A comparison of entrapped and covalently bonded laccase: Study of its leakage, reusability, and the catalytic efficiency in TEMPO-mediated glycerol oxidation

Article abstract of DOI:10.1080/10242422.2017.1384467

This article presents the comparison for reusability and leakage between entrapped and covalently bonded laccase and their performances towards the selective oxidation of glycerol. The reusability of immobilized laccase enzyme was studied by reacting a batch of immobilized laccase with ABTS for 15 cycles. The investigation of the leakage of immobilized laccase was carried out by storing the immobilized laccase in acetate buffer solution for 32 days. The data show that the retained enzyme activities of entrapped and covalently bonded enzyme after being reused for eight cycles were well above 60% and the leakages after storing for a month in the acetate buffer at 4 °C were well below 15%. The entrapped laccase coupled with TEMPO was found to perform better and gave a two-fold higher yield of glyceraldehyde and glyceric acid in the selective oxidation of glycerol compared to covalently bonded laccase. Hence, physical entrapment of laccase would be a suitable immobilization method in the laccase-mediated selective oxidation of glycerol.

Full text of DOI:10.1080/10242422.2017.1384467

Biocatalysis and Biotransformation
ISSN: 1024-2422 (Print) 1029-2446 (Online) Journal homepage: http://www.tandfonline.com/loi/ibab20
A comparison of entrapped and covalently bonded
laccase: Study of its leakage, reusability, and the
catalytic efficiency in TEMPO-mediated glycerol
oxidation
Chi Shein Hong, Cindy Chin Yee Lau, Chun Yi Leong, Gek Kee Chua & Sim Yee
Chin
To cite this article: Chi Shein Hong, Cindy Chin Yee Lau, Chun Yi Leong, Gek Kee Chua & Sim
Yee Chin (2017): A comparison of entrapped and covalently bonded laccase: Study of its leakage,
reusability, and the catalytic efficiency in TEMPO-mediated glycerol oxidation, Biocatalysis and
Biotransformation, DOI: 10.1080/10242422.2017.1384467
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Date: 18 October 2017, At: 09:52

