Enzyme and Microbial Technology 34 (2004) 78–82

Effect of �-cyclodextrin-polysucrose polymer on thestability properties of soluble trypsin

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

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

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

Received 24 October 2002; accepted 8 September 2003

Abstract

Polymerized sucrose, previously modified with mono-6-hexylenediamino-6-deoxy-�-cyclodextrin, was evaluated as thermoprotectantadditive for trypsin. Maximal thermal stabilization was achieved by using highly substituted polysaccharides. The catalytic properties ofthe enzyme were not affected by the presence of the polymer. The optimum temperature for trypsin activity was increased by 10◦C whenadding the�-cyclodextrin containing polysaccharide. The enzyme thermostability was increased from 48.5 to 64.7◦C in the presence ofthe additive. Trypsin was also 22-fold more stable against thermal incubation at 50◦C when adding the polysaccharide. The protease wassixfold more resistant to autolytic inactivation at alkaline pH in the presence of the�-cyclodextrin-polysucrose polymer.© 2003 Elsevier Inc. All rights reserved.

Keywords:Trypsin;�-Cyclodextrin; Polymerized sucrose; Enzyme stability; Supramolecular interactions

1. Introduction

Highly thermostable enzymes able to catalyze reactionsin homogeneous systems are required for several indus-trial and biotechnological applications. Strategies for ther-mostabilizing enzymes in aqueous media have been mainlyaddressed to produce genetically[1,2], chemically [3–6]or enzymatically[7] modified enzyme variants, as well asto use water-soluble polyols as thermoprotectant additives[8,9]. Among these, the latter appears as the most economicapproach, taking into account the simplicity and low costof this method. In this regard, carbohydrates are the polyolsused most successfully as additives for stabilizing enzymesin homogeneous systems, considering both the number ofreports and the improvement on enzyme thermal stabilityachieved[9].

Cyclodextrins (CDs) are cyclic non-reducing oligosac-charides composed of 6 (�CD), 7 (�CD) or 8 (�CD)�-(1–4)-linked d-glucopyranose units in the4C1 chairconformation, with a hydrophobic central cavity and a hy-drophilic outer surface[10]. CDs are capable to includea wide variety of hydrophobic guest compounds, such as

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

several amino acids side chains located at the surface ofproteins [11]. Recently, we reported new strategies forimproving the thermal stability properties of enzymes bychemical or enzymatic conjugation with several CD deriva-tives [7,12–14]. In addition, we demonstrated that the sta-bilizing effect conferred to these conjugated enzymes wasmediated by the occurrence of CD–protein supramolecularinteractions at the surface of the enzyme[14,15].

Attending to these facts, here we propose a novel approachfor increasing enzyme functional stability by means of theinduction of multipoint supramolecular interactions at theprotein surface of enzymes in the presence of CD-containingpolymers. Regarding this, the present work describes theinfluence of�CD-polysucrose polymers on the resistance oftrypsin (EC 3.4.21.4) to thermal and autolytic inactivationin aqueous solutions.

2. Materials and methods

2.1. Materials

Bovine pancreatic trypsin (30 U/mg versus BAEE[16],3.0 × 10−2 katal/kg versus casein[17]), NaBH4, m-NaIO4and N-�-benzoyl-l-arginine ethyl ester hydrochloride

0141-0229/$ – see front matter © 2003 Elsevier Inc. All rights reserved.doi:10.1016/j.enzmictec.2003.09.003

M. Fernandez et al. / Enzyme and Microbial Technology 34 (2004) 78–82 79

(BAEE) were obtained from Merck. Polymerized sucrose(Ficoll 70) and CM-Shephadex C-25 were obtained fromPharmacia Biotech.�CD was purchased from Amaizo. Allother chemicals were of analytical grade.

2.2. Preparation ofβCD-polysucrose polymers

Mono-6-hexylenediamino-6-deoxy-�CD was previ-ously obtained by treating mono-6-O-tosyl-�CD [18] with1,6-hexylenediamine in DMF[19]. The product was puri-fied by cation exchange chromatography on CM-ShephadexC-25 (NH4

+ form) and characterized by conventional NMRtechniques. Polymerized sucrose was oxidized by dissolv-ing 100 mg of the polymer in 5 ml of H2O and treated with400 mg ofm-NaIO4 under continuous stirring at 4◦C in thedark for 2 h. The oxidation reaction was stopped by adding100�l of ethylene glycol and stirred for 1 h, and furtherdialyzed against distilled H2O. For the preparation of the�CD-containing polymers, portions of 10, 20, 30 and 50 mgof mono-6-hexylenediamino-6-deoxy-�CD were added to5 ml aliquots of the activated polymer and then treated with20 mg of NaBH4 for 4 h under continuous stirring. Themodified polymer solutions were further dialyzed againstdistilled H2O and finally lyophilized.

