Biocatalysis and Biotransformation, September
December 2009; 27(5
6): 360
366

ORIGINAL ARTICLE

Immobilized copper-ion affinity adsorbent based on a cross-linked

b-cyclodextrin polymer for adsorption of Candida rugosa lipase

ELIF YILMAZ, MEHMET SEZGIN, & MUSTAFA YILMAZ

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Department of Chemistry, Faculty of Arts and Science, Selcuk University, Konya, Turkey
(Retrived 3 December 2008; revised 7 April 2009; accepted 6 August 2009)

Abstract
Lipase from Candida rugosa was immobilized on a b-cyclodextrin-based polymer by adsorption and subsequent cross-
linking with epichlorohydrin (EP-CD). The ligand iminodiacetic acid (IDA) was then bonded with the cross-linked b-
cyclodextrin (EP-CD-IDA). This affinity adsorbent was further chelated with Cu2 for the purpose of binding affinity and
stability. The properties of the immobilized lipase were assayed and compared with those of the free enzyme. Results showed
that 266 mg protein with an activity of 17.85 U was bound per gram of matrix, giving 188% of the specific activity of the free
enzyme and a total recovered activity of 79.7% under the optimum conditions. The pH and thermal stabilities of lipase were
Biocatal Biotransformation 2009.27:360-366.

improved after immobilization on the b-cyclodextrin-based polymer (EP-CD-IDA-Cu2 ). In addition, experimental results
indicated that the residual activity of the immobilized lipase was 50% after eight cycles of reuse.

Keywords: Lipase immobilization, Candida rugosa lipase, epichlorohydrin, iminodiacetic acid, b-cyclodextrin

Introduction cosmetics and perfumery industries (Hung et al.

Enzyme immobilization is the most commonly used
strategy to impart the desirable features of conven-
tional heterogeneous catalysts onto biological cata-
lysts (Moreno & Sinisterra 1994; Won et al. 2005).
Besides enhanced stability, immobilization is also
known to offer several advantages such as reusability,
ease of product separation, greater control over
catalysis and process economics (Rucka &
Turkiewicz 1990; Matsumoto et al. 1998).
Lipase (EC 3.1.1.3, triacylglycerol acylhydrolase)
is an important enzyme in biological systems, where it
catalyzes the hydrolysis of triacylglycerol to glycerol
and fatty acids. The enzyme is distributed among
higher animals, plants and micro-organisms, in which
it plays a key role in lipid metabolism. Depending on
the nature of the substrate and reaction conditions,
lipases catalyze a wide range of enantio- and regiose-
lective reactions such as hydrolysis, esterification,
transesterification, aminolysis and ammoniolysis.
The versatility of lipase-catalyzed reactions has
made them a unique industrial biocatalyst in drug
synthesis, food and flavor making, and recently in the
2003). Lipases have been immobilized on various
supports by physical adsorption, covalent bind-
ing, ionic interactions or by entrapment (Madeira
Lau et al. 2000; Mateo et al. 2000b, 2002, 2003;
Kilinc et al. 2002, 2006; López-Serrano et al. 2002;
Amorim et al. 2003; Noureddini et al. 2005; Rahman
et al. 2005; Yesiloglu 2005; Dincer & Telefoncu 2007;
Yujun et al. 2007; Guisan 2006). The extent of
enzyme stabilization depends on the choice of im-
mobilization process and selection of support.
Cyclodextrins (CDs) are natural products pro-
duced from starch by different bacterial species that
have been successfully used to improve enzyme
activity and to increase the reaction rate in
enzyme-catalyzed reactions in organic solvents
(Szejtli 1988). They are a family of chiral cyclic a-
(1,4)-linked D-glucose oligomers with six, seven or
eight glucose units, corresponding to a-, b- and g-
homologs, possessing a toroidal conformation in the
solid state and in solution. The internal hydrophobic
cavity and the external hydrophilic rim of chemically
modified CDs render them ideal for modeling
enzyme
substrate binding (Hamilton et al. 2000).

