Process Biochemistry 39 (2004) 535–539

�-Chymotrypsin stabilization by chemical conjugation withO-carboxymethyl-poly-�-cyclodextrin

Michael Fernándeza, Maria L. Villalongaa, Alex Fragosob, Roberto Caob,Maysa Bañosb, Reynaldo Villalongaa,∗

a Enzyme Technology Group, Center for Biotechnological Studies, University of Matanzas,Autopista a Varadero Km 3 1/2, C.P. 44740, Matanzas, Cuba

b Laboratory of Bioinorganic Chemistry, Faculty of Chemistry, Havana University, Havana 10400, Cuba

Received 11 November 2002; accepted 8 February 2003

Abstract

Bovine pancreatic�-chymotrypsin was covalently modified with theO-carboxymethyl poly-�-cyclodextrin (M= 1.3× 104, 40% COOHgroups) using a water-soluble carbodiimide as a coupling agent. The conjugate prepared by this procedure retained high proteolytic andesterolytic activity and contained about 74% carbohydrate by weight of transformed protein. The optimum temperature for�-chymotrypsinwas increased by 5◦C after this transformation. The thermostability of the polymer–enzyme adduct was also increased by 5◦C. The conjugateprepared was also more resistant to thermal inactivation at different temperatures, ranging from 45 to 55◦C. Additionally, the modified enzymewas 11-fold more stable at pH 9.0. The direct influence of supramolecular interactions between the hydrophobic amino acid residues locatedat the surface of the protease and the attached polycyclodextrin moieties on�-chymotrypsin stabilization was demonstrated.© 2003 Elsevier Ltd. All rights reserved.

Keywords:�-Chymotrypsin; Cyclodextrin; Modified enzyme; Enzyme thermostability; Supramolecular interaction

1. Introduction

During the last few decades, several methodologieshave been investigated to increase the thermostability ofindustrial enzymes, in order to achieve improved opera-tional performance[1–4]. Amongst these, methods basedon chemical manipulation of the protein structure con-stitute the most effective when the catalysts are used todigest insoluble or macromolecular substrates, or when thediffusion-determining reaction rate in the homogeneousstate is higher than that in the heterogeneous state[5].

Historically, carbohydrate compounds have been exten-sively used as modifying agents for enzymes[2,6,7]. Thisselection has been inspired by naturally occurring glycoen-zymes, in which the glycosidic residues play an importantrole in the stability properties of these enzymes[8]. In thisregard, the covalent attachment of ionic[9–11]and non-ionic[6,7,12]polysaccharides, as well as oligosaccharide moieties

∗ Corresponding author. Tel.:+53-45-26-1251; fax:+53-45-25-3101.E-mail address:reynaldo.villalonga@umcc.cu (R. Villalonga).

[13] has been established as a useful tool for increasing thethermal stability to enzymes.

In the present work, we describe the stabilization ofbovine pancreatic�-chymotrypsin (EC 3.4.21.1) by chem-ical modification with the O-carboxymethyl derivativeof poly-�-cyclodextrin (CPCD). This polymer is highlywater-soluble and can easily be prepared by a two reactionstep process. The interest in this type of polymers is alsobased on the ability of cyclodextrins to form stable inclu-sion complexes with hydrophobic compounds[14], suchas amino acid residues located at the protein surface ofenzymes. In this regard, we have evaluated the influenceof this kind of supramolecular interaction on the thermalstability of the polysaccharide–enzyme complex prepared.

2. Experimental

2.1. Materials

Bovine pancreatic �-chymotrypsin (45 U mg−1),N-�-acetyl-l-tyrosine ethyl ester hydrochloride (ATEE),

0032-9592/$ – see front matter © 2003 Elsevier Ltd. All rights reserved.doi:10.1016/S0032-9592(03)00069-4

536 M. Fernandez et al. / Process Biochemistry 39 (2004) 535–539

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC)and Fractogel EMD BioSEC (S) were obtained from Merck(Darmstadt, Germany).�-Cyclodextrin was purchased fromAmaizo (Indiana, USA) and used as received. All otherchemicals were of analytical grade.

