International Journal of Biological Macromolecules 35 (2005) 89–95

Activity and stability of laccase in conjugation with chitosan

Gary Delanoy a , Qing Li a , Jian Yu b, ∗
a Department of Molecular Bioscience and Bioengineering, Honolulu, HI 96822, USA
b Hawaii Natural Energy Institute, University of Hawaii at Manoa, 1680 East-West Road, Honolulu, HI 96822, USA

Received 18 November 2004; received in revised form 10 January 2005; accepted 11 January 2005

Abstract

Laccase is one of a few enzymes that can directly reduce oxygen into water under ambient conditions, while oxidizing a variety of aromatic
compounds. Its conjugation with chitosan generates a pH-sensitive functional biomaterial that changes its solubility in response to pH
variation. The molecular conjugation between laccase and chitosan of different molecular mass was investigated with a carbodiimide reaction
to understand the mechanism of the enzyme’s activity loss during conjugation. With 81–93% laccase being conjugated, a moderate activity loss
(16–28% less than the initial activity) was observed in conjugation solution. A second severe activity loss (63–78% less than the conjugated
activity) occurred during a cycle of phase change consisting of precipitation, centrifugation and re-dissolution of the enzyme–chitosan
conjugates. The chitosan molecular size has little effect on the first moderate activity loss in the conjugation reaction, but visible effect on the
substantial activity loss associated with phase change. Small chitosan molecules gave high residual activity. The conjugated laccase exhibited
a high stability in the following repeated phase changes and had the same temperature and pH profile as those of free laccase. Compared
to free laccase, the conjugated laccase had a similar affinity (Km ), but reduced turnover (kcat ) that was adversely affected with increase of
molecular mass of chitosan.
© 2005 Elsevier B.V. All rights reserved.

Keywords: Chitosan; pH-sensitive biomaterials; Protein conjugation; Laccase assay; Enzyme immobilization

1. Introduction for example, is a biocatalyst that can change its solubility in

Chitin, a linear carbohydrate composed of ␤-1,4-linked N-
acetylglucosamine residues, is an abundant marine biopoly-
mer. Millions of tons of chitin are generated annually by
marine zooplankton (copepods) and crustaceans, such as
shrimps and crabs [1]. The deacetylated derivative of insolu-
ble chitin, chitosan, becomes soluble in acidic solution when
the degree of deacetylation is more than 65–70% and exhibits
interesting functions as a heavy metal adsorbent, bioadhe-
sive, emulsion stabilizer, antimicrobial agent, and transport
enhancer of drug delivery [2–5]. The dissolved chitosan is a
cationic polyelectrolyte that can be conjugated with proteins
to make functional biomaterials with novel physiochemical
and biological properties [6]. A chitosan–enzyme conjugate,
∗ Corresponding author. Tel.: +1 808 956 5873; fax: +1 808 956 2336.
E-mail address: jianyu@hawaii.edu (J. Yu).
response to variation of environmental pH [7].
Laccase (EC1.10.3.2) is a multi-copper-containing en-
zyme that catalyzes the oxidation of various aromatic com-
pounds, such as phenols and anilines by reducing molecular
oxygen to water [8]. It is one of a few enzymes that can re-
duce dioxygen to water under ambient conditions. Among
more than 60 laccases detected in the resources of plants, in-
sects, bacteria and fungi, the laccase from fungus Trametes
versicolor has been studied for molecular properties and en-
zymology [9]. The laccase molecule is a dimeric or tetrameric
glycoprotein and each monomer contains four copper atoms
that are bound to three redox sites (types 1, 2 and 3). Sub-
strate oxidation and oxygen reduction are carried out at these
separated sites. Because of its broad substrate spectrum, lac-
case is one of the potential biocatalysts for bioremediation
of contaminated soils and water [9]. Other applications in-
clude bleaching and decolorization in textile and paper man-
ufacturing, removing phenolic compounds from beverages,

