Enzyme and Microbial Technology 31 (2002) 543–548

Chemical conjugation of trypsin with monoaminederivatives of cyclodextrins

Catalytic and stability properties

Michael Fernándeza, 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, Matanzas, CP 44740, Cuba

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

Received 13 December 2001; received in revised form 23 April 2002; accepted 10 May 2002

Abstract

Bovine pancreatic trypsin was chemically modified by the mono-6-amino-6-deoxy derivatives of�-, �-, and�-cyclodextrin, using1-ethyl-3-(3-dimethylaminopropyl) carbodiimide as a coupling agent. The enzyme–cyclodextrin conjugates contained about 2 mol ofoligosaccharide per mole of trypsin. The specific esterolytic activity and the affinity of trypsin for substrate were improved by theattachment of the cyclodextrin residues. The thermostability of the enzyme was increased in about 14–17.2◦C after modification. Theconjugates prepared were also more stable against thermal incubation at different temperatures ranging from 45 to 60◦C. In comparisonwith native trypsin, the cyclodextrin–enzyme complexes were about 5.5- to 8.2-fold more resistant to autolytic degradation at pH 9.0.© 2002 Elsevier Science Inc. All rights reserved.

Keywords: Trypsin; Cyclodextrin; Modified enzyme; Enzyme thermostability

1. Introduction

From the industrial point of view, processes catalysed bythermoresistant enzymes are very desired[1]. In this regard,there are several important technological aspects favouredby carrying out enzymatic reactions at high temperatures,such as (a) increase of reaction rate and operational stability,(b) the shift of thermodynamic equilibrium, (c) increasedsolubility of reactants and products, (d) decreased viscosityof the reaction medium, and (e) reduced microbial contam-ination [2,3].

Several methods based on protein engineering[4], immo-bilisation in solid supports[5], isolation from thermophilicorganisms[6], the use of additives[7], and chemical mod-ification with polymeric [8,9] and low-molecular weightcompounds[10] have been reported to be successful forpreparing thermostable enzymes. However, among thesestrategies, chemical modification of enzymes appears tobe the most promising approach for preparing thermore-sistant enzymes able to work in homogeneous systems[9].

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

Undoubtedly, carbohydrates are one of the most com-monly used modifying agents for enzymes[8,9,11,12]. Thisselection has been inspired by naturally occurring glycoen-zymes, in which the glycosidic chains play a relevant rolein the stability behaviour of these proteins[13]. In thissense, many reports have been devoted to the modification ofenzymes by ionic[8,14,15]and non-ionic polysaccharides[9,12], as well as with mono- and oligosaccharide residues[12,16].

The aim of the present work is to evaluate the noveluse of monoamino derivatives of cyclodextrins (CD),named mono-6-amino-6-deoxy-�-CD (�CDNH2), mono-6-amino-6-deoxy-�-CD (�CDNH2), and mono-6-amino-6-deoxy-�-CD (�CDNH2), as enzyme modifiers. In thisregard, the synthesis of several trypsin–CD conjugates is re-ported, as well as the behaviour of these modified enzymesagainst several denaturing conditions.

CDs are versatile cyclic oligosaccharides than can forminclusion complexes with a great number of hydrophobicguest compounds[17]. As a result, they have been exten-sively used in different applications such as drug deliv-ery, enzyme mimics and chiral chromatography[17,18,19].However, this type of compound is an alternative, whichdoes not seem to have been used for enzyme modification.

0141-0229/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved.PII: S0141-0229(02)00151-5

544 M. Fernandez et al. / Enzyme and Microbial Technology 31 (2002) 543–548

2. Materials and methods

2.1. Materials

Bovine pancreatic trypsin, Fractogel EMD BioSEC (S),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochlo-ride (EDAC) andN-�-benzoyl-l-arginine ethyl ester hy-drochloride (BAEE) were obtained from Merck (Darmstadt,Germany). CDs were purchased from Amaizo (USA) andused as received. CM-Sephadex C-25 was purchased fromPharmacia Biotech (Uppsala, Sweden). All other chemicalswere of analytical grade.

2.2. Synthesis of CD derivatives

The CD derivatives were obtained by treating the corre-sponding mono-6-O-tosyl derivative[20] with 35% aqueousammonia[19]. The modified oligosaccharides were purifiedby ion exchange chromatography on CM-Sephadex C-25(NH4

+ form). The purity and identity of these productswas checked by TLC,1H and 13C NMR and positive-ionFAB-MS.

