Process Biochemistry 101 (2021) 137–146

Contents lists available at ScienceDirect

Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Laccase immobilized polyacrylamide-alginate cryogel: A candidate for

treatment of effluents
Rukiye Yavaşer *, Arife Alev Karagözler
Chemistry Department, Faculty of Arts and Sciences, Aydın Adnan Menderes University, Aydın, Turkey

A R T I C L E I N F O A B S T R A C T

Keywords: Enzyme immobilization is a promising technique for several purposes including biotechnology, medicine,
Laccase biochemistry and environment. Laccase is one of the intensely studied enzyme in immobilization research. In this
Cryogel study, laccase enzyme from Trametes versicolor was immobilized on glycidyl methacrylate (GMA) functionalized
Polyacrylamide-alginate
polyacrylamide-alginate cryogel (PAG). Characterization of the cryogel was carried out by Fourier transform
Olive mill wastewater
Effluent
infrared spectrometry, environmental scanning electron microscopy, energy dispersive X-ray analysis, surface
Decolorization area analysis by Brunauer-Emmett-Teller technique and swelling test. Laccase enzyme was immobilized cova­
Dephenolization lently and maximum loading (68.7 ± 1.45 mg/g) occurred at pH 3.0 and 25 ◦ C. Activities of free and immo­
bilized laccase enzymes were measured using three substrates, 2,2ˈ-azinobis-(3-ethylbenzothiazoline-6-sulfonate,
syringaldazine and guaiacol. Optimum pH, optimum temperature and kinetic parameters were calculated with
each substrate. Stability of immobilized laccase enzyme due to temperature rise, storage duration and repeated
use was demonstrated to be higher than free laccase enzyme. Immobilized laccase was also utilized for decol­
orization of dyes, dephenolization of olive mill wastewater (OMW) and treatment for a textile effluent. It was
demonstrated that immobilized laccase enzyme removed 70.0 ± 1.2 % of phenolic compounds in OMW, suc­
cessfully. Additionally, 55.6 ± 0.72 % of removal of dyes from textile wastewater and 93.3–99.1 % decolor­
ization of some selected dyes in solution was observed.

1. Introduction molecular oxygen to water [5]. Immobilization of laccase allows the

Enzyme immobilization is an efficient process offering several im­
provements for enzymatic applications. Immobilization may provide
enhanced structural and catalytic stability, thermostability and dura­
bility to enzymes and allows separation of enzyme and product rapidly
from the reaction medium and ensure repeated use of the support ma­
terial [1]. The choice of immobilization method, support material and
application of immobilized enzyme is crucial for an efficient immobili­
zation process. Covalent binding is one of the intensely preferred pro­
cedure for immobilization where the enzyme is firmly bound without
leakage from the support [2].
Laccase enzyme (EC 1.10.3.2) is a member of oxidoreductases class
and widely used in industrial areas including dye, textile, baking, paper,
fruit juice processing and wastewater treatment [3]. This
copper-containing biocatalyst displays low substrate specifity and have
the ability to oxidize a wide range of substrates including phenols, ar­
omatic amines, ascorbate, and lignin [4]. Oxidation of the substrate by
laccase is carried out by concomitant four monoelectronic reduction of
enzyme to be more stable against temperature and pH changes, storage
and operation conditions [4].
Cryogels became a widely-studied body of research since 1970 with
the synthesis of the first cryogel based on polyvinyl alcohol (PVA) [6].
These hydrogel-type materials that are usually fabricated as monolithic
columns with soft, spongy, macroporous and low-cost properties [7].
They are synthesized at sub-zero temperatures and cryogelation com­
bines both crystallization of the bulk solvent to generate interconnected
macropores and polymerization of monomer(s) resulting a porous gel
after thawing [8,9]. The main mechanism for transportation in such
macroporous structures is convection, therefore, mass transport resis­
tance is minimized [10]. Cryogels have broad applications in separation,
purification, tissue engineering and biomedical sciences [11]. In recent
years, cryogels have gained remarkable attention as novel supports
enabling attachment of enzymes.
Increasing population, industrialization and urbanization cause ris­
ing environmental pollution since large amounts of wastes are disposed.
Dyes, effluents, phenol and its derivatives are hazardous chemicals