BIOCATALYSIS AND BIOTRANSFORMATION, 2017
https://doi.org/10.1080/10242422.2017.1384467
RESEARCH ARTICLE
A comparison of entrapped and covalently bonded laccase: Study of its
leakage, reusability, and the catalytic efficiency in TEMPO-mediated
glycerol oxidation
Chi Shein Honga , Cindy Chin Yee Laua , Chun Yi Leonga , Gek Kee Chuaa
and Sim Yee China,b
b
aFaculty of Chemical & Natural Resources Engineering, Universiti Malaysia Pahang, Gambang, Pahang, Malaysia; Centre of Excellence
for Advanced Research in Fluid Flow, Faculty of Chemical & Natural Resources Engineering, Universiti Malaysia Pahang, Gambang,
Pahang, Malaysia
ABSTRACT
ARTICLE HISTORY
Received 14 March 2017
Revised 10 September 2017
Accepted 19 September 2017
This article presents the comparison for reusability and leakage between entrapped and cova-
lently bonded laccase and their performances towards the selective oxidation of glycerol. The
reusability of immobilized laccase enzyme was studied by reacting a batch of immobilized lac-
case with ABTS for 15 cycles. The investigation of the leakage of immobilized laccase was carried
out by storing the immobilized laccase in acetate buffer solution for 32 days. The data show
that the retained enzyme activities of entrapped and covalently bonded enzyme after being
reused for eight cycles were well above 60% and the leakages after storing for a month in the
acetate buffer at 4 ꢀC were well below 15%. The entrapped laccase coupled with TEMPO was
found to perform better and gave a two-fold higher yield of glyceraldehyde and glyceric acid in
the selective oxidation of glycerol compared to covalently bonded laccase. Hence, physical
entrapment of laccase would be a suitable immobilization method in the laccase-mediated
selective oxidation of glycerol.
KEYWORDS
Laccase; selective oxidation;
immobilization; leakage;
reusability
Abbreviations: A: Absorbance; ABTS: 2,20-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid);
GlAc: Glyceric acid; Gled: Glyceraldehyde; HPLC: High-performance Liquid Chromatography; e:
Extinction coefficient; TEMPO: 2,2,6,6-Tetramethylpiperidine-1-oxyl.
1. Introduction
Enzyme USA Co. Ltd. for colour enhancement in tea)
(Osma et al. 2010). The other potential uses of laccases
are in the areas of biosensors, waste detoxification,
effluent decolourization, nanobiotechnology, cosmet-
ics, synthetic chemistry, and bioremediation of food
industry wastewater (Mayer and Staples 2002;
Oxidoreductive enzyme laccases (EC 1.10.3.2) or cop-
per-containing polyphenol oxidase, with a molecular
mass around 70 kDa, are widely distributed in fungi,
plants, bacteria, and insects. They can oxidize a variety
of substrates such as diamines, polyphenols, and some
inorganic compounds using molecular oxygen as the
electron acceptor (Piontek et al. 2002; Claus 2004). The
excellent properties of laccases including high activity,
selectivity, and specificity allow them to react in the
chemical processes under mild environmental condi-
tions (Koschorreck et al. 2009). The unique set of bene-
ficial features makes laccase a very useful industrial
biocatalyst. Laccases have various potential applica-
tions in different industrial processes such as pulp and
paper (e.g. Novozym 51003 from Novozymes, Denmark
for paper pulp delignification), textiles (e.g. IndiStar
from Genencor Inc., Rochester for denim finishing),
and food industries (e.g. LACCASE Y120 from Amano
ꢀ
Rodrıguez Couto et al. 2007), in which many of them
were patented (Si 2001; Maupin-Furlow et al. 2013).
Laccase applications are hindered by some practical
problems and the common perceptions are sensitivity
to process conditions, high cost of isolation and purifi-
cation, and structure instability (Krajewska 2004; Yang
et al. 2016). Improvement of the enzyme features, viz.
protein engineering, microbiology, and chemistry of
protein, are crucial (Cirino and Arnold 2002; Reetz
et al. 2006). Besides those mentioned, an apparently
old technique – immobilization has been revealed as
the most adopted stabilization method to overcome
these limitations and allow enzyme reutilization (Hong
et al. 2015). Enzyme immobilization can be categorized
CONTACT Gek Kee Chua
chua@ump.edu.my
Universiti Malaysia Pahang, Faculty of Chemical and Natural Resources Engineering, Lebuhraya Tun
Razak, Kuantan 26300, Malaysia
ß 2017 Informa UK Limited, trading as Taylor & Francis Group