Correspondence: Mustafa Yilmaz, Department of Chemistry, Faculty of Arts and Science, Selcuk University, Konya-42075, Turkey.
Tel: 90-332-2232774. Fax: 90-332-2410106. E-mail: myilmaz42@yahoo.com

ISSN 1024-2422 print/ISSN 1029-2446 online # 2009 Informa UK Ltd

DOI: 10.3109/10242420903242805
 Properties of CRL immobilized to b-cyclodextrin-based polymer
Supports modified by a multidentate chelat-
ing agent, such as iminodiacetic acid (IDA) or
nitrilotriacetic acid, can be used for affinity adsorp-
tion after being further charged with cations.
In the literature (Klibanov 1983; Mateo et al.
2000a, 2001, 2007) when these new multifunctional
supports were used, the physical adsorption of the
protein was also the first step of the immobilization.
For example, IDA-Eupergit (without Cu2 adsorbed
on the support) was unable to immobilize penicillin G
acylase at pH 7. The same support, but with Cu2
adsorbed on the IDA groups, was also unable to
immobilize the enzyme in the presence of imidazole.
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However, immobilization of the enzyme was quanti-
tative when the enzyme was offered to the Cu2
support at pH 7 and in the absence of imidazole.
In our previous work (Ozmen et al. 2009),
Candida rugosa lipase (CRL) was immobilized
through covalent bonding on a b-CD-based polymer
chemically modified with different activating agents,
such as glutaraldehyde and hexamethylene diisocya-
nate (HMDI). The use of different activation agents
allowed the immobilization of CRL using different
Biocatal Biotransformation 2009.27:360-366.
properties. It was observed that the specific activity
of the immobilized lipase with glutaraldehyde was
62.75 U mg-protein 1, which is 28.13 times higher
than that of the immobilized lipase with HMDI.
In the present work, b-CD was chosen as a
support material for lipase immobilization and IDA
as a ligand was bound to the cross-linked b-CD.
Cu2 ions were then chelated with the ligand-bound
adsorbent. The properties of CRL immobilized to
this support are described.
Materials and methods
Materials
CRL, Bradford reagent, 99% pure bovine serum
albumin (BSA), p-nitrophenylpalmitate (p-NPP)
and epichlorohydrin (EP) were supplied by Sigma
Chemical Co. (St. Louis, MO) and used as received.
b-CD, N,N-dimethyl formamide (DMF), acetone
and ethanol were bought from Merck (Darmstadt,
Germany). All aqueous solutions were prepared with
deionized water that had been passed through a
Millipore (Bedford, MA, USA) Milli-Q Plus water
purification system. All other chemicals were of
analytical grade and used without further purification.
Synthesis of b-CD copolymer
b-Cyclodextrin
epichlorohydrin copolymer (EP-
CD) was synthesized according to a modified method
of Komiyama & Hirai (1987), as follows. b-CD (2 g),
0.5 g of soluble starch and 5 mL of 20% (w/v) NaOH
were combined in a beaker and the mixture vigorously
361
stirred at 50
608C until the reactants had dissolved. A
total of 2.8 mL of EP was added dropwise into this
solution; EP-CD was formed in 30 min. After washing
five or six times with Milli-Q water, the polymer was
dried at 1008C under reduced pressure and ground to
about 60 mesh. The polymer was stored in a desicca-
tor before use.
Treatment of EP-CD support with IDA
EP-CD-IDA was synthesized according to a mod-
ified method of Armisen et al. (1999). EP-CD
(3.5 g) was incubated in 6.96 mL of 0.1 M sodium
borate/2 M IDA, pH 9.0, at 258C for 24 h under
very gentle stirring. The support was washed with an
excess of distilled water and stored at 48C.
Loading Cu(II) onto the EP-CD-IDA support
EP-CD
IDA-Cu2 was synthesized according to a
modified method of Armisen et al. (1999). A 3.15 g
portion of EP-CD-IDA was incubated in 20 mL of
distilled water containing 0.63 g CuSO4 with very
gentle stirring. After 2 h, the support was washed with
an excess of distilled water and stored at 48C. This
treatment should modify 100% of the IDA groups in
the support. The preparation of EP-CD-IDA-Cu2
metal-ion affinity adsorbent is illustrated in Scheme 1.
Immobilization of enzyme on the support
(EP-CD-IDA-Cu2 )
A 3 g portion of support (EP-CD-IDA-Cu2) was
suspended in 27 mL of enzyme solution (1 mg mL1
based on crude lipase, equivalent of 70 mg mL1
based on protein analysis) in 50 mM sodium phos-
phate (pH 7.0) and the mixture was incubated at
258C and stirred at 150 rpm for 3 h. After that, the
immobilized lipase was washed thoroughly with
50 mM phosphate-buffered saline (PBS). Immobi-
lized enzyme was analyzed for bound lipase activity.
The preparation was then lypophilized and stored at
48C until use.
Determination of enzyme activity
Activity of the free and immobilized lipase was assayed
using 14.4 mM p-NPP in 2-propanol as substrate.
The reaction, consisting of 1 mL of 50 mM phosphate
buffer (pH 7.0 for immobilized lipase) containing
25 mg of immobilized lipase or 0.1 mL of free lipase
(concentration as above), was initiated by adding
1 mL of substrate and mixed for 5 min at 308C. The
reaction was terminated by adding 2 mL of 0.5 N
Na2CO3 followed by centrifuging at 4000 rpm
(2028g) for 10 min. The increase in the absorbance
at 410 nm produced by the release of p-nitrophenol in
 362 E. Yilmaz et al.
O
O
OH Cl
+