0141-8130/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijbiomac.2005.01.003
90 G. Delanoy et al. / International Journal of Biological Macromolecules 35 (2005) 89–95
and biosensing a variety of organic and inorganic compounds
[10–12]. Conjugating laccase and chitosan generates a new
type of functional biopolymers exhibiting novel features that
are not possessed by the individual components.
Different from the conventional immobilization of lac-
case on pre-formed solid supports [13] or physically trapped
in gels [14], conjugating laccase and chitosan occurs in a so-
lution by forming covalent bonds between the protein and
polysaccharide molecules. Once bound, the molecules are
strongly coupled and distributed uniformly in the conjugate
aggregates or gels. Since the protonation of amino groups on
the chitosan backbone is dependent on solution pH, the lac-
case conjugate becomes a pH-sensitive biomaterial and can
adjust its aggregation state in response to pH variation. The
conjugates, therefore, can undergo repeated phase change
with little loss of enzyme proteins, a useful feature for novel
applications in bioremediation, organic synthesis, biosensing
and immunoenzyme assays. Laccase is a glycosylated pro-
tein with a typical molar mass ranging from 60–80 kDa and
15–20% (wt) of equal amounts of N- and O-linked carbohy-
drate [15]. It is a challenge to attach additional large carbo-
hydrate polymers onto the glycosylated proteins [6]. Since
N-type of glycosylation occurs exclusively on the nitrogen
atoms of asparagine residues and O-glycosylation on the oxy-
gen atoms of hydroxyls of serine and theronine residues, the
carbodiimide reaction provides an efficient means to form
amide bonds between the primary amino groups of chitosan
and the carboxylic residues of aspartic and glutamic acids
[7].
One major problem in enzyme conjugation or immobiliza-
tion is the loss of enzyme activity, which is attributed to many
factors involving enzyme, polymer, reagents and process con-
ditions. Chitosan has two fundamental parameters, the degree
of deacetylation and the molecular mass. These two proper-
ties influence chitosan’s solubility, functionality, and reac-
tivity [16] and hence the properties of enzyme conjugates.
Commercially available chitosans are heterogeneous, poorly
characterized biopolymers with the degree of deacetylation
more than 70% and the molecular weights typically ranging
from 50 to 2000 kDa [17]. This work investigated the effect
of molecular size of chitosan on laccase conjugation and the
activity loss in conjugation process. The laccase conjugates
were further evaluated for activity stability, temperature and
pH profiles and kinetic parameters.
2. Materials and methods
␣-Chitosans of low, medium and high molecular weight
were purchased from Fluka Chemie AG (Buchs, Swiz-
erland), ICN Biomedical Inc. (Aurora, OH, USA) and
Sigma & Aldrich (St. Louis, MO, USA). Laccase from
fungus Trametes versicolor and its substrate 2,2 -azino-
bis(3-ethylthiazoline-6-sulfonate) (ABTS) were purchased