2
C. S. HONG ET AL.
into entrapment, encapsulation, adsorption, covalent
bonding, and self-immobilization (Brady and Jordaan
2009; Hong et al. 2015; Bilal et al. 2017). Entrapment is
the easiest immobilization method in that it only
involves the physical retention of the enzyme in the
porous solid matrix with no alteration of the enzyme
native structure and the procedure is less tedious than
other methods (Bailey and Ollis 1986; Lu et al. 2007).
Covalent attachment, on the other hand, is a method
which binds the enzyme to the carrier via chemical
bonds that might perturb the enzyme native structure
when the process is run in batch mode. Unfortunately,
Arends et al. (2006) only studied the rate of the LMS
in oxidizing glycerol with free laccase, while
Liebminger et al. (2009) only briefly compared the
same system with covalently bonded laccase in terms
of stability for a long-term reaction. Therefore, this
paper examines two different immobilization methods
and the practical problems of both immobilized
enzymes such as stability, reusability, and efficiency of
immobilized laccase incorporated with TEMPO towards
the selective oxidation of glycerol. It is hoped that the
outcomes of the study would assist the industry in
selecting a proper immobilization method for the LMS
in oxidizing glycerol.
ꢀ
(Duran et al. 2002). The immobilization yield depends
on the immobilization technique and material. For
example, Asgher et al. (2017) revealed that immobiliza-
tion yield of laccase entrapped in agar–agar matrix
was 79.7% while the immobilization of laccase on
magnetic chitosan microspheres obtained 24% yield of
protein coupling and an enzyme activity of 322.6 units
per gram of support (Jiang et al. 2005). Some of the
supports like carbon nanotubes have a long and com-
plicated procedure to prepare, hence the enzyme
structure may be disrupted during the immobilization
process (Feng and Ji 2011). Furthermore, the stability
of the immobilized enzyme highly depends on the
immobilization technique.
The use of laccase has been extended by the intro-
duction of laccase-mediator system (LMS) to overcome
the limitation of its redox potential. The mechanistic
details of LMS catalyzed oxidations have been
reported in detail (Christopher et al. 2014; Moilanen
et al. 2014). To date, the abundancy of glycerol (a by-
product of biodiesel production) has prompted the
development of new applications. In this context, the
resulting surplus of glycerol has been used as a sub-
strate in LMS for selective oxidation (Liebminger et al.
2009) to attain a series of value added intermediates.
This LMS is a greener approach compared to the con-
ventional oxidation method with noble metals in gly-
cerol oxidation (Porta and Prati 2004; Wang et al.
2015). The efficient utilization of glycerol with LMS,
however, requires extensive studies to provide suffi-
cient evidence for practical usage in industries.
Previous studies on LMS focused more on pre-treat-
ment of paper and pulp (Moilanen et al. 2014) and
dye decolourization (Soares et al. 2001). Since oxidiz-
ing glycerol using LMS involves a series of reactions
and the transfer of electrons among oxygen, laccase,
mediator, and substrates, the reaction is complex.
Hence, investigating the method of immobilizing lac-
case and its reusability in the complex reaction is vital.
In an industrial setting, besides robustness of the pro-
cess, enzyme reusability and its storage stability are
also important factors to be considered, especially
2. Materials and methods
2.1. Enzymes and chemicals
Laccase from Trametes versicolor (ꢁ10 U/mg) was pur-
chased from Sigma-Aldrich, Germany without further
purification. Alumina pellets and gelatine were pur-
chased from Sigma-Aldrich, Germany. Glycerol was
obtained from Fisher Scientific, United Kingdom.
2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO), poly-
ethylene glycol (PEG) (MW ¼ 4000), glutaraldehyde, cal-
cium chloride (CaCl2) were bought from Merck,
Germany. 2,20-Azino-bis(3-ethylbenzothiazoline-6-sul-
phonic acid) (ABTS) was procured from Roche,
Germany while sodium alginate was provided by R&M
chemicals.
2.2. Laccase immobilization
2.2.1. Entrapment by sodium alginate
Two grams of gelatine and 0.5 g of PEG was added to
100 mL of 2.0% (w/v) of sodium alginate solution. One
thousand units of laccase were then added and mixed
thoroughly for 10 min at 25 ꢀC. The mixture was with-
drawn using a sterile 5-mL syringe and extruded
through a 21 gauge (0.51 mm inner diameter) needle
into 2.0% (w/v) of CaCl2 solution. Beads formed imme-
diately when the droplets of the mixture touched the
surface of the solution. Beads were left unstirred to
harden in the CaCl2 solution at 4 ꢀC for 2 h, after which
the CaCl2 solution was removed by filtration and the
beads were washed twice with distilled water. The
beads were subsequently incubated in 100 mL of 0.6%
(w/v) glutaraldehyde solution and stirred at 4 ꢀC for
2 h. The beads were washed several times with phos-
phate buffer (pH 7) and kept at 4 ꢀC in acetate buffer
(pH 4.5). The method was adapted from Wang et al.
(2008).