β-CD EP-CD

O
OH
O N
+ OH OH
NH
OH OH O

β-EP-CD
O β-EP-CD-IDA
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IDA

Cu2+

O-

N Cu2+ E

O-
OH O
Biocatal Biotransformation 2009.27:360-366.

β-EP-CD-IDA-Cu2+

Scheme 1. Scheme for preparation of immobilized metal-ion b-CD affinity adsorbent.

the enzymatic hydrolysis of p-NPP was measured in a (Madrid, Spain) protein dye reagent concentrate.
Shimadzu UV-160A (Tokyo, Japan) spectrophot- BSA was used as the standard. This indicated that
ometer. A molar extinction coefficient (o410) of the crude CRL powder was only 7% protein. The
15 000 M 1 cm 1 for p-nitrophenol was used amount of immobilized protein was calculated from
(Lotrakul & Dharmsthiti 1997). One unit (U) of lipase the difference between initial amount of enzyme and
activity was defined as the amount of enzyme neces- enzyme released during the immobilization process.
sary to hydrolyze 1 mmol of p-NPP per minute under
the conditions of assay (Winkler & Stuckmann 1979).
Recovered activity (%) was calculated according to Physico-chemical properties of free and immobilized
Yang et al. (2009). All results are the average of lipase
triplicate measurements. Effect of pH on activity. The effect of pH on activity of
Lipase activity (U g-support1 ) free and immobilized lipase was assayed in 50 mM
phosphate buffer over a pH range from 4.0 to 9.0 by
Activity of immobilized lipase
 using the standard activity assay procedure above
Amount of immobilized lipase (Hung et al. 2003; Deng et al. 2004; Tumturk et al.
Specific activity (U mg-protein1 ) 2007; Vaidya et al. 2007, 2008).
Activity of immobilized lipase
 Effect of temperature on activity. The rates of thermal
Amount of protein loaded inactivation of the free and immobilized lipase were
Recovered activity (%) studied in the temperature range 25
608C. Both
Total activity of immobilized lipase forms of enzyme were incubated in PBS (50 mM,
 100 pH 7.0) for 20 min at different temperatures before
Total activity of free lipase determining the activity at its optimum reaction
temperature. Relative activities were calculated as
above and plotted against temperature.
Protein assay
Protein content was determined following the Thermal stability. Free and immobilized lipase pre-
method of Bradford (1976) using the Bio-Rad parations were stored in the phosphate buffer solutions
 Properties of CRL immobilized to b-cyclodextrin-based polymer 363
Table I. Activity of the free and immobilized lipase under optimum reaction conditions.