2.3. Assays

The esterolytic activity of native and modified trypsin wasdetermined at 25◦C in 67 mM Tris–HCl buffer, pH 8.0, us-ing BAEE as substrate[16]. One unit of esterolytic activ-ity was defined as the amount of enzyme that hydrolyses1.0�mol of BAEE per minute at 25◦C. Proteolytic activitywas determined as described by Laskowski[17] using milkcasein as substrate. One unit of proteolytic activity, katal,is defined as the amount of enzyme that releases one moleof tyrosine per second at 25◦C. Total carbohydrates weredetermined by the phenol-sulfuric acid method[20] usingglucose as standard. Protein concentration was estimated asdescribed by Lowry et al.[21] using bovine serum albuminas standard. The amount of�CD residues attached to thepolymer was determined by colorimetric titration with phe-nolphthalein[22].

2.4. Kinetics of thermal inactivation

Trypsin preparations were incubated at different temper-atures ranging from 45 to 70◦C in 50 mM sodium acetatebuffer, pH 5.0 (0.04 mg protein/ml), in the presence and ab-sence of the polysaccharide solutions. Aliquots were re-moved at scheduled times, chilled quickly, and assayed forenzymatic activity.

2.5. Optimum temperature

The esterolytic activity of trypsin preparations (0.04 mgprotein/ml) was measured at different temperatures ranging

from 35 to 80◦C in the presence and absence of the polysac-charide solutions (0.7 mg polymer/ml). The correspondingvalues of optimum temperature were calculated from Arrhe-nius plots.

2.6. Thermal stability profile

Trypsin preparations were incubated at scheduledtemperatures in 50 mM sodium acetate buffer, pH 5.0(0.04 mg protein/ml), in the absence and presence of thepolysaccharide solutions (0.7 mg polymer/ml). Aliquotswere removed after 10 min of incubation, chilled quicklyand assayed for esterolytic activity.

3. Results

Four�CD-polysucrose polymers were prepared by treat-ing the periodate-oxidized polysaccharide with differentamounts of the amino-cyclodextrin under reductive con-ditions. The �CD content in these polysaccharides wasestimated as 18, 32, 51 and 116 mol of�CD per mol ofpolymer, respectively.

The influence of both modified and non-modified polysac-charides on the thermal inactivation of trypsin at 50◦C isreported inFig. 1. We defined the thermoprotective effect ofthe polysaccharides as the ratio between the half-life times(t1/2) of trypsin in the presence and in the absence of eachadditive. The thermal resistance of trypsin at this temper-ature was slightly increased in the presence of the nativepolysaccharide. On the contrary, a remarkable stabilizationwas conferred to the enzyme when adding the�CD-modifiedpolymers. Interestingly, this stabilizing effect increases whenthe amount of�CD attached to the polymeric chains in-creases, achieving the maximal thermal stabilization for themost substituted polymer (116 mol of�CD per mol of poly-mer). This polysaccharide was then selected for further ex-periments.

Fig. 1. Effect of�CD content in the modified polysucrose polymers onthe thermal stabilization of trypsin at 50◦C.

80 M. Fernandez et al. / Enzyme and Microbial Technology 34 (2004) 78–82

Fig. 2. Effect of�CD-polysucrose polymer concentration on the thermalstabilization of trypsin at 50◦C.

In order to determine the optimum concentration of thishighly substituted polymer for trypsin stabilization, thet1/2of the enzyme at 50◦C was determined in the presence ofdifferent concentrations of the additive. As is illustrated inFig. 2, t1/2 values increase progressively when the concen-tration of the polymer increases, reaching maximal stabiliza-tion at values higher than 0.7 mg/ml polymer concentration.Consequently, this value of optimum polymer concentrationwas selected for further experiments.

The functional properties of trypsin in the presence ofthe selected additive at 0.7 mg/ml concentration were fur-ther evaluated. A physical mixture of�CD and polymerizedsucrose having the same molar composition of the preparedpolymer was tested as control in all experiments further de-scribed. The catalytic properties of the protease were notaffected by adding the�CD-polysucrose polymer. The spe-cific esterolytic and proteolytic activities, as well as the cat-alytic constants, remained without variations in the presenceand absence of the polysaccharide.

Fig. 3 depicts the behavior of the esterolytic activity oftrypsin preparations as a function of temperature. The opti-mum temperature for catalytic activity of the enzyme was

Fig. 3. Optimum temperature for trypsin in the absence (�) andpresence of�CD-polysucrose polymer (�) and control (�) solutions(0.7 mg polymer/ml).

Fig. 4. Thermal stability profile of trypsin in the absence (�) andpresence of�CD-polysucrose polymer (�) and control (�) solutions(0.7 mg polymer/ml).

increased from 55 to 61◦C after adding the control mixture.On the other hand, an increase of about 10◦C was deter-mined for trypsin in the presence of the�CD-polysucrosederivative.

Fig. 4 depicts the thermal stability profile of trypsin af-ter 10 min of incubation at different temperatures in theabsence and presence of the additives. The enzyme prepa-rations containing both the�CD-polysucrose derivative andthe control mixture were more stable at temperatures higherthan 45◦C, in comparison with the free enzyme (Fig. 4).This stabilizing effect was remarkably higher when addingthe �CD-polysucrose polymer to trypsin solutions. In thisregard, the value ofT50, defined as the temperature at which50% of the initial activity was retained, was increased fortrypsin from 48.5◦C to about 64.7◦C after addition of themodified polymer. On the contrary, the value ofT50 fortrypsin was only increased by about 4◦C in the presence ofthe control mixture.