2.2. Synthesis of the modifying polymer

�-Cyclodextrin (5 g) was dissolved in 50 ml of 10% (w/v)NaOH and 10 ml of epichlorohydrin were added. The systemwas vigorously stirred for 8 h, another 5 ml of epichlorohy-drin added with stirring and the mixture kept overnight atroom temperature. The solution was concentrated to about15 ml and precipitated by addition of cold ethanol (500ml). The gummy precipitate was crushed several times withethanol in a mortar until a fine precipitate was obtained. Theprecipitate was then washed again with ethanol and acetoneand dried under high vacuum overnight. The yield was 4 g.

Two grams of the above polymer were further dissolvedin 50 ml 5% (w/v) NaOH and 2 g ofmonochloroacetic acidwere added. The system was vigorously stirred for 24 h,neutralized with 2 M HCl, concentrated to about 15 ml andcooled to 4◦C. The precipitated NaCl was filtered off and thesupernatant was precipitated by addition of cold ethanol (500ml). The gummy precipitate was crushed several times withethanol in a mortar until a fine precipitate was obtained. Theprecipitate was then washed two more times with ethanoland acetone and dried under high vacuum overnight. Theyield was 1.5 g.

The molecular weight of CPCD was determined by gelpermeation chromatography on Fractogel EMD BioSEC (S)(1.6× 100 cm) calibrated with dextran standards. The degreeof carboxymethylation was determined by potentiometrictitration [15].

2.3. Preparation ofα-chymotrypsin–CPCD complex

For coupling the activated polymer to�-chymotrypsin,30 mg EDAC were added to a reaction mixture containing10 mg of protease and 50 mg of the polymer, dissolved in 5ml 50 mM sodium phosphate buffer, pH 6.0. The solutionswere stirred for 1 h atroom temperature and for 16 h at 4◦C,and further applied to a gel filtration column Fractogel EMDBioSEC (S) (2.6× 60 cm), equilibrated in 20 mM sodiumacetate buffer, pH 5.0, containing 100 mM NaCl. The frac-tions containing the polysaccharide–enzyme complex werepooled and kept at 4◦C.

2.4. Assays

Esterolytic activity of native and modified�-chymotryp-sins was determined at 25◦C in 50 mM sodium phosphatebuffer, pH 7.0 using ATEE as substrate[16]. One unit ofesterolytic activity is defined as the amount of enzyme thathydrolyses 1.0�mol of ATEE per minute at 25◦C. Prote-olytic activity was determined as described by Laskowski

[17] using milk casein as substrate. One unit of proteolyticactivity, katal, is defined as the amount of enzyme that re-leases 1 mol of tyrosine per second at 25◦C. Total carbohy-drates were determined by a phenol–sulphuric acid method[18] using glucose as standard. Protein concentration wasestimated as described by Lowry et al.[19] using bovineserum albumin as standard. The degree of modification ofamino groups was determined by measuring free aminogroups with o-phtalaldehyde using glycine as standard[20].

2.5. Optimum temperature

The activities of native and CPCD-modified�-chymotry-psin preparations were measured at different temperaturesranging from 20 to 67◦C. The corresponding values of op-timum temperature were calculated from Arrhenius plots.

2.6. Thermal stability profile

Native and modified enzyme preparations were incubatedat different temperatures in 50 mM sodium acetate buffer,pH 5.0. Aliquots were removed after 10 min of incubation,diluted in cold 50 mM sodium phosphate buffer, pH 7.0, andassayed for esterolytic activity. The values of T50 for bothenzyme forms, defined as the temperature at which 50% ofthe initial activity was retained, were determined from theplots of residual activity (%) versus temperature.

2.7. Kinetics of thermal inactivation

Native and modified�-chymotrypsin were incubated attemperatures ranging from 45 to 55◦C in 50 mM sodiumacetate buffer, pH 5.0. Aliquots were removed at sched-uled times, chilled quickly and assayed for enzyme activ-ity. Half-life times were calculated from the first-order rateconstants of inactivation, which were obtained from linearregression in logarithmic coordinates.

2.8. Resistance to alkaline pH

Native and modified�-chymotrypsin preparations wereincubated at 35◦C in 50 mM Tris–HCl buffer, pH 9.0.Aliquots were removed at different times, diluted in 50 mMsodium phosphate buffer, pH 7.0 and further assayed for es-terolytic activity.

