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Process Biochemistry 40 (2005) 2091–2094

Stabilization of a-chymotrypsin by chemical modification

with monoamine cyclodextrin

Michael Fernandeza, Alex Fragosob, Roberto Caob,Reynaldo Villalongaa,*

aEnzyme Technology Group, Center for Biotechnological Studies, University of Matanzas, Matanzas, C.P. 44740, CubabLaboratory of Bioinorganic Chemistry, Faculty of Chemistry, University of Havana, Havana 10400, Cuba

Received 10 October 2003; received in revised form 3 July 2004; accepted 16 July 2004

Abstract

Bovine pancreatic a-chymotrypsin was chemically modified with mono-6-amino-6-deoxy-b-cyclodextrin. The modified enzymes

contained about 2 mol of oligosaccharide per mol of protein and retained full proteolytic and esterasic activity. The optimum temperature

for a-chymotrypsin was increased by 5 8C and its thermostability was enhanced by about 6 8C after modification. The glycosylated enzyme

turned markedly more resistant to thermal inactivation at 50 8C and retained 70% of the original activity when pre-incubated at pH 9.0 for

180 min as compared to a complete inactivation seed for the unmodified protease.

# 2004 Elsevier Ltd. All rights reserved.

Keywords: a-Chymotrypsin; Aminated b-cyclodextrin; Modified enzyme; Enzyme stability

1. Introduction

There is considerable interest in developing new methods

that can be used to increase the functional stability of an

enzyme by manipulation of its protein structure. Artificial

glycosylation of enzymes by chemical derivatization with

carbohydrate compounds has been successfully employed as

a tool for increasing enzyme stability and preparing more

efficient biocatalysts for industrial applications [1–6]. These

strategies have been supported by the fact that in nature

glycoproteins are more stable than similar non-glycosylated

counterparts [7]. Neutral and ionic polysaccharides con-

stitute the carbohydrate most commonly used for enzyme

modification [8–12]. However, glycosylation of enzymes

with macromolecular substances often yield adducts with

reduced catalytic activity. For this reason, the evaluation of

new carbohydrate derivatives as modifying agents for

enzymes receives considerable attention in biotechnology.

* Corresponding author. Tel.: +53 45 26 1251; fax: +53 45 25 3101.

E-mail address: reynaldo.villalonga@umcc.cu (R. Villalonga).

0032-9592/$ – see front matter # 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.procbio.2004.07.023

In previous reports, the use of cyclodextrin (CD)

derivatives to modify enzymes has been proposed [13–15].

CDs are a group of cyclic oligosaccharides containing 6, 7

or 8 /-(l!4)-linked D-glucopyranose units in the 4C1 chair

conformation which are named a-, b-, and g-CDs,

respectively. The structure of these molecules resembles

a truncated annular cone with a central cavity, which is

hydrophobic in nature and has the appropriate size to

include a wide variety of hydrophobic compounds and

aromatic residues of proteins [16]. The stability of these

guest–host complexes has been extensively studied due to

their potential applications in pharmacology, enzyme

mimicking and chromatography [16]. Recently, the

supramolecular-mediated stabilization of bovine pancrea-

tic trypsin using several monoactivated CD derivatives as

well as CD-grafted polysaccharides was reported [13–

15,17].

In this paper, we describe the chemical modification of

chymotrypsin with mono-6-amino-mono-6-deoxy-b-CD

derivative and the effects of this glycosylation on the

catalytic and stability properties of the protease.

M. Fernandez et al. / Process Biochemistry 40 (2005) 2091–20942092

Table 1

Structural and catalytic properties of native and CD-modified a-chymo-

trypsin preparations

Property a-chymotrypsin a-chymotrypsin–CD

Carbohydrate content (%, w/w) – 9.1

Proteolytic activity (katal/kg) 5.6 � 10�2 5.4 � 10�2

Esterase activity (U/mg) 45.0 44.8

2. Experimental

2.1. Materials

Bovine pancreatic a-chymotrypsin (45 U/mg), N-a-

acetyl-L-tyrosine ethyl ester hydrochloride (ATEE), 1-

ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC),

NaBH4 and Fractogel EMD BioSEC (S) were obtained

from Merck (Darmstadt, Germany). b-CD was purchased

from Amaizo (USA) and used as received. All other

chemicals were analytical grade.

