8.5. Enzyme was immobilized at pH 7 as described inMethods, with a protein loading of 25 mg protein/g of support.Inactivation of enzyme preparations was carried out at 55 °C.

Figure 4. Thermal stability of invertase Sepabeads EC-EP3-PEI preparations immobilized at different pH values; enzymeload was 25 mg of protein/g of support: (2) immobilized at pH8.5; (9) immobilized at pH 7.0; (b) immobilized at pH 5.5.Inactivation of immobilized enzyme was performed at pH 4.5and 55 °C

Table 1. Effect of pH on Oligomeric Structure of SolubleInvertase from S. cerevisiaea

relative oligomer concn (% of total)b

pH valueoctamer and

hexamer tetramer dimer

5.5 90 7 38.5 42 46 12

a The invertase was dissolved in 5 mM sodium phosphate atthe desired pH value at an enzyme concentration of 5 mg/mL andkept for at least 1 h at 4 °C before the gel filtration experiment.b The calculations were performed from the Activity associated toeach peak in the GFC chromatograms divided by the total Activityrecovered (that corresponds to around 100% of the injectedactivity).

Biotechnol. Prog., 2002, Vol. 18, No. 6 1223

Effect of Enzyme Concentration during Immobi-lization on Stability Properties of the Final Prepa-ration. Considering the influence of the oligomericstructure of the enzyme on its final stability properties,we have prepared preparations of the enzyme startingfrom very different situations, using pH 8.5 as im-mobilization pH value: one having maximum loadingcapacity and another one having only 1% of the maxi-mum loading capacity, using enzyme solutions withdifferent concentration. The stability of the poorly loadedenzyme preparation prepared with diluted enzyme wasfound to be lower than that of the highly loaded prepara-tion prepared with highly concentrated enzyme at tem-peratures ranging from 45 to 60 °C (e.g., see Figure 5),even though the enzyme concentration during the inac-tivation experiment was similar.

Moreover, two highly loaded preparations were pre-pared starting from two very different enzyme concentra-tions. To prepare these preparations, 250 mg of protein(determined by Bradford’s method) was offered to 10 gof PEI support under two very different situations. Inthe first one, the enzyme was contained in only 82 mL ofbuffer, while in the other preparation we used 4200 mLof buffer. Again, a clear effect of the enzyme concentrationduring preparation on the immobilized enzyme stabilitywas found at all temperatures studied (Figure 6).

When we prepared immobilized enzyme preparationsloaded with only 2.5 mg of protein/mL of support butusing high and low concentration in the immobilizationsuspension, results were very similar to those described

in Figure 6. Therefore, the differences in stability werenot a result of the enzyme concentration in the gel butof the enzyme concentration during immobilization.

Again, these differences could be correlated with thedifferent oligomerization degree of the invertase; Table2 shows that at the highest concentration employed,invertase exhibited mainly tetramers, whereas at lowconcentration the percentage of dimers was much higher.Therefore, tetramers seemed to be more stable, afteradsorption on this PEI-coated support, than dimers,perhaps through some stabilization effects from a higherdegree of protein-protein interaction (Poltorak et al.,1998), plus a stabilization of this multimeric structureby the PEI-coated support (Mateo et al., 2000a).

The fact that the concentration of enzyme duringimmobilization was the key to the final properties of thebiocatalysts (independently from the enzyme load) sug-gested that the oligomer of the enzyme should be formedprior to the immobilization process.

Optimized Protocol for Invertase ImmobilizationOn PEI. From our results, We suggest the followingoptimal and easy protocol for invertase immobilizationon supports coated with PEI: (1) dissolve 5 g of the crudeinvertase powder (375 mg of total protein) supplied byNovo in 85 mL of 5 mM sodium phosphate at pH 8.5; (2)add 10 g of PEI-Sepabeads previously equilibrated in thesame buffer; (3) permit the immobilization to proceed for16 h at 4 °C; (4) wash the immobilized preparations withan excess of distilled water or the desired buffer (prefer-ably at pH 4.5-5); (5) keep at 4 °C (enzyme activityremained fully unaltered after 3 months).

Characterization of Optimal Enzyme Prepara-tion. This optimal immobilized invertase preparationkeeps unaltered its activity after incubation at pH 4.5and 50 °C during 1 week in the absence of substrate(Figure 7), while the soluble enzyme lost around 40% ofactivity in this time period. The presence of substrateeven promoted a certain stabilization of the enzyme (seein Figure 8 the stabilizing effect of 40% substrates orproducts).