from Fluka Chemie AG. The laccase–chitosan conjugation
agent N-(3-dimethylaminopropyl)-N -ethylcarbodiimide hy-
drochloride (ECD), buffer salts, acids and bovine serum albu-
min (BSA) were purchased from Sigma & Aldrich. Dextran
polysaccharide polymer standards (MW 181 Da to 410 kDa)
were obtained from Polymer Standards Service (Warwick,
RI, USA).
2.1. Chitosan modification and characterization
A chitosan working solution of 10 g/L was prepared by
dissolving the polymers in 1% (wt) acetic acid, filtering
through 0.2 ␮m membrane and storing at 4 ◦ C for later use.
The molecular mass and distribution of one chitosan (ICN
Biomedical Inc.) was modified with ultrasonication. Approx-
imately 20 mL of chitosan solution was treated with a soni-
cator (Model 100, Fisher Scientific, Houston, TX, USA) at a
power output of 3–4 W for 5 h. After being filtered through
a 0.2 ␮m membrane filter, the polycationic polymer was pre-
cipitated out from the filtrate at neutral pH by adding 100 mM
Tris buffer (pH 8). The precipitates were collected by cen-
trifugation at 5000 rpm, washed two times with Tris buffer,
and freeze-dried for later use. The molecular mass of chi-
tosans from different vendors and prepared in this study were
measured with size exclusion chromatography (SEC, Shi-
madzu, Japan). The SEC was equipped with a RID detector
and SB804 HQ + SB805 HQ columns (Shodex, Munich, Ger-
many) to measure polysaccharides of MW 10–1000 kDa. The
chitosan molecules were eluted with a sodium acetate buffer
(pH 4.5) at 1 mL/min and the molecular mass was calibrated
with standard dextran solutions in 1% (wt) acetic acid.
2.2. Chitosan–laccase conjugation
The conjugation solution contained chitosan 8.8 g/L, lac-
case powder 0.1 g/L, and ECD 2 mM, and had a pH of 4.75.
Conjugation was started by adding ECD and shaken gently
with a wrist action shaker at room temperature (∼20 ◦ C) for
4 h. After conjugation reaction, the laccase conjugates un-
derwent a complete cycle of phase change as follows. The
conjugates were first precipitated out from the solution by
adding 100 mM Tris buffer (pH 8), centrifuged at 5000 rpm
at 4 ◦ C, and washed three times with 2 mL Tris buffer to re-
move the unbound enzyme proteins. The wet pellets were
finally re-dissolved in 100 mM acetate buffer (pH 4.0) to the
same volume of conjugation solution and stored at 4 ◦ C. Dur-
ing the process, aliquots of the solutions were withdrawn to
measure the protein content and enzyme activity. Controls of
laccase-only or chitosan-only were run in parallel.
2.3. Protein and enzyme assays
Free or conjugated laccase was pre-dissolved in 100 mM
acetate buffer (pH 4). The protein concentration was mea-
sured with a Bradford microassay kit (BioRAD, Richmond,
CA, USA). An aliquot of solution (0.8 mL) was mixed with
the reagent solution (0.2 mL) and incubated at room temper-
ature for 15 min. The absorbance at 595 nm was measured
 G. Delanoy et al. / International Journal of Biological Macromolecules 35 (2005) 89–95 91