2.3. Preparation of trypsin–CD conjugates

Thirty milligrams of EDAC were added to reaction mix-tures containing 20 mg of trypsin dissolved in 10 ml of50 mM potassium phosphate buffer, pH 6.0, and 100 mgof each CD derivatives. The solutions were stirred for 1 hat room temperature and for 16 h at 4◦C, and further ap-plied to a gel filtration column Fractogel EMD BioSEC(S) (2.6 cm× 60 cm), equilibrated in the same buffer made100 mM of NaCl. The active fractions containing carbohy-drates were pooled and kept at 4◦C.

2.4. Assays

Esterolytic activity of native and modified trypsins wasdetermined at 25◦C in 67 mM Tris–HCl buffer, pH 8.0 usingBAEE as substrate[21]. One unit of esterolytic activity isdefined as the amount of enzyme that hydrolyses 1.0�molof BAEE/min at 25◦C. Total carbohydrates were determinedby the phenol–sulfuric acid method[22] using glucose asstandard. Protein concentration was estimated as describedby Lowry et al.[23] using bovine serum albumin as standard.

The association constants between BAEE and the nativeCDs were determined by1H NMR spectrometry[24] usinga Bruker AC 250 spectrometer at 250.13 MHz apparatus. A0.01 M solution of the corresponding CD in D2O at pD 8.0and 25◦C was titrated with BAEE at different BAEE:CDmolar ratios (usually from 0 to 2) and the induced chemicalshift differences of the inner H-3 protons of CD were deter-mined. The values of the association constants were deter-mined by least-squared fitting of the experimental values tothe theoretical equation obtained considering 1:1 complex-ation stoichiometry[24].

2.5. Thermostability

Native and modified trypsin preparations were incubatedat different temperatures in 20 mM sodium acetate buffer,pH 5.0. Aliquots were removed after 10 min incubation, di-luted in cold 0.1 M Tris–HCl buffer, pH 8.0, and assayed foresterolytic activity.

2.6. Thermal inactivation

Native and modified trypsin preparations were incubatedat different temperatures ranging from 45 to 70◦C in 50 mMsodium acetate buffer, pH 5.0. Aliquots were removed atscheduled times, chilled quickly, and assayed for enzymaticactivity. The first-order rate constants of inactivation,ki ,were obtained from linear regression in logarithmic coordi-nates. The activation free energy of inactivation (�Gi) forall enzyme forms was calculated according to the followingequation:

ki =(

kBT

h

)exp

(−�Gi

RT

)

whereki is the first-order inactivation rate constant (h−1),kB the Boltzmann’s constant (J/K),h the Planck’s constant(J h), R the gas constant (J/mol K) andT is the absolutetemperature.

2.7. Autolysis

Native and modified trypsin forms were incubated at 35◦Cin 50 mM Tris–HCl buffer, pH 9.0. Aliquots were removedat different times, diluted in 0.1 M sodium acetate buffer, pH5.0, and further assayed for esterolytic activity.

3. Results

The synthesised CD derivatives (Fig. 1), in which one�-d-glucopyranose residue contained an amino group at

Fig. 1. Structure of the mono-6-amino-6-deoxy derivatives of�, � and�CD.

M. Fernandez et al. / Enzyme and Microbial Technology 31 (2002) 543–548 545

Table 1Structural and catalytic properties of native and CD-modified trypsins

Enzyme form Parameters

CD content(mol/mol enzyme)

Specificactivity (U/mg)

Km (�M)

Trypsin – 36.0 35.5Trypsin–�CD 2 50.4 20.0Trypsin–�CD 2 52.2 23.8Trypsin–�CD 2 57.6 24.3

C-6 position, were previously tested for esterolytic activitytowards BAEE as described for trypsin. Under these con-ditions, none of these oligosaccharides showed esterolyticactivity. The activated CDs were further attached to bovinepancreatic trypsin using EDAC as coupling agent.

The carbohydrate content of the�CDNH2-, �CDNH2-and�CDNH2-modified trypsins was estimated as 8, 9.5 and11% by weight of transformed enzyme, respectively. Thesevalues represented an amount of 2 mol of oligosaccharidesattached to each mole of protein in the CD–enzyme com-plexes, as is shown inTable 1.

Fig. 3. Kinetics of thermal inactivation of native (A) and modified trypsin with�CDNH2 (B), �CDNH2 (C) and�CDNH2 (D) derivatives at 45◦C (×),50◦C (�), 55◦C (�) and 60◦C (�).