BIOCATALYSIS AND BIOTRANSFORMATION
3
defined as 1 lmol ABTS oxidized per minute under the
stated assay condition.
2.2.2. Covalent attachment on alumina support by
cross-linking agent
Laccase immobilization using covalent bonding was
2.4.2. Enzyme reusability test
ꢀ
adapted from Rodrıguez Couto et al. (2007). In brief,
alumina pellets were silanized at 45 ꢀC for 20 h in a 2%
(v/v) solution of aminopropyltriethoxysilane in acetone.
The silanized supports were washed once with acet-
one and silanized again for 24 h. Then, they were
washed several times with deionized water and air
dried. In the second stage, the alumina pellets were
treated with 2% (v/v) aqueous glutaraldehyde (50% v/
v) for 2 h at room temperature, washed again with
deionized water, and air dried. These supports were
immersed in 100 mL of laccase solution (10 U/mL) for
48 h at room temperature. The supports were washed
numerous times with 0.05 M phosphate buffer (pH 7)
to remove the unbound proteins and kept at 4 ꢀC until
further use.
The reusability of immobilized laccase was studied for
15 cycles. Three sample vials, each containing approxi-
mately 0.1 g covalently bonded laccase enzyme, were
supplemented with 1.9 mL of buffer solution and
1.0 mL of ABTS solution. The immobilized laccase was
allowed to catalyze the oxidation of ABTS for 75 min
(Mohd Zain et al. 2010). The immobilized laccases
were filtered, washed, and placed into a new batch of
ABTS solution for the second cycle. The experiment
was repeated to test the reusability of entrapped lac-
case by substituting the covalently bonded laccase
with the entrapped enzyme. The activity of laccase
was assayed as described in the Section 2.4.1. The
retained activity was calculated using Equations (1)
and (2).
Absorbance of mixture
Unit of enzymes
¼
(1)
(2)
ðnÞ
2.3. Selective oxidation of glycerol by immobilized
laccase coupled with TEMPO
e
Unit of enzyme
ðnÞ
ð Þ
Retained activity % ¼
Unit of enzyme
The reaction was performed in an incubator with the
orbital shaking system. The reaction mixture (total
volume ¼ 50 mL) consisted of 100 mM of glycerol,
30 mM of TEMPO, and 0.1 g of immobilized laccase in
100 mM of sodium acetate buffer (pH 4.5). The stirring
speed was set at 180 rpm. The reaction temperature
and pH were controlled at 25 ꢀC and pH 4.5, respect-
ively. One hundred microlitres of sample were taken
hourly for the first 3 h and the final sample at the 24th
h. Nine hundred microlitres of 10% (v/v) of acetic acid
was added into the sample immediately to stop the
reaction. Analysis of samples was conducted as
described in the Section 2.4.4.
ðn¼1Þ
2.4.3. Leakage test
Leakage test was carried out for a period of 32 days
for both covalently bonded and entrapped laccase.
Immobilized laccases were stored in acetate buffer (pH
4.5) and a sample of the buffer solution was collected
every day for the first week and then every 5 days
thereafter. One hundred microlitres of the sample buf-
fer solution were added to 1.9 mL of 0.1 M of acetate
buffer. One millilitre of ABTS solution was added and
the activity of laccases was assayed as described in
the Section 2.4.1. The sample analysis was repeated
three times to obtain an average value. In the period
of study, all samples were stored at 4 ꢀC. The percent-
age of leakage was calculated based on Equations (3)
and (4).
2.4. Analytical methods
2.4.1. Spectrophotometric enzymatic assay
Laccase activity was determined using ABTS as sub-
strate and performed spectrophotometrically with
UV–VIS spectrophotometer (light path ¼ 1 cm) at room
temperature. The oxidation of ABTS by laccase was
monitored at 415 nm with a molar absorption value of
36,000 Mꢂ1 cmꢂ1. The assay mixture for the blank con-
sisted of 1.9 mL of 0.1 M of acetate buffer (pH 4.5),
0.1 mL of pure water, and 1.0 mL of 0.5 mM of ABTS
solution. In free enzyme assay, pure water was
replaced by 0.1 mL of laccase, while 1 g of immobilized
laccase was used to replace pure water in the immobi-
lized enzyme assay. One unit (U) of the enzyme was
ð Þ
Units of enzyme leaked U ¼
Absorbance
ð
Þ
ꢃ dilution factor ꢃ volume of buffer ml
(3)
e
ð Þ
Leakage % ¼
Units of enzyme leaked Þ ꢂ Units of enzyme leaked
ð
ðd¼1Þ
d
Units of enzyme leaked
ðd¼1Þ
ꢃ 100%
(4)