Protein loaded Protein bound Lipase activity Specific activity Recovered

(mg g-support 1) (mg g-support1) (U g-support1) (U mg-protein 1) activity (%)a

Free lipase

2.49b 35.6c

Immobilized lipase 0.63 0.266 17.85 67.1 79.7
a
Recovered activity (%)(total activity of immobilized lipase/total activity of free lipase used for immobilization)100. bActivity of the free
lipase per mL. cAccording to the Bradford method the protein percentage of the lipase commercial powder was 7%.

(50 mM, pH 7.0) at 608C for 2 h, respectively.
Samples were periodically withdrawn for activity
assay. The residual activities were determined as
above.
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contained in the enzyme whereas Cu2, Ni2 and
Zn2 ions preferentially coordinate with nitrogen,
oxygen and sulfur.
Adsorption experiments of CRL on EP-CD
IDA-

Cu2 were carried out as described above; crude

Reuse of the immobilized lipase
The stability of immobilized lipase on repeated use
was also examined by eight cycles of successive
repetition of the p-NPP hydrolysis assay. After each
activity determination, the immobilized lipase was
washed with buffer (PBS, 50 mM, pH 7.0) and
reintroduced into a fresh medium.
Biocatal Biotransformation 2009.27:360-366.
CRL at 1.0 mg mL1 (70 mg protein mL 1) with an
initial activity of 2.49 U mL1 was applied to the
adsorption.
Table I shows the activities of free and the
immobilized lipase under the optimum reaction
conditions. In comparison with the free enzyme,
the activity of the immobilized enzyme was en-
hanced by binding to EP-CD-IDA-Cu2, having
188% of the specific activity of the free enzyme,

although as only 42% of the protein was immobi-

Results and discussion
Previously, a cross-linked b-CD with IDA as the
ligand was chelated with different metal ions to
evaluate the affinity performance of a-amylase (Liao
& Syu 2005). It was observed that Cu2 gave the
best results and also very high capacity both in
adsorption as well as desorption.
In the current work a cross-linked b-CD-based
polymer (EP-CD) treated with IDA was prepared
because EP-CD alone shows a poor affinity toward
CRL. The cross-linked b-CD with IDA as the ligand
was chelated with Cu2 ions to increase the binding
affinity of CRL. Mateo et al. (2002, 2007) prepared
an IDA-Cu2-epoxy support and immobilized
CRL. They observed that the immobilized lipase
showed high activity. According to Tishchenko
(2003), Ag and Hg2 are likely to bind with sulfur
lized, the net recovery of activity was 79.7%.
Scanning electron micrographs comparing the
IDA-treated support with that containing the im-
mobilized lipase showed that the latter had a much
smoother surface compared with the irregular sur-
face cavities of the IDA-treated polymer (Figure 1).
The loss of the grainy appearance in the matrix
suggested a fairly uniform covering with the enzyme.
Effect of pH and temperature on the activity of
immobilized enzyme
The catalytic activity of the immobilized lipase was
investigated at different pH values. pH is one of the
most influential parameters altering enzyme activity
in an aqueous medium. Immobilization is likely to
result in a conformational change of the enzyme,

Figure 1. SEM micrographs of IDA-treated support (a) and lipase immobilized support (b) (500).
 364 E. Yilmaz et al.

which can change the catalytic activity, while 100

localized influences of the matrix itself can also

Relative Acitivity (%)