3. Results and discussion

3.1. Synthesis and structural properties of the conjugate

The synthesis of CPCD-modified�-chymotrypsin wascarried out in three steps, as depicted inScheme 1. Thefirst step involved the cross-linking of�-cyclodextrin withepichorohydrin in alkaline medium, to give a randomlypolymerized material with an average molecular mass of

M. Fernandez et al. / Process Biochemistry 39 (2004) 535–539 537

Scheme 1. Preparation of the conjugate�-chymotrypsin–CPCD.�CD represents�-cyclodextrin.

1.3× 104, as determined by gel filtration chromatography.In the second step, the above obtained polymer was treatedwith monochloroacetic acid in alkaline medium with theaim of attaching carboxymethyl moieties to the remaininghydroxyl groups. The IR spectrum of this product showedthe characteristic ?C=O band at 1695 cm−1. Potentiometrictitration of the –OCH2COOH groups with alkali gave adegree of substitution of 40% (mol mol−1 d-glucose) forCPCD.

Finally, CPCD was covalently linked to bovine pancreatic�-chymotrypsin using EDAC as a coupling agent. The car-bohydrate content in the conjugate prepared was estimatedas 74% by weight of transformed protein. The extent of mod-ification of the protein, assessed by determining the amountof free amino groups before and after the polysaccharide waslinked to the enzyme molecule[20], was estimated as 26%(mol mol−1 protein) for the�-chymotrypsin–CPCD adduct.According to the amino acid sequence of this protease[21],this result showed that four amino groups out of 17 of theenzyme were modified with the polymer.

3.2. Catalytic properties

The specific proteolytic activity of the polysaccharide-modified enzyme was 5.9× 10−2 kat kg−1, which repre-sents about 105% of the original caseinolytic activity of�-chymotrypsin. The esterolytic activity of�-chymotrypsintoward ATEE was slightly increased after modification withthe polymer: the enzyme–CPCD complex showed about115% of the initial activity toward ATEE. In the light of theseresults, it could be inferred that the active conformation ofthe enzyme was not affected by the covalent attachment ofCPCD.

The optimum temperature for esterolytic activity of�-chymotrypsin was increased by about 5◦C after mod-ification with CPCD, as illustrated inFig. 1. This resultsuggests that the conformational rigidity of the enzyme

protein structure was increased after the attachment of thepolymer, then requiring a higher temperature for expressingits maximum esterolytic activity[22].

3.3. Stability properties

From the practical point of view, thermotolerance is oneof the most important characteristics to be considered forapplying enzymes in industrial processes[1]. In the presentwork, two different types of experiments were carried outto determine the influence of the chemical attachment ofCPCD on�-chymotrypsin thermostability.

First, the thermal stability profile of free and modified�-chymotrypsin was examined by incubation for 10 min atdifferent temperatures (Fig. 2). Under these conditions, themodified enzyme was more stable at temperatures higherthan 45◦C, in comparison with native�-chymotrypsin. Con-sequently, the value of T50 was increased by about 5◦C for�-chymotrypsin after modification with CPCD.

In another set of experiments, the kinetics of thermalinactivation at a variety of temperatures, ranging from 45

Fig. 1. Optimum temperature of native (�) and CPCD-modified�-chymotrypsin (�).

538 M. Fernandez et al. / Process Biochemistry 39 (2004) 535–539

Fig. 2. Thermal stability profile of native (�) and CPCD-modified�-chymotrypsin (�).

to 55◦C, was determined for CPCD-modified enzyme andcompared with those obtained for free�-chymotrypsin.The attachment of the polysaccharide chains to the surfaceamino groups of�-chymotrypsin resulted in a noticeableresistance of the enzyme against inactivation at these tem-peratures (Table 1). The half-life times calculated for theconjugate were 2.6–12.1 fold higher than those correspond-ing to the native counterpart. This effect was maximal at50◦C, the temperature at which an increase of 6.7 kJ mol−1

in the standard free energy of thermal inactivation process,calculated as previously described[10], was estimated forthe CPCD-modified enzyme.

In the light of the results showed above (Fig. 2, Table 1) itcan be inferred that the attached polysaccharide chains serveto maintain the active conformation of the enzyme afterthermal treatment. This conformational stabilization shouldbe mainly associated with the intramolecular cross-linkingoriginated in the modified enzyme by the covalent multi-point attachment of the polymer chains[23,24]. In addition,the polyanionic nature of the modifying polysaccharidecould also promote the formation of multipoint salt bridgesat the protein surface, then reducing its polypeptide chainmobility after exposure at elevated temperatures. A simi-lar stabilizing effect was previously described for enzymesmodified with water-soluble anionic and cationic polyelec-trolytes[9,25,26].