4
C. S. HONG ET AL.
These results are better than the one obtained by
Asgher et al. (2017) where they reported that the
retained activity of laccases entrapped in gelatin after
eight cycles of reactions were 22.8%. In this study, the
retained activity for covalently bonded laccase after
eight cycles of reuse was also comparable to 60% as
reported by Diao et al. (2002), who covalently immobi-
lized the laccase on the activated carboxylated polyvi-
nyl alcohol carrier. Hence, the reusability of the laccase
enzymes immobilized using these two methods in this
study is high.
The disintegration of alginate beads might be one
of the reasons that lower the retained activities at the
end of the 15th cycle of reaction. Possible reasons for
disintegration to occur are poor immobilization han-
dlings such as the presence of bubbles in the beads
and the pH of storage buffer. Since sodium alginate
dissolves slowly in water, air tends to enter the solu-
tion during the stirring process, causing bubbles to
form in the solution. The formation of bubbles might
also occur if the alginate solution is not properly with-
drawn using the syringe. When bubbles are present in
the beads, the chance of leakage increases as the
polymerization and cross-linking bonds are weak due
to the obstruction of bubbles. According to Babu et al.
(2010), alginate beads are stable at low acidic pH but
undergo swelling and disintegration at intestinal pH
(pH 6–7.4). Since beads are highly dependent on the
pH of the medium, phosphate buffer with a pH of 7
might not be a good storage buffer and causes the
disintegration (Sabyasachi 2017).
A smaller diameter of beads could promote mass
transfer and minimize bead disintegration or rupture
due to the formation and accumulation of gas.
However, bead size is limited by the viscosity of the
solution and the diameter of the syringe needle.
Alginate beads are formed immediately when calcium
is introduced to the alginate in the soluble form. The
gels produced are usually inhomogeneous and have a
structure that is stronger at the exterior than that of
the interior. This is because when alginate and calcium
ions were in contact with each other, gelation occurs.
Since the alginate solution was introduced as a droplet
into the high calcium ions solution, gelation on the
surface of the droplet was faster than that inside the
droplet. Gelation of alginate solution inside the droplet
require diffusion of calcium ions through the gel
(Roopa and Bhattacharya 2008; Bhushan et al. 2015;
Hong 2016).
2.4.4. Analysis of oxidation products
Glycerol oxidation products were analyzed by high per-
formance liquid chromatography (Agilent 1260 Infinity
Liquid Chromatography, United State) equipped with
DAD and RID. A Phenomenex Rezek ROA column
(300 ꢃ 7.8 mm) was used with 3 mM of H2SO4 as a
mobile phase. The temperature of column and RID
were fixed at 75 and 35 ꢀC, respectively. DAD wave-
length was set at 210 nm. Ten microlitres of sample
was injected into the system with 0.5 mL/min of flow
rate. Identification of components was performed by
comparison of their retention times with the standards.
2.5. Data analysis
All experiments were performed in triplicates and their
mean values were taken into consideration for
calculation.
3. Results and discussion
3.1. Yield and morphology of immobilized laccase
Immobilization yield was determined as described in
the Section 2.3 in which the yield for both entrapment
and covalent bonding method were 90 and 94%,
respectively. Figure 1 shows the structural morpholo-
gies of alumina pellet only and immobilized laccase on
alumina pellet. Under 5k magnification, the surface
morphology of the alumina pellets was considerably
altered. A noticeable roughness on the surface of alu-
mina pellets after enzyme immobilization is possibly
due to the laccase that was randomly bound and filled
the alumina pellets (Figures 1(c,d)). Micropores in a
range of 200–400 nm were reduced to approximately
100 nm in 50k magnification (Figures 1(a,b)), suggest-
ing that laccase was immobilized on the surface and
covered a part of the pores. In general, these results
demonstrate that the laccase enzyme was effectively
immobilized on the surface of alumina pellets.
3.2. Reusability of immobilized laccase
The retained activities of covalently bonded and
entrapped laccase are illustrated in Figure 2. The per-
centage of retained activity was calculated with
respect to the initial activity. It can be observed that
the retained activity of entrapped laccase was slightly
higher compared to the covalently bonded laccase for
the first eight cycles of reactions (65.1 and 62.4%,
respectively). However, the retained activities for cova-
lently bonded and entrapped laccase after being
reused for 15 cycles were 45.7 and 33.5%, respectively.
Gelation or the strengthening of gel depends on
diffusion rate of calcium ions through the gel. In this
study, the gelation kinetic was extremely rapid,
observed by an instantaneous sol-gel transition at the

BIOCATALYSIS AND BIOTRANSFORMATION
5
Figure 1. FESEM image in 50k magnification for (a) alumina and (b) immobilized laccase on Alumina pellet; in 5k for (c) alumina
and (d) immobilized laccase on Alumina pellet.
surface of the alginate. Glutaraldehyde (GA) was used
as a crosslinker for the enzyme to make the enzyme
molecules aggregate and thus reducing their leakage
(Kumar et al. 2017). Bhushan et al. (2015) demon-
strated an improvement in the immobilization effi-
ciency and a 2-fold increase of activity by using GA as
the crosslinker. They revealed that the entrapped
enzyme after crosslinking with GA was stable with
reduced diffusion characteristics. Besides that, different
alginate sources also affect the mechanical properties
of alginate gel. Mancini et al. (1999) studied the mech-
anical properties of several guluronic and mannuronic
alginate gels at different concentrations by using
stress relaxation and uniaxial compression method.
They reported that mannuronic alginates formed softer
and more elastic gel than that of guluronic alginates