80
affect properties such as pH optimum. Figure 2
shows that maximum enzyme activity was exhibited 60
at pH 7.0 with both free and immobilized enzyme,
indicating the absence of either effect on the enzyme 40
during adsorption (Ozturk et al. 2007). The princi-
immobilized lipase
ple of this binding method is that amino acid 20
free lipase
residues such as aspartic acid (Asp), glutamic acid
0
(Glu), histidine (His), cysteine (Cys) or lysine (Lys) 20 30 40 50 60
on the protein surface can specifically interact Temperature (°C)
through non-binding lone-pair electrons with the
metal ion. It is recognized that the imidazole group Figure 3. Effect of reaction temperature on the relative activity of
free ( ) and immobilized lipase ( ).
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of His, the thiol group of Cys and the indoyl group of

tryptophan (Trp), in particular, contribute to bind-
ing with immobilized metal ions (Hemdan et al.
1989; Jiang & Hearn 1996; Tishchenko et al. 2003).
The effect of temperature in the range 25
608C on
the activity of free and immobilized lipases at pH 7.0
is shown in Figure 3. Immobilization shifted the
optimum temperature for lipase activity from 358C
for the free enzyme to nearly 458C, due to either
conformational limitations on enzyme movement
Biocatal Biotransformation 2009.27:360-366.
lipase was incubated for 2 h at 608C and the enzyme
activity was measured at various time intervals after
cooling the enzyme to 308C. The free lipase lost its
initial activity within about 80 min at 608C, while
the immobilized lipase retained about 79% of its
initial activity after 120 min of heat treatment at
608C. Improvement in thermal stability of an
immobilized enzyme typically reflects the interaction
between the enzyme and support preventing

as a result of multipoint interaction between the

enzyme and the support or improved substrate
diffusion at high temperature. Similar changes in
the optimum temperature have also been reported in
analogous studies of enzyme immobilization (Ye
et al. 2005, 2007). In our previous study it was
found that the optimum temperature for immobi-
lized CRL on CD polymer with cross-linked HMDI
was also approximately 458C (Ozmen et al. 2009).
Thermal stability
Figure 4 shows the thermal stability of the free and
immobilized lipase at 608C. Free or immobilized
100
80
Relative activity (%)
conformational transitions of the enzyme at high
temperatures.
Repeated use of the immobilized lipase
Reusability of immobilized enzymes is very impor-
tant economically, and increased stability can make
the immobilized enzyme more advantageous than
its free counterpart. To investigate reusability, the
immobilized enzyme was washed with PBS (50 mM,
pH 7.0) after each catalysis run and reintroduced
into a fresh p-NPP solution for another reaction at
308C. Figure 5 shows that more than 50% of the
immobilized lipase activity was maintained after
eight reuse experiments. This shows that this
immobilized lipase can be used successfully for
100

80
Relative activity (%)

60

60
40
40

20
20

0 0
3 4 5 6 7 8 9 0 20 40 60 80 100 120
pH Time (min)
Figure 2. Effect of substrate pH on the relative activity of free Figure 4. Thermal stability of free ( ) and immobilized
( ) and immobilized lipase ( ). lipase ( ).

100
Relative Activity (%)
80
60
40
20
0 Totowa (NJ): Humana Press Inc. pp. 107
128.
0 1 2 3 4 5
Reuse
Figure 5. Reusability of the immobilized lipase.
Table II. Evaluation of the desorption performance of CRL
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bound to EP-CD-IDA-Cu2 adsorbent.
Lipase activity Activity after desorption with
(U g-support 1) imidazole (U g-support 1)
17.85 1.2690.20a
a Klibanov AM. 1983. Immobilized enzymes and cells as practical
Mean9standard deviation of three measurements.
industrial applications requiring long-term reaction
stability.
To further inspect the stability of the metal-ion
Biocatal Biotransformation 2009.27:360-366.
affinity adsorbent thus prepared, the reutilization of
the support after repeated adsorption
desorption
operations of lipase was examined (Table II). The
support was easily regenerated using imidazole as
washing solution. Imidazole competes with CRL for
coordination with Cu2, eventually replacing the
enzyme in a reversible manner. The results showed
that CRL could be successfully adsorbed to EP-CD-
IDA-Cu2 and desorbed using an imidazole wash
treatment.
Acknowledgements
We thank the Scientific Research Foundation of
Selcuk University, Konya, Turkey (BAP Grant Num-
ber 08101024) for financial support of this work.
Declaration of interest: We report no conflicts of
interest. We alone are responsible for the content and
writing of the paper.
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