Fig. 2. Thermal stability profile of native (�) and modified trypsin with�CDNH2 (�), �CDNH2 (�) and �CDNH2 (×) derivatives.

Table 1 reports the catalytic properties of the preparedadducts. The specific esterolytic activity of the�CDNH2–,�CDNH2– and �CDNH2–enzyme complexes representedabout 140, 145 and 160% of the original enzymatic activityof trypsin. In addition, the attachment of CD moieties en-hanced the affinity of the enzyme for the substrate BAEE:native and�CDNH2-, �CDNH2- and �CDNH2-modified

546 M. Fernandez et al. / Enzyme and Microbial Technology 31 (2002) 543–548

Table 2Half-life times of native trypsin and trypsin–CD conjugates at different temperatures

Temperature (◦C) Half-life (min)

Trypsin Trypsin–�CD Trypsin–�CD Trypsin–�CD

45 60± 3 131± 4 139± 5 186± 850 16± 1 88 ± 2 75 ± 1 108± 455 8.0± 0.7 50± 1 55 ± 1 57 ± 260 5.3± 0.1 38± 1 37 ± 2 40 ± 1

trypsins gaveKm values of 35.5, 20.0, 23.8 and 24.3�M, re-spectively. However, the optimum pH for enzymatic activityof trypsin (8.0) remained the same after modification.

The thermal stability profile of native and CD-modifiedtrypsins, determined for the esterolytic activity retained af-ter heating the enzymes at different temperatures for 10 min,is shown inFig. 2. The modified enzymes are more stableat temperatures higher than 45◦C, in comparison with thenative counterpart. The values ofT50, defined as the tem-perature at which 50% of the initial activity was retained,was increased in about 17.2, 14 and 17◦C after modificationwith �, � and�CDNH2 derivatives, respectively.

Fig. 3 reports the kinetics of thermal inactivation of allenzyme forms exposed at different temperatures from 45 to60◦C. During 1 h of incubation, all enzyme preparations lostactivity progressively with time according to a first-orderkinetics, but the half-life of the transformed enzymes weremarkedly higher than that of native trypsin (Table 2).

As can be seen fromTable 2, the resistance to heat in-activation was higher at 60◦C for all the CD–trypsin com-plexes. This stabilising effect corresponds to an increase inabout 5.3–5.6 kJ/mol for the free energy of activation of thethermal inactivation processes at this temperature.

The time course of autolysis for native and CD-modifiedtrypsins at pH 9.0 is shown inFig. 4. Under these condi-tions, all enzyme forms showed similar autolytic patterns.However, the rate of self-digestion of the CD–trypsin com-plexes was noticeably lower than the corresponding to thenative form. In this sense, the half-life of the enzyme modi-fied by the�, � and�CDNH2 derivatives were 6.0-, 5.5- and

Fig. 4. Kinetics of autolytic degradation for native (�) and modifiedtrypsin with �CDNH2 (�), �CDNH2 (�) and�CDNH2 (×) derivatives.

8.2-fold higher than the corresponding to the non-modifiedtrypsin, respectively.

4. Discussion

Several modification methods have been previously re-ported for stabilising trypsin: covalent cross-linking[25],attachment of ionic [26,27] and non-ionic polymers[12,28,29], and chemical transformation with low-molecularweight compounds[10,30]. Here we described the noveluse of several amino–CD as modifying agents for trypsin.

The covalent attachment of CD residues to trypsin resultedin noticeable advantages regarding its functional stabilityand catalytic behaviour. In this sense, the esterolytic activityof trypsin was increased after chemical transformation withthe CD residues. This phenomenon could be attributed to thehigher affinity for BAEE showed by the prepared adducts.In fact, the Michaelis constant were reduced, relative to thenative form, in about 1.5–1.8-fold after modification withthe oligosaccharide residues.

This change in the affinity for substrate could be associ-ated with the fact that the CD residues, located at the pro-tein surface of trypsin, can form inclusion complexes withcompounds containing aromatic groups, like BAEE[17]. Inthis regard, we determined by1H NMR that the associationconstants for the inclusion complex between the BAEE andunmodified�, � and�CD in D2O at pD 8.0 and 25◦C were420, 180 and 100 M−1, respectively. This kind of inclusioncomplex might increase the concentration of BAEE at themicroenvironment of the active site, increasing the affinityof the modified forms for the substrate by shifting the equi-librium to the formation of the Michaelis complex.