6
C. S. HONG ET AL.
Figure 2. The retained activities of entrapped and covalently bonded laccase for 15 cycles. Data were reported as the average of
three replicates (symbols: ᭿¼ entrapment; ꢄ¼ covalent bond).
by the asymptotic stress relaxation constant. Since the
source of sodium alginate was unable to be identified
for this study, it is presumed to be guluronic alginate
as revealed by the experimental results. Another pos-
sible reason for the decrease in the retained activity is
the degradation of laccase for both immobilization
methods. Enzyme at its optimum temperature might
not be stable for a long period as well. The exposure
time would be another factor that influences the effi-
ciency of the enzyme at a certain temperature (Hong
2016).
storage at 4 ꢀC. At this temperature, laccase is inactive
and hence prolonging its shelf life.
On the other hand, entrapped laccase has low leak-
age because the enzyme is physically restricted within
a network or matrix. With GA, the leakage is presumed
to be minimized, yet, leakage of the enzyme through
large pores of the matrix over a long period is still
possible. Some of the enzymes gradually diffuse
towards the gel outer shell and eventually leak into
the medium. Mancini et al. (1999) suggested that the
swelling of the beads might affect the diffusivity of
certain molecules through the pore of the beads.
Fernandez-Lorente et al. (2015) reported that most
soluble enzymes have high stability with high concen-
trations of PEG. With this interesting antecedent, the
fixation of PEG on enzyme surface would be a viable
stabilization strategy as in the case of enzyme entrap-
ment in this study. In contrast, the enzyme immobi-
lized through covalent binding provides a strong and
stable enzyme attachment, thus reducing the detach-
ment of enzyme from the support. As a consequence,
the enzyme leakage is low for both methods.
3.3. Leakage of immobilized laccase
Figure 3 illustrates the leakage of both covalent
bonded and entrapped laccase enzymes increased
over time. The leakages at day 32 were 14.5 and
13.9% for covalent bonded and entrapped laccase
enzyme, respectively. Since the leakage for both
immobilization methods is less than 15% in a month,
it could be considered as low leakage. The low leakage
level obtained might be due to the application of opti-
mum storage temperature of 4 ꢀC, which is suitable to
be used to store both immobilized laccase enzymes.
Storage temperature of 4 ꢀC was in agreement with
3.4. Oxidation of glycerol by immobilized laccase
coupled with TEMPO
ꢀ
the investigation by Moreno-Perez et al. (2016). In their
study, they reported that the immobilized laccase still
retained 95–100% of its initial value after 10 days of
Valuable chemical intermediates produced from the
selective oxidation of glycerol include glyceraldehyde,

BIOCATALYSIS AND BIOTRANSFORMATION
7
Figure 3. The leakage of laccase from its respective support over a period of 32 days. Data were reported as the average of three
replicates (symbols: ᭿¼ covalent bond; ᭢¼ entrapment).
Figure 4. Conversion of glycerol (free enzyme: ᭡; entrapped laccase: ꢄ; covalent bonded laccase: ᭜) and concentration of the oxi-
dation products (free enzyme:
¼ Gled,
¼ GlAc; entrapped laccase:
¼ Gled,
¼ GlAc; covalent bonded laccase:
¼ Gled,
¼ GlAc). Data were reported as the average of two replicates for entrapped and covalent bonded laccase; average
of three replicates for free enzyme.
glyceric acid, tartronic acid, and mesoxalic acid (Werpy
and Petersen 2004). Primary hydroxyl groups would be
selectively oxidized to glyceraldehyde (Gled) which is
an intermediate for carbohydrate metabolism and
standard for the comparison of chiral molecules (D- or
L-). Further oxidation would yield glyceric acid (GlAc).
It is used in treating some skin disorders. In addition,
GlAc in its ester form associated with a quaternary
ammonium salt can be used as a biodegradable fabric
softener (Behr et al. 2008).
Figure 4 illustrates the conversion of glycerol as
well as the concentration of the oxidation products for