The optimal temperature for the immobilized prepara-tion was higher than that of the soluble enzyme using3.5% sucrose as substrate (from 53 to almost 57 °C)(Figure 9). Using 40% sucrose (similar to the sucroseconcentration currently used in the industry) the optimaltemperature of the enzyme preparation increased to 70°C.

The reaction courses in the hydrolysis of 40% sucroseat 50 and 60 °C (industrial conditions) catalyzed bysoluble and immobilized preparation was studied. Figure10 shows the improvement of the reaction course by usingthe immobilized preparation. Ten reaction cycles couldbe carried out at 50 °C without significant decreases ofthe biocatalyst activity.

Figure 5. Effect of enzyme load on the thermal stability ofimmobilized Sepabeads EC-EP3-PEI invertase: (b) loading of0.3 mg protein/g support; (9) loading of 30 mg protein/g ofsupport. Immobilization was carried out at pH 8.5. as describedin Methods. Inactivation of enzyme preparations was carriedout at pH 4.5 and 55 °C. Enzyme concentration during inactiva-tion was 0.025 mg protein/mL of suspension.

Figure 6. Effect of enzyme concentration during immobiliza-tion on the thermal stability of immobilized invertase; theenzyme preparations were immobilized at same enzyme loadper unit of support but with different initial enzyme concentra-tion: (b) 0.1 mg protein/mL; (9) 5 mg protein/mL. Immobiliza-tion was carried out at pH 8.5 as described in Methods.Inactivation of enzyme preparations was carried out at pH 4.5and 55 °C. Enzyme concentration during inactivation was 0.025mg protein/mL of suspension.

Table 2. Effect of Enzyme Concentration on OligomericStructure of Soluble Invertase from S. cerevisiaea

relative oligomer concn (% of total)b

enzyme concn(mg/mL)

octamer andhexamer tetramer dimer

5 42 46 120.25 15 30 55

a The invertase was dissolved in 5 mM sodium phosphate atpH 8.5 and kept for at least 1 h at 4 °C before the gel filtrationexperiment. b The calculations were performed from the Activityassociated to each peak in the GFC chromatograms divided bythe total Activity recovered (that corresponds to around 100% ofthe injected activity).

1224 Biotechnol. Prog., 2002, Vol. 18, No. 6

We must bear in mind that this invertase preparationpresented a mixture of several oligomeric forms, and fromour results, those conditions that favored the presenceof tetramers should improve the performance of theimmobilized preparation. This could explain the clearmultiphasic profile of the inactivation experiments; thepresence of several subpopulations seemed to be clear.

Reuse of PEI Support. By washing the PEI invertasepreparation with 3 M NaCl pH 3 at 22 °C, all of theprotein can be removed from the support for reuse. Toregenerate the support after invertase inactivation, theenzyme preparation may also be washed in HCl at pH1.5-2 and 40 °C at low ionic strength. Alternatively,saturated solutions of guanidine or milder conditions athigher temperature could be used to release all of theproteins from the support. This support could be reusedfor 5 cycles without any decrease in enzyme loadingcapacity.

The support of the preparations used during 10 cyclesat 50 °C (see above) also could be reused, once all the

adsorbed protein was fully released from the supportusing protocols similar to those described above.

DiscussionThe results obtained in this paper are an improvement

in some aspects (activity, stability) when compared to thepreviously described using other immobilization protocols(Tumturk et al., 2000; Akgol et al., 2001; Arica et al.,2000; Ettalibi and Beratti, 2001; Tanriseven and Dogan,2001; Erginer at el, 2000; D’Souza and Godbole, 2002;Vrabel et al., 1997).

Results seem to suggest that invertase immobilized onPEI-Sepabeads at different conditions presented differentoligomeric structures with different stability properties.The adsorption on PEI-Sepabeads, a polymeric andflexible bed, seems to be able to stabilize the differentoligomeric forms of the invertase (Scheme 1), making itpossible to keep that structure under acidic or dilutedconditions, which results in an increment of the enzymestability.

In this way, to the previous advantages described forthis support, this ability of the polymer-coated matrix tostabilize complex multimeric structures should be takeninto account even for basic research.

AcknowledgmentThe authors gratefully recognize the financial support

and the supply of matrices from Resindion SRL. The

Figure 7. Thermal inactivation of different invertase prepara-tions at pH 4.5 and 50 °C: (9) optimal preparation; (b) solubleenzyme. Enzyme concentration during inactivation was 0.025mg protein/mL of suspension. Other specifications as describedin Methods.