Furthermore, another mechanism might contributein the present case to the observed thermostabilization.

Table 1Half-life times of native and CPCD-modified�-chymotrypsin prepara-tionsa

Temperature (◦C) Half-life (min)

�-Chymotrypsin �-Chymotrypsin–CPCD

45 243± 11 634± 1550 20.6± 0.7 250± 952 10.0± 0.8 49± 255 3.3± 0.5 14.4± 0.8

a Average of three measurements.

Cyclodextrins are able to form inclusion complexes withhydrophobic compounds[14]. Therefore, the occurrence ofsupramolecular interactions, involving the protein surfacehydrophobic amino acid residues and the attached cyclodex-trin moieties, could also be associated with the increasedthermostabilization shown by the conjugate. To assesswhether these interactions are present in this case a strategybased on comparing the behavior of both�-chymotrypsinforms at 50◦C in the presence and in the absence of asaturated 1-adamantanol solution (about 10−4 M−1) wasemployed. This molecule forms a highly stable 1:1 inclu-sion complex with cyclodextrins[27] (K∼104 M−1) andits presence in the incubated enzyme solution is expectedto disrupt any supramolecular interactions occurring in thepolymer-modified enzyme.

The protective effect of the polymer on the thermal resis-tance of the modified conjugate at 50◦C was reduced afteraddition of 1-adamantanol (Fig. 3). The half-life time of themodified enzyme was only 1.5-fold increased in the presenceof the guest 1-adamantanol with respect to the native coun-terpart. In contrast, an increase of about 12.1-fold was foundfor the modified enzyme without addition of 1-adamantanol.That means that the conjugate is more thermostable in theabsence of 1-adamantanol, which suggests the direct influ-ence of the supramolecular interactions on the thermal resis-tance conferred to the conjugate. Although the contributionof each of the cited possible interactions to the increase inthermostability cannot be directly measured with these ex-periments, it seems clear that they act together to producean overall stabilizing effect.

Fig. 4 reports the time-course of inactivation of nativeand CPCD-modified enzyme at pH 9.0. It is well knownthat the catalytic activity of�-chymotrypsin is drasticallyreduced at values of pH higher than 8.5, due to the break-age of the stabilizing salt bridge between the N-terminusof the B-chain (Ile 16) and the Asp 194 side chain[28].The polymer–enzyme complex was significantly more sta-ble against incubation at this value of pH than the native

Fig. 3. Kinetics of inactivation of native (�) and CPCD-modified�-chymotrypsin (�) at 50◦C in the presence (· · · · · · ) and the absence(—) of saturated 1-adamantanol.

M. Fernandez et al. / Process Biochemistry 39 (2004) 535–539 539

Fig. 4. Kinetics of inactivation at pH 9.0 for native (�) and CPCD-modified �-chymotrypsin (�).

counterpart (Fig. 4). This is revealed by the 11-fold increasein the half-life time of this process. This result suggests thatthe attached polymer chains protect the active enzyme con-formation at alkaline pH, avoiding the breakage of the citedsalt bridge in�-chymotrypsin.

4. Conclusion

The novel use of a cyclodextrin polymer as a modify-ing agent for enzymes is reported. The data reported hereinfor the conjugate�-chymotrypsin–CPCD demonstrated theeffectivity of this strategy for improving stability to thisprotein against thermal treatment and incubation at alka-line pH. Additionally, the direct influence of supramolecu-lar interactions cyclodextrin–protein on the thermoresistanceshowed by the conjugate was demonstrated. Attending tothese results, the induction of supramolecular interactions atthe surface of enzymes by their covalent modification withcyclodextrin-containing polymers might be a useful tool forimproving the functional stability of these biocatalysts.

Acknowledgements

This research was supported by the International Foun-dation for Science, Stockholm, Sweden, and the Orga-nization for the Prohibition of Chemical Weapons, TheHague, The Netherlands, through a grant to R. Villalonga(Grant F/3004-1). Financial support from The Third WorldAcademy of Sciences (Grant 01-279 RG/CHE/LA) is alsogratefully acknowledged.

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