with a spectrophotometer (Beckman Coulter DU530, Fuller- Table 1

ton, CA, USA) and the protein content was calibrated with Molecular mass of commercial chitsoans
standard solutions of bovine serum albumin ranging from 0 Source Classification MW MN Polydispersity Peak
to 20 ␮g/mL. The linear regression coefficient (r2 ) was 0.99 (kDa) (kDa) (kDa)
or above. The laccase activity was measured with colorimet- Fluka Medium 505 384 1.3 513
ric kinetics. A 2 mL assay solution contained 200 mM ABTS Aldrich High 490 332 1.5 542
Aldrich Medium 442 222 2.0 539
and 50 mM Britton & Robinson buffer (pH 3.0). Oxidation Fluka Low 432 180 2.4 539
of ABTS by laccase was monitored as absorbance increase at Aldrich Low 375 164 2.3 525
420 nm (ε = 36 mM−1 cm−1 ) at 20 ◦ C [18]. One unit of lac- ICN Low 214 74 2.9 453
case activity was defined as 1 ␮mol of product from the ABTS ICN a 131 47 2.8 119
oxidation per min. The effect of free chitosan (not conjugated Aldrich Oligomer 1.87 1.25 1.5 1.15
aObtained in this study by ultrasonication treatment of ICN’s low
with enzyme) on the enzyme assay was measured by adding
a predetermined amount of chitosan into the assay solution molecule weight chitosan.
before the enzyme was introduced.
biopolymer solution. Table 1 shows the big difference in
2.4. Stability of the conjugated laccase molecular weight of the same category from different suppli-
ers. Since low molecular weight chitosan is usually prepared
The effect of pH on free and conjugated laccase was mea- by breaking down the natural large molecules in harsh condi-
sured with enzyme assay at different pH levels, pH 1.2–2.2 in tions [19], the polydispersity increases with decline in molec-
potassium chloride/hydrochloric acid buffer (50 mM) and pH ular size. Ultrasonication broke down the large biopolymers
2.0–8.0 in Britton & Robinson buffer (50 mM). Temperature effectively and did not increase the sample’s polydispersity.
has two major effects on laccase activity, the effect on en- Fig. 1 shows that the peak (453 kDa) of large molecules in
zymatic kinetics and the effect on protein structure. To mea- ICN’s low molecular weight sample was actually eliminated.
sure the effect on structures of conjugated laccase, free and
conjugated laccase were incubated in 100 mM acetate buffer 3.2. Laccase activity assay
(pH 4.0) at temperatures ranging from 30 to 80 ◦ C for 1 h.
Aliquots of enzyme solutions were taken for activity assay at Laccase activity was assayed by measuring the oxidation
room temperature. In this way, the assay reveals the structural rate of ABTS and hence possibly affected by molecular dif-
stability of free or conjugated enzyme, rather than the kinetic fusion rates of substrate to and product from the enzyme.
effect of different temperatures. The stability of conjugated The presence of large molecules of chitosan in the enzyme
laccase in repeated phase change of precipitation and dissolu- conjugates, even though many of them might be in free form
tion was also measured. One cycle of phase change included: and not conjugated with laccase proteins, might increase the
(a) precipitation of the conjugation solution with 100 mM Tris viscosity of the solution and reduce the mass transfer or ox-
buffer (pH 8.0), (b) centrifugation at 5000 × g for 10 min, and idation rate. This might cause a bias to the measurement of
(c) dissolution of the wet pellets into 100 mM acetate buffer intrinsic laccase activity. Table 2 shows that both molecular
(pH 4.0). The dissolved laccase–chitosan conjugate was as- size and concentration of chitosan had significant adverse ef-
sayed for the residual activity as described above. fects on activity assay. In general, large molecules and high
concentrations resulted in low laccase activity that could drop
2.5. Kinetic parameters of the conjugated laccase as much as 50%, in comparison with the absence of chitosan
macromolecules. Little adverse effect, on the other hand, was
The kinetics of ABTS oxidation by free and conju- observed with a chitosan oligomer (MW 1870 Da, about 10
gated laccase was compared in the enzyme assay solu- glucosamine residues) at the same concentration level. This
tion with ABTS concentration ranging from 5 to 40 ␮M. fact implies that the high viscosity of large molecules at high
Time course of absorbance increase at 420 nm was mon- concentrations was a leading factor causing the apparent ac-
itored for oxidation product. The reaction rates were ob- tivity loss.
tained from the slopes of time course and simulated with the Chitosan is a well-known adsorbent of heavy metals in
Michaelis–Menton equation. The kinetic parameters were es- aqueous solution [2] and may also denature laccase by strip-
timated from Lineweaver–Burke plots. ping Cu(II) from the active sites of the enzyme even though
the cooper ions are embedded in proteins and complexed with
amino acid residues [20]. This possibility was investigated by
3. Results and discussions adding Cu(II) into the assay solution to saturate the adsorption
capacity of chitosan before laccase was added. The results are
3.1. Chitosan characterization and modification shown in Fig. 2. Compared to the controls of laccase-only,
the external copper ions alleviated to some extent the loss
Commercial chitosan is usually classified as low, medium of activity at the moderate chitosan dosage of 50 mg/L, but
and high molecular weight based on the viscosity of the was less efficient with high chitosan dosage. The results in
92 G. Delanoy et al. / International Journal of Biological Macromolecules 35 (2005) 89–95

Fig. 1. SEC chromatograms of commercial and modified low MW chitosans. The x-axis represents the retention time (min) of chitosan molecules; the large
molecules appear first followed by small ones.

Table 2
Effects of dosage and molecular weight (low, medium and high) of free chitosan on laccase activity assay
Chitosan dosage (mg/L) Laccase activitya

Oligomer MW (1.87 kDa) LMW MW (375 kDa) MMW MW (442 kDa) HMW MW (490 kDa)
0 1.00 1.00 1.00 1.00
25 0.99 1.05 1.10 0.95
50 1.16 0.83 0.86 0.60
250 1.09 0.53 0.5 0.54
500 0.95 0.51 0.53 0.48
a Laccase activity relative to nil chitosan in the assay solution.