The relationship between the association constants for theCD–BAEE complexes and the decrease in the Michaelisconstant of the conjugates prepared supports this hypothe-sis. In fact, a lowerKm of the modified forms of trypsincorresponds to higher association constant of the complexBAEE–CD.

In addition, the attachment of CD moieties could per-turb the water activity and arrangement in the vicinity ofthe active site, changing the substrate binding ability ofthe enzyme. In this regard, three clusters of ordered watermolecules have been identified in trypsin that connect theactive site and the binding pocket with the bulk solvent

M. Fernandez et al. / Enzyme and Microbial Technology 31 (2002) 543–548 547

region [31]. Analysing similar patterns in other serineproteases, the authors concluded that such channels pro-vide conserved mechanisms for water displacement by thesubstrate.

Chemical modification of trypsin by CDs resulted in a re-markable thermal stabilisation. This effect was reflected ei-ther by the increased values ofT50, as well as by the higherhalf-life at different temperatures showed by the preparedconjugates. In comparison with the results previously re-ported by Murphy and Fágáin[10] and Villalonga et al.[27],the strategy described here seems to be a more effective toolfor improving the thermal stability of trypsin.

Since protein aggregation plays a determinant role inthe thermal inactivation mechanism of trypsin[32], an im-portant factor contributing to the heat resistance shown bythe modified enzymes could be the hydrophilisation of theprotein surface by the oligosaccharide moieties. This hy-drophilisation effect prevents the formation, during the ther-moinactivation processes, of new intra- and intermolecularinteractions, as has been postulated by Mozhaev et al.[30].On the other hand, the masking of the hydrophobic aminoacid residues at the surface of trypsin by the CD moietiescan also prevent the energetically unfavourable interactionof these amino acid residues with the surrounding watermolecules[30].

In view of our results, it is also interesting to mention thefindings of Cooper[33], who reported that CDs when addedto a protein solution reduce its mean unfolding temperature.In this regard, the author concluded that CDs form inclu-sion complexes with the side chains of buried hydrophobicamino acid residues of the protein, shifting the equilibriumin favour of the unfolded polypeptide. In our case, it hasbeen demonstrated that the attachment of some CD residuesto the enzyme surface reduced its thermal unfolding by sta-bilisation of the folded conformation of the protein. This isconfirmed by the residual activity showed by the modifiedtrypsins after heat treatment.

In addition to the factors cited above, the stabilisation ofthe native conformation of trypsin can also be associatedto the formation of intramolecular inclusion complexes be-tween the attached CDs and the aromatic amino acid residueslocated in the vicinity of the modification points[17]. In thissense, it is expected that the hydrophobic nature of these“cross-links” can contribute to the increased stability showedby the CD-modified trypsin at high temperatures. In fact, inglobular proteins it has been previously demonstrated thatthe stabilising effect caused by the hydrophobic interactionsincreases with the increase of temperature[34].

It is well known that autolytic processes are involvedin all denaturation mechanisms of proteases, because theunfolded protein molecules are more prone to autolysisthan the corresponding folded form[35]. That is the reasonfor which the development of new methods for stabilisingproteases against autolysis receives considerable attention.In the present work, a noticeable reduction of the rate ofself-digestion at pH 9.0 was induced for trypsin by chemical

modification with the CD derivatives. This stabilisationcould be associated with the steric hindrance caused by thebulky CD residues to the cleavage sites in trypsin. Thishypothesis is supported by the higher resistance to autolysisshowed by the�CD–trypsin complex (8�-d-glucopyranoseresidues in�CD), in comparison with the correspond-ing to the enzyme modified by the other CD derivatives(6 and 7 �-D-glucopyranose residues in� and �CDs,respectively).

5. Conclusion

In the present work, we have reported the chemical mod-ification of trypsin by the monoamine derivatives of�, �and�CD. The effectiveness of this strategy has been demon-strated by the improved stability and catalytic propertiesshowed by the synthesised conjugates. From the practicalpoint of view, the increased resistance of CD–trypsin con-jugates to autolysis and temperature is very interesting. Ac-cording to the results described in this paper, we proposethis modification procedure as a useful tool for improvingthe functional and stability properties of enzymes.

Acknowledgments

This research was supported by the International Foun-dation for Science, Stockholm, Sweden, and the Organisa-tion for the Prohibition of Chemical Weapons, The Hague,The Netherlands, through a grant to R. Villalonga (GrantF/3004-1).

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