The time course of thermal inactivation of trypsin prepa-rations in the range of temperatures between 45 and 70◦Cis reported inTable 1. No significant differences were foundfor trypsin in the absence and presence of the control mix-ture (data not shown). On the other hand,t1/2 evaluated ateach temperature was significantly increased for the enzymein the presence of the�CD-polysucrose polymer. This sta-bilizing effect was remarkably higher at 50◦C, temperature

Table 1Half-life times of trypsin in the absence and presence of�CD-polysucrosepolymer

Temperature (◦C) Half-life time (min)

Trypsin Trypsin+ �CD-polysucrose

45 60± 3 378± 1650 15.6± 0.4 347± 1055 8.0± 0.7 90.1± 0.460 5.3± 0.1 43.0± 0.470 – 20.5± 0.3

M. Fernandez et al. / Enzyme and Microbial Technology 34 (2004) 78–82 81

Fig. 5. Kinetics of autolytic degradation of trypsin at pH 9.0 in theabsence (�) and presence of�CD-polysucrose polymer (�) and control(�) solutions (0.7 mg polymer/ml).

at whicht1/2 of trypsin was 22-fold higher after addition ofthe �CD-containing polymer. The thermal stabilization ofthe enzyme in the presence of the additive corresponds toan increase of 8.4 kJ/mol.

Fig. 5 depicts the autolytic behavior of trypsin prepara-tions at pH 9.0 and 30◦C. As can be seen, the enzyme wasnot protected against autolytic degradation at alkaline pH inthe presence of the control mixture. On the contrary, the ad-dition of the�CD-polysucrose polymer to trypsin solutionsprevents the autolytic inactivation of the protease. In thissense,t1/2 of the enzyme preparation containing the modi-fied polysaccharide was sixfold higher than that correspond-ing to the free enzyme preparation.

4. Discussion

Polyols have been successfully used as additives for im-proving the thermal stability of enzymes in aqueous andnon-aqueous media[8,9]. In general, high concentrationsof polyols ranging from 1 to 5 M are required to achievethis goal [9]. In this work, we described the novel use of�CD-containing polymers as thermoprotectant additives forenzymes. In this sense, a remarkable thermal stabilizationwas conferred to bovine pancreatic trypsin in the presenceof low concentration of the polyol (about 2 mM as sucrose).This effect was achieved without affecting the esterolyticand proteolytic properties of trypsin, suggesting that boththe structure of the active site of the enzyme and the dif-fusion mechanism of substrates were not disturbed by thepresence of the polymer.

Thermostabilization of enzymes in the presence of polyolshas been associated with several mechanisms, i.e., the directspecific or not specific interaction between the additive andthe polypeptide enzyme structure[23], increase in the freeenergy of protein denaturation[24] and changes in the struc-ture of water[25]. In the present study, it is evident that theinfluence of the additives on the stability properties of trypsin

was also mediated by the presence of the�CD moieties inthe macromolecular structure of the polysaccharide, accord-ing to the data reported for the control experiments (Figs. 3and 4). This fact was also supported by the results reported inFig. 1, in which the stabilizing effect conferred to trypsin wasdirectly associated with the amount of�CD attached to thepolymers.

A possible explanation for this phenomenon could bethe formation of multipoint supramolecular associationsbetween the�CD residues attached to the polymeric chainsand the hydrophobic amino acid residues located at thesurface of trypsin. According to this mechanism, the poly-meric molecules could increase the rigidity of the enzymestructure through non-covalent multipoint cross-links, re-ducing the protein chain mobility of trypsin and then in-creasing its optimum temperature and its resistance to heattreatments. Recently, we demonstrated that a similar mech-anism was involved in the thermal stabilization showedby enzymes chemically modified with CD derivatives and�CD-containing polymers[14,15].

It is well known that the inactivation of trypsin at al-kaline pH constitutes a self-degradation process[26] andthis phenomenon often limits the use of trypsin in sev-eral practical applications. Interestingly, a noticeable sta-bilization was conferred to trypsin in the presence of the�CD-containing polymer. On the contrary, no significanteffect was showed when adding the control mixture, sug-gesting that the modified polysaccharide protects the en-zyme against autolytic degradation. This effect could alsobe mediated by the supramolecular association mechanismproposed above. Consequently, the�CD-polysucrose chainscould cover the enzyme surface, masking the potential cleav-age sites on the trypsin molecules.

5. Conclusions

In this work, we propose the novel use of a�CD-containingpolymer as additive for improving the functional stabilityof enzymes in aqueous solutions. Independent of any mech-anistic conclusions, the remarkable stabilization conferredto trypsin in the presence of the�CD-polysucrose poly-mer is relevant from a practical point of view. Experimentsare now in progress to generalize this method of enzymestabilization.

Acknowledgments

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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