Table 2 and Fig. 2 together reveal that the enzyme assay can
measure the intrinsic laccase activity in the presence of chi-
tosan around 25 mg/L or lower. At concentrations above this
level, a moderate or significant bias to enzyme activity assay
exits. The apparent activity loss, because of this bias, may
not reflect the real denaturation of enzyme proteins, but more
likely the non-biological factors, such as solution viscosity
and molecular mass transfer.
Fig. 2. The effects of chitosan concentration and external Cu(II) concentra-
tion on the assay of laccase activity.
3.3. Laccase conjugation
Laccase was conjugated with dissolved chitson molecules
in a buffer solution (pH 4.75). The conjugates and free chi-
tosan molecules were precipitated out from the solution by
adjusting the pH to neutral. Table 3 gives the results of lac-
case conjugation with chitosans of different molecular mass
in comparison with laccase-only. Both protein and activity
of laccase were measured and are shown in Table 3. In or-
der to avoid the interference from proteinaceous impurities
bound with chitosan, the amount of laccase protein conju-
gated was calculated from the difference of two measure-
ments, the initial protein concentration (PI ) before conjuga-
tion and the residual protein concentration (PR ) in the su-
pernatant after the precipitation and centrifugation. Based on
proteins, 81–93% of the initial laccase was effectively con-
jugated with chitosan and collected with precipitation and
centrifugation. Little effect of chitosan molecular mass on
conjugation was observed.
Based on enzyme activity, however, a considerable effect
of chitosan molecular mass on laccase conjugation was ob-
served (see AE in Table 3). The residual activity of conjugated
laccase, compared to the initial activity, declined from 23 to
11%, while chitosan molecular mass increased from 131 to
505 kDa. We followed the activity loss step by step during
the conjugation. At the end of a 4-h conjugation reaction,
 G. Delanoy et al. / International Journal of Biological Macromolecules 35 (2005) 89–95 93

Table 3
Conjugation of laccase with chitosans of different molecular weight (triplicates)
Chitosan (kDa) Protein Laccase activity
a
PI (␮g/mL) b
PR (␮g/mL) c
PE (%) Ad (U/mL) Ac e (%) Aub f (U/mL) Ab g (U/mL) Ap h (%) AE i (%)
0j 15.1 15.1 0 32 0 – – – –
131 15.1 2.5 ± 0.8 83 23 −28 3.2 ± 0.7 7.4 ± 0.6 −63 23
214 15.1 1.1 ± 0.3 93 25 −22 2.8 ± 0.9 6.2 ± 0.2 −72 19
432 15.1 2.9 ± 0.9 81 27 −16 2.5 ± 0.4 4.4 ± 0.7 −82 14
505 15.1 1.4 ± 0.2 91 23 −28 2.3 ± 0.3 3.6 ± 0.6 −83 11
a Initial laccase protein concentration in conjugation solution.
b Residual protein in conjugation solution after laccase–chitosan conjugates were recovered by precipitation at pH 7 and centrifugation.
c Protein conjugation efficiency from the difference of free protein before and after conjugation: ((1 − (PR /PI )) × 100%.
d Total Laccase activity (bound and unbound with chitosan) in the conjugation solution after a 4-h conjugation but before conjugates precipitation.
e Overall activity loss due to conjugation with chitosan: −(1 − (A/32)) × 100%.
f Activity of the unbound laccase in the supernatant after precipitation and centrifugation.
g Activity of conjugated laccase re-dissolved in 1% (wt) acetic acid.
h Activity loss of conjugated laccase during a cycle of phase change: −Ab /(A − Aub ) × 100%.
i Overall efficiency of laccase conjugation relative to the initial free laccase activity (Ab /A) × 100%.
j Free laccase without chitosan.

but before precipitation, aliquots of conjugation solutions,
containing conjugates, free laccase and free chitosan, were
assayed (A). Compared with the laccase activity (32 U/mL)
in the enzyme-only control, a moderate enzyme activity of
16–28% was lost (Ac ) during the coupling reaction. Because
chitosan polymers, together with the enzyme, were brought
into the assay solution at about 44 mg/L, this apparent activ-
ity loss was equivalent to the measurement bias (Table 2). It
implies that little enzyme activity was lost during the con-
jugation reaciton due to the covalent bonds formed with the
carboxylic residues of protein. After a cycle of phase change,
including precipitation, centrifugation and re-dissolution of
the conjugates in the same volume of acetate buffer, the ac-
tivity of conjugated laccase was measured (Ab ) as well as the
activity of unbound laccase in the supernatant (Aub ). The ac-
tivity loss (Ap ) after one cycle of phase change is estimated
with Eq. (1):