Figure 8. Thermal inactivation of PEI-Sepabeads-preparationat 62 °C at pH 4.5 in buffer (9), buffer supplemented withsucrose 40% (b), or buffer supplemented with 20%/glucose/20%fructose (2). Experiments were performed as described inMethods.

Figure 9. Effect of T on the activity of soluble (b) andimmobilized invertase (9) at pH 4.5. Experiments were per-formed as described in Methods.

Figure 10. Reaction course of hydrolysis of 40% sucrose usingsoluble (b) or immobilized (9) invertase; 1 g of immobilizedinvertase (or the equivalent soluble enzyme) was added to 5 Lof sucrose. Experiments were performed as described in Meth-ods.

Scheme 1. Likely Mechanism of Protein Structure“Freezing” during Immobilization of MultimericEnzymes on PEI-Coated Supports

Biotechnol. Prog., 2002, Vol. 18, No. 6 1225

authors gratefully recognize the kind donation of theenzyme from Novo. This work has been partially foundedby the Spanish CICYT (project BIO2000-0747-C05-02).We thank a PhD fellowship from Universidad Industrialde Santander, Colombia for R.T.; a PhD fellowship forJ.M.P., and a postdoctoral fellowship from CAM and aI3P- BPG2001 fellowship for M.F. from CSIC. We thankDr. Ramirez (Novo) and Dr. Hidalgo (ICP-CSIC) for theirinteresting suggestions.

References and NotesAkgol, S.; Kacar, Y.; Denizli, A.; Arica, M. Y. Hydrolysis of

sucrose by invertase immobilized onto novel magnetic poly-(vinyl alcohol) microspheres. Food Chem. 2001, 74, 281-288.

Andersson, M. M.; Hatti-Kaul, R. Protein stabilising effect ofpolyethyleneimine. J. Biotechnol. 1999, 72, 21-31.

Arica, M. Y.; Senel, S.; Alaeddinoglu, N. G.; Patir, S.; Denizli,A. Invertase immobilized on spacer arm attached poly-(hydroxyethyl methacrylate) membrane: preparation andproperties. J. Polym. Sci. 2000, 75, 1685-1692.

Bahar, T.; Tuncel, A. Immobilization of invertase onto cross-linked poly (p-chloromethylstyren) beads. J. Appl. Polym. Sci.2002, 83, 1268-1279.

Batista-Viera, F.; Brena, B.; Luna, B. Reversible immobilizationof soybean amylase on phenylboronate-agarose. Biotechnol.Bioeng. 1988, 31, 711-713.

Batista-Viera, F.; Barbieri, M.; Ovsejevi, K.; Manta, C.; Carlsson,J. A new method for reversible immobilization of thiolbiomolecules based on solid-phase bound thiosulfonate. Appl.Biochem. Biotechnol. 1991, 31, 175-195.

Batista-Viera, F.; Manta, C.; Carlsson, J. Solid-phase thiosul-fonates for reversible immobilization of thiols. Appl. Biochem.Biotechnol. 1994, 44, 1-14.

Blanco, R. M.; Calvette, J. J.; Guisan, J. M. Immobilization-stabilization of enzymes. Variables that control intensity ofthe trypsin (amine)-agarose (aldehyde) multi-point covalentattachment. Enzyme Microb. Technol. 1988, 11, 353-359.

Bradford, M. M. A rapid and sensitive method for the quanti-fication of microgram quantities of protein utilizing theprinciple of protein-dye binding. Anal. Biochem. 1976, 72,248-254.

Brena, B. M.; Ovsejevi, K.; Luna, B.; Batista Viera, F. Thiolationand reversible immobilization of sweet potato amylase onthiosulfonate-agarose. J. Mol. Catal. 1993, 84, 381-390.

Brena, B. M.; Ryden, L. G.; Porath, J. Immobilization ofgalactosidase on metal-chelate substituted gels. Biotechnol.Appl. Biochem. 1994, 19, 217-231.

Bryjak, J. Storage stabilization of enzyme activity by poly(ethylene-imine). Bioprocess Eng. 1995, 13, 177-181.

Chibata, I.; Tosa, T.; Sato, T. Biocatalysis: immobilized cellsand enzymes. J. Mol. Catal. 1986, 37, 1-24.

Chu, F. K.; Watorek, W.; Maley, F. Factors affecting theoligomeric structure of yeast external invertase. Arch. Bio-chem. Biophys. 1983, 223, 543-555.

D’Souza, S. F., Godbole, S. S. Immobilization of invertase onrice husk using polyethylenimine. J. Biochem. Biophys.Methods 2002, 52, 59-62.