8
C. S. HONG ET AL.
Figure 5. Proposed reaction mechanism on oxidation of glycerol by immobilized laccase/TEMPO system.
achieving nearly 27% of glycerol conversion and the
the interval studied. In this study, the reaction was
only performed up to 24 h, so only the products
attained within the reaction period were reported. It
can be seen that free laccase performed the best after
24 h of reaction, with nearly 40% glycerol conversion
and the highest amount of Gled and GlAc attained.
This result is in agreement with Liebminger et al.
(2009) who reported that the conversion of glycerol is
higher for free enzyme than immobilized enzyme
within the same reaction period. Free enzyme is
homogeneous with the reactants in the solution, thus
eliminating all the possible mass transfer resistances.
This is noticeable from the glycerol conversion and the
products concentrations achieved by the free enzyme
for the first 3 h of reaction in Figure 4. The glycerol
conversion of free enzyme was lower compared to the
entrapped laccase, but the products concentrations
achieved were higher. This is possibly due to the mass
transfer resistance experienced by the entrapped lac-
case, where the products of oxidation required some
times to transport out from the alginate beads and
detected in the external reaction mixture.
final concentrations for Gled and GlAc were 2-fold
greater than that of covalently bonded laccase. The
Gled concentration attained by the entrapped laccase
at 24 h of reaction is comparable to the one obtained
by free enzyme. However, the GlAc achieved was only
a quarter of that attained by the free enzyme. The effi-
ciency of the immobilized laccase coupled with
TEMPO in the reaction with glycerol was highly
dependent on the active site of laccase as laccase
would regenerate TEMPO. Figure 5 shows the pro-
posed reaction mechanism, which reveals the reaction
between immobilized laccase, TEMPO, and substrate.
Unexpectedly, the entrapped laccase with TEMPO gave
a high concentration of Gled and GlAc. The diffusion
limits of the entrapped laccase did not seem to be a
barrier for TEMPO to react with laccase. Covalently
bonded laccase in which the laccase was bonded on
the surface of the support was supposed to have a
better interaction with TEMPO in oxidizing glycerol
due to lesser mass transfer resistance. Nonetheless,
this is not the case.
Figure 4 shows that entrapped laccase had better
performance than covalently bonded laccase,
During covalent bonding of laccase onto the alumina
support, free amino group of the laccase is used for

BIOCATALYSIS AND BIOTRANSFORMATION
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http://orcid.org/0000-0001-7828-8217
http://orcid.org/0000-0002-5933-1600
http://orcid.org/0000-0002-9302-6917
9
bonding and this includes the N-terminal of the enzyme.
According to Morozova et al. (2007), a decreased contri-
bution of free electron pair from nitrogen atom would
cause an increase in the length of the Cu–N bond and
reduce the redox potential of the T1 site of laccase
enzyme. Since T1 site of the laccase is the primary
acceptor of electron from the reducing substrate, it
determines the catalysis efficiency of the enzyme.
Therefore, the performance of covalently bonded lac-
case was not as good as entrapped laccase which was
only physically retained in the alginate beads.
Immobilization by entrapment seems to be a more
suitable immobilization technique compared to cova-
lent bonding in coupling with TEMPO to oxidize gly-
cerol into Gled and/or GlAc. The procedure of laccase
entrapment is less tedious and more environmentally
friendly, and a complete immobilization only took
around half a day compared to the covalent method
that took a week. Moreover, it consumed less energy
as it could be conducted at room temperature (25 ꢀC).
ORCID
Chi Shein Hong
Cindy Chin Yee Lau
Chun Yi Leong
Gek Kee Chua
Sim Yee Chin
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Disclosure statement
Fernandez-Lorente G, Lopez-Gallego F, Bolivar JM, Rocha-
Martin J, Moreno-Perez S, Guisan JM. 2015. Immobilization
of proteins on highly activated glyoxyl supports: dramatic
increase of the enzyme stability via multipoint immobiliza-
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oxidation by laccase/2,2,6,6-tetramethylpiperidine-1-oxyl
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kinetic study. Malaysia: Universiti Malaysia Pahang.
No potential conflict of interest was reported by the authors.
Funding
This work was financially supported by the Ministry of
Higher Education Malaysia under the Fundamental Research
Grant Scheme [RDU 130101] and Graduate Research Scheme
[GRS 140303] by Universiti Malaysia Pahang.

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