Ab
−Ap = − × 100 (1)
A − Aub
where (A − Aub ) refers to the maximum activity of bound lac-
case and Ab refers to the actually measured activity of bound
laccase. The laccase activity loss (Ap ) ranged from 63 to 83%
based on the conjugated laccase activity (A − Aub ), which
was much higher than the assay bias as revealed above. Fur-
thermore, the severe activity loss that occurred during phase
change was affected by the molecular weight of chitosan. The
smaller the chitosan molecule, the less the enzyme activity
loss. Because of this major activity loss, the overall resid-
ual activity (AE ) of laccase–chitosan conjugates was also af-
fected by the molecular size of chitsoan. Small molecules
of chitosan are beneficial to high residual activity of laccase
conjugates.
Water soluble carbodiimide is widely used in conjugation
reactions for peptide synthesis in a pH range of 4.5–7.5 [21].
It facilitates the formation of amide bonds between a car-
boxylic acid residue and an amine residue, giving a so-called
‘zero-length’ cross-linking since the amide linkage does not
add a spacer molecule [21]. Covalent binding of the enzyme
to the cationic polymer has a number of advantages, such as
the formation of strong bonds between enzyme and polymer,
allowing the enzyme–polymer conjugate to be resistant to
changes in pH, temperature, and ionic conditions. The co-
valent linkages may also reduce the enzyme activity if the
bonds are formed at or close to the active sites causing space
hindrance by the large chitosan molecules and other adverse
effects. This might not be the main reason of the apparent
activity loss since little activity loss was observed during
conjugation reaction. The phase change, however, involved
precipitation, centrifugation and re-dissolution, and applied
a drag force on large laccase–chitosan conjugates (65 kDa
plus 100–500 kDa). Compared to big chitosan molecules, the
small biopolymers might generate a smaller drag force on the
protein and result in less activity loss.
3.4. Stability of the conjugated laccase
Fig. 3 shows the residual activity of conjugated laccase
during the repeated cycles of precipitation, centrifugation and
dissolution. It is very interesting to note that the residual ac-
tivity did not decline continuously with the repeated phase
change. It seems that once a laccase protein was conjugated in
a right form or pattern with chitosan molecule(s), the external
drag forces in phase change might not affect the protein even
with repeated precipitation, centrifugation and dissolution. A
stable residual activity could thus be reserved. On the other
hand, those proteins conjugated in a wrong pattern lost their
activity in the first cycle of phase change as shown above.
Further studies at the nano scale on enzyme–biopolymer con-
jugation shall provide insights into this phenomenon.
Figs. 4 and 5 show the profiles of temperature and pH of
conjugated laccase and compare them with the profiles of free
laccase. For easy comparison, the profiles are relative to the
maximum activity of individual laccase and laccase conju-
94 G. Delanoy et al. / International Journal of Biological Macromolecules 35 (2005) 89–95

Table 4
Kinetic parameters of ABTS oxidation by laccase conjugated with chitosans
of different molecule weight
Chitosan (kDa) Km (mM) kcat a (min−1 )
Free laccase 42.3 15.1
131 95.0 10.1
214 49.8 4.81
432 85.2 6.05
505 45.2 3.84
a Calculated with a laccase molecular weight of 65 kDa.

3.5. Kinetics of the conjugated laccase

Table 4 gives the values of kinetic parameters of ABTS

Fig. 3. Residual activity of conjugated laccase after repeated phase change.
One cycle of phase change included conjugate precipitation (pH 4–7), cen-
trifugation/washing, and re-dissolution into acetate buffer (pH 4). The lac-
case activity was measured after each cycle.
oxidation by free and conjugated laccase. The value of
kcat , or turnover of laccase, is calculated from laccase
protein content and an average molar mass of 65 kDa.
Obviously, the turnover of laccase declined compared to
free enzyme and was adversely affected by the chitosan
molecular size. In contrast, the value of Km or the affin-
ity of ABTS to laccase was not affected very much,
or no clear trend was observed with chitosan molecular
size.

Acknowledgement

This work was supported primarily by the ERC program of

Fig. 4. pH Profiles of free laccase and conjugated laccase with chitosans of
different molecular mass. Each profile is relative to the enzyme activity at
pH 3.
the National Science Foundation under Award Number EEC-
9731725. GD was supported by a graduate studentship of the
Marine Bioproducts Engineering Center at the University of
Hawaii.

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but its intrinsic biocatalytic property is reserved.
Fig. 5. Temperature profile of free laccase and conjugated laccase with chi-
tosans of different molecular mass. Each profile is relative to the enzyme
activity at 40 ◦ C.
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