Erginer, R.; Toppare, L.; Alkan, S.; Bakir, U. Immonilization ofinvertase in functionalized copolymer matrices. React. Funct.Polym. 2000, 45, 227-233.

Esmon, P.; Esmon, B.; Schauer, I.; Taylor, A.; Schekman, R.Structure, assembly and secretion of octameric Invertase. J.Biol. Chem. 1987, 262, 4387-4394.

Ettalibi, M.; Baratti, J. C. Sucrose hydrolysis by thermostableimmobilized inulinases from Aspergilus ficuum. EnzymeMicrob. Technol. 2001, 28, 596-601.

Fernandez-Lafuente, R.; Rodriguez, V.; Mateo, C.; Penzol, G.;Hernandez-Justiz, O.; Irazoqui, G.; Villarino, A.; Ovsejevi, K.;Batista, F.; Guisan, J. M. Stabilization of multimeric enzymesvia immobilization and post-immobilization techniques. J.Mol. Catal. B: Enzymatic 1999, 7, 181-189.

Gianfreda, L.; Scarfi, M. R. Enzyme stabilization: state of theart. Mol. Cell. Biochem. 1991, 29, 1-28.

Guisan, J. M.; Alvaro, G.; Fernandez-Lafuente, R.; Stabilizationof heterodymeric enzyme by multi-point covalent immobiliza-tion: penicillin G acylase from Kluyvera citrophila. Biotech-nol. Bioeng. 1993, 42, 464.

Gupta, M. M. Thermostabilization of proteins. Biotechnol. Appl.Biochem. 1991, 14, 1-11.

Hartmeier, W. Immobilized biocatalysts-from simple to complexsystems. TIBTECH 1985, 3, 149-153.

Katchalski-Katzir, E. Immobilized enzymes: Learning from pastsuccesses and failures. TIBTECH 1993, 11, 471-478.

Kenedy, J. F.; Melo, E. H. M.; Jumel, K. Immobilized enzymesand cells. Chem. Eng. Prog. 1990, 45, 81-89.

Kern, G.; Schulke, N.; Schmid, F.; Jaenicke, R. Stability,quaternary structure, and folding of internal, external andcore-glycosylated invertase from yeast. Protein Sci. 1992, 1,120-131.

Klibanov, A. M. Immobilized enzymes and cells as practicalcatalysts. Science 1983, 219, 722-727.

Mateo, C.; Abian, O.; Fernandez-Lafuente, R.; Guisan, J. M.Reversible enzyme Immobilization via very strong and non-distorting ionic adsorption on support-polyethyleneiminecomposites. Biotechnol. Bioeng. 2000a, 8, 98-105.

Mateo, C.; Abian, O.; Fernandez-Lafuente, R.; Guisan, J. M.Increase in conformational stability of enzymes immobilizedon epoxy-activated supports by favoring additional multipointcovalent attachment. Enzyme Microb. Technol. 2000a, 26,509-515.

Poltorak, O. M.; Chukhary, E. S.; Torshin, I. Y. Dissociativethermal inactivation, stability and activity of oligomericenzymes. Biochemistry (Moscow) 1998, 63, 360-369.

Rosevear, A. Immobilised biocatalysts: A critical Review. J.Chem. Technol. Biotechnol. 1984, 34B, 127-150.

Royer, G. P. Immobilized enzymes as catalyst. Catal. Rev. 1980,22, 29-73.

Reddy, A. V.; MacColl, R.; Maley, F. Effect of oligosaccharideson oligomeric structures of external, internal and deglycosy-lated invertase. Biochemistry 1990, 29, 2482-2487.

Tammi, M.; Ballou, L.; Taylor, A.; Ballou, C. Effect of glycosy-lation on yeast invertase oligomer stability. J. Biol. Chem.1987, 262, 4395-4401.

Tanriseven, A.; Dogan, S. Immobilization of invertase withincalcium alginate gel capsules. Proc. Biochem. 2001, 36, 1081-1083.

Trimble, R. B.; Maley, F. Subunit structure of external invertasefrom Saccharomyces cerevisiae. J. Biol. Chem. 1977, 252,4409-4412.

Tumturk, T.; Arslan, F.; Disli, A.; Tufan, Y. Immobilization ofinvertase attached to a granular dimer acid-co-alkyl polyamine.Food Chem. 2000, 69, 5-9.

Vrabel, P.; Polakovic, M.; Godo, S.; Bales, V.; Docolomansky,P.; Gemeiner, P. Influence of immobilization on the thermalinactivation of yeast invertase. Enzyme Microb. Technol.1997, 21, 196-202.

Accepted for publication September 20, 2002.

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