2.2. Preparations of the enzyme–CD conjugate

The activated CD was obtained by treating the correspond-

ing mono-6-O-tosyl derivative [18] with 35% aqueous

ammonia [19]. The modified oligosaccharide was purified

by ion exchange chromatography on CM-Sephadex C-25

(NH4+ form). The purity and identity of this product, CDN, was

checked by TLC, 1H and 13C NMR and positive-ion FAB–MS.

The conjugate a-chymotrypsin–CD was further prepared

by adding 50 mg EDAC to a reaction mixture containing

50 mg of the activated oligosaccharide and 10 mg of

protease dissolved in 5 ml of 50 mM sodium phosphate

buffer (pH 6.0). The solution was stirred for 1 h at room

temperature and for 16 h at 4 8C. The solution was further

dialyzed against 20 mM sodium acetate buffer, pH 5.0, and

then applied to a gel filtration column Fractogel EMD

BioSEC (S) (2.6 � 60 cm), equilibrated in the same buffer

made 100 mM NaCl. The fractions containing the poly-

saccharide–enzyme complex were pooled and kept at 4 8C.

2.3. Assays

The esterase activity of native and modified a-

chymotrypsins was determined at 25 8C in 50 mM sodium

phosphate buffer, pH 7.0 using ATEE as substrate [20]. One

unit of esterase activity is defined as the amount of enzyme

that hydrolyses 1.0 mmol of ATEE per minute at 25 8C.

Proteolytic activity was determined as described by Zhong

et al. [21] using milk casein as substrate. Total carbohydrates

were determined by a phenol-sulphuric acid method [22]

using glucose as standard. Protein concentration was

estimated as described by Lowry et al. [23] using bovine

serum albumin as standard.

2.4. Optimum temperature

Native and modified enzyme preparations were assayed

for esterase activity at scheduled temperatures in 50 mM

sodium phosphate buffer, pH 7.0 using ATEE as substrate.

2.5. Thermal stability profile

Native and modified enzyme preparations were incubated

at scheduled temperatures in 50 mM sodium acetate buffer,

pH 5.0. Aliquots were removed after 10 min incubation,

chilled quickly, and assayed for enzymic activity.

2.6. Kinetics of thermal inactivation at 50 8C

Native and modified enzyme preparations were incubated

at 50 8C in 50 mM sodium acetate buffer, pH 5.0. Aliquots

were removed at scheduled times, chilled quickly, and

assayed for enzymatic activity.

2.7. Kinetics of inactivation at alkaline pH

Native and modified enzyme preparations were incubated

at 30 8C in 50 mM tris–HCl buffer, pH 9.0. Aliquots were

removed at different times, diluted in 50 mM sodium

phosphate buffer, pH 7.0, and assayed for esterase activity.

3. Results and discussion

In this paper, CDN was covalently attached to bovine

pancreatic a-chymotrypsin using EDAC as coupling agent.

Aspartic and glutamic acids side chain carboxy residues are

the expected sites for aminated carbohydrate chemical

linkage. Table 1 suggests that the modified enzyme

contained two mol of CD per mol of protein. This amount

of carbohydrate, corresponding to the glyco-building units

of D-glucose in CD, was lower when compared with the

amount attached to the lysine residues of a-chymotrypsin by

modification with mono-6-formyl-b-CD and mono-6-suc-

cinyl-6-deoxy-b-CD [24]. Although the number of lysine

residues is similar to the total amount of acidic amino acid

residues, this result was not surprising. The three-dimen-

sional structure of a-chymotrypsin shows that all lysine

residues are located at the protein surface, but the majority of

the aspartic and glutamic acid residues are buried into the

enzyme structure, forming the catalytic pocket and chelating

the essential Ca2+ ion [25].

The specific activity toward casein and ATEE was not

significantly reduced concerning a-chymotrypsin activity

after glycosylation, suggesting that the microenvironment of

the catalytic site was not affected by the attached CD

moieties.

Fig. 1 reports the influence of temperature on the esterase

activity of native and modified enzymes. Optimum

temperature for a-chymotrypsin was increased from 50 to

56 8C after the attachment of the CD residues. This fact

reflects that the modification conferred rigidity to the

M. Fernandez et al. / Process Biochemistry 40 (2005) 2091–2094 2093

Fig. 1. Optimum temperature profile of native (*) and CD-modified a-

chymotrypsin (*).Fig. 3. Kinetics of thermal inactivation of native (*) and CD-modified a-

chymotrypsin (*) at 50 8C.

conformation structure of the modified enzyme, requiring

then higher temperatures for expressing its maximum

catalytic activity [3].

The effect of CD modification on the thermal stability of

a-chymotrypsin was tested through two different set of

experiments. Fig. 2 shows the thermostabiliry profile for

both enzyme forms after 10 min incubation at different

temperatures ranging from 35 to 60 8C. The conjugate

showed improved resistance to denaturation at temperatures

higher than 45 8C.

Consequently, the value of T50, corresponding to the

temperature at which the enzyme retained 50% of the initial

activity, was increased by about 5 8C for a-chymotrypsin

after chemical glycosylation with the CD derivative.

The kinetics of thermal inactivation for native and

modified proteases at 50 8C is reported in Fig. 3. Both a-

chymotrypsin forms lost activity with time of incubation

according to a biphasic inactivation mechanism [3], but the

CD-modified protease retained as much as 70% of the

activity as compared to barely 12–13% for the unmodified

enzyme. This stabilization was associated with an increase

of 5.0 kJ/mol in the free energy of the thermal inactivation

process at this temperature.

Fig. 2. Thermal stability profile of native (*) and CD-modified a-chymo-

trypsin (*).

The improved thermostability showed by the CD-

modified a-chymotrypsin could be associated with the

formation of new stabilizing hydrogen bonds at the protein

surface of the enzyme as well as by the masking of the

hydrophobic clusters located at the surface of the enzyme,

preventing their unfavourable interaction with the surround-

ing water molecules [26]. Additionally, the ability of CDs to

form host–guest interactions with aromatic residues of

proteins could originate non-covalent intramolecular cross-

links mediated by supramolecular associations. A supra-

molecular-mediated mechanism for thermal stabilization

has been reported for other enzymes modified with CD

derivatives [27,28].

Fig. 4 shows the stability behaviour of native and CD

modified a-chymotrypsin preparations following incubation

at pH 9.0. a-chymotrypsin is inactivated under alkaline

conditions by the disruption of a saline bridge formed

between the N-terminus amino acid of the B-chain (Ile 16)

and the Asp-194 side chain [25], as well as by autolytic

degradation. As is illustrated in Fig. 4, the chemical

glycosylation of this protease with the aminated CD

derivative conferred noticeable stabilization at pH 9.0.

In this particular pH range at the longer but one time of

Fig. 4. Kinetics of inactivation at pH 9.0 for native (*) and CD-modifieda-

chymotrypsin (*).

M. Fernandez et al. / Process Biochemistry 40 (2005) 2091–20942094

pre-incubation (150 min), native enzyme retained only

about 5% of activity as compared to more than 75% in the

case CD-linked protease. This significant resistance to

alkaline inactivation could be caused by both the

supramolecular-mediated conformational stabilization men-

tioned above, as well as by the steric hindrance provoked by

the attached carbohydrate residues which could markedly

decrease the autodegradation processes in a-chymotrypsin.

4. Conclusions

The chemical modification of a-chymotrypsin by the C-6

monoamine derivative of b-CD was described. The

effectiveness of this approach has been demonstrated by

the high catalytic activity retained as well as by the increased

resistance showed by the synthesized conjugate against

thermal and alkaline inactivation. According to these results,

this modification procedure has been shown to be a useful

tool for improving the functional and stability properties of

enzymes.

Acknowledgment

This research was supported by the Third World

Academy of Sciences, through a grant to R. Villalonga

(Grant 01-279 RG/CHE/LA).

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