* Corresponding author.
E-mail addresses: ryavaser@adu.edu.tr (R. Yavaşer), akaragozler@adu.edu.tr (A.A. Karagözler).

https://doi.org/10.1016/j.procbio.2020.11.021
Received 24 July 2020; Received in revised form 10 November 2020; Accepted 19 November 2020
Available online 24 November 2020
1359-5113/© 2020 Elsevier Ltd. All rights reserved.
R. Yavaşer and A.A. Karagözler Process Biochemistry 101 (2021) 137–146

Table 1 a water purification equipment (Millipore, Simplicity UV, France) with a

Composition and properties of cryogels synthesized using different quantities of specific resistivity of 18.2 MΩ cm.
AAm, MBAAm, alginate and water.
Combination AAm MBAAm Alginate Distilled Observation
(g) (g) solution water 2.2. Experimental
(1.0 %, mL) (mL)

1 1.835 0.3 – 36.0 Non-porous 2.2.1. Fabrication and characterization of polyacrylamide-alginate (PAG)
2 0.4 1.7 – 30.0 Weak cryogels
3 0.395 0.0286 3.0 17.0 Weak Different concentrations of AAm, MBAAm, alginate and solvent were
4 0.395 0.0424 3.0 17.0 Weak
used whereas GMA, APS and TEMED concentrations (Table 1) were
5 0.395 0.0848 3.0 17.0 Non-porous
6 0.395 0.1058 3.0 12.0 Weak constant in order to optimize the combination of PAG cryogel with
7 0.395 0.1058 3.0 7.0 Elastic, spongy, elastic, porous and robust properties. Combination 7 was
robust, determined to be the most convenient cryogel for the study. PAG cryogel
porous was fabricated using polymerization solution containing 0.395 g (5.56
mmol) acrylamide and 3.0 mL of sodium alginate solution (1.0 %, w/v)
threating the environment due to their toxicity for humans and as monomers, 0.1058 g/7.0 mL of distilled water (0.69 mmol), MBAAm
ecosystem [12,4]. Thus, search for new techniques and materials is as cross linker and GMA (25.0 μL). After cooling the solution in an ice
conducted to combat these wastes. bath to +4 ◦ C with continuous stirring, polymerization was initiated
Olive mill wastewater (OMW) is a by-product generated during olive with addition of 20.0 mg APS and 25.0 μL of TEMED. A plastic syringe
oil production and is a complex mixture composed of various constitu­ (total volume: 5.0 mL, inner diameter: 0.8 cm) was filled with 3.2 mL of
ents such as phenolic compounds, salts, suspended solids, sugars, min­ the solution and kept at − 12 ◦ C for 24 h for cryogelation. Cryogel was
eral nutrients and water. Despite the beneficial effects of phenolic thawed and washed by passing 200 mL of distilled water through the
compounds of OMW as antioxidant and antibacterial, it is also a major cryogel column in order to remove unreacted components. A control
pollutant of water, soil, and air. OMW is an ecological threat that needs cryogel without GMA (PA) was also produced.
to be tackled to reduce its impact owing to high toxic organic load, low Structural characterization of PAG cryogel was conducted by Fourier
pH value and high BOD and COD [13]. Although OMW is attempted to Transform Infrared spectrophotometry (FTIR), Environmental Scanning
be remediated by methods such as physical adsorption, chemical Electron Microscopy (ESEM), Energy Dispersive X-Ray (EDX) Analysis
oxidation, ozonation and extraction; low-efficiency, low stability, high and Brunauer-Emmett-Teller (BET) techniques. FTIR spectrum of PAG
cost and undesirable by-products limit the applicability of these tech­ cryogel was obtained by analyzing the pellet of 0.01 g of dried cry­
niques. Thus, enzymatic remediation pathways are being investigated. ogel:0.1 g of KBr, using an FTIR spectrophotometer (Varian FTS 7000,
Free laccase and immobilized laccase enzymes have become one of the US). Morphological properties of PAG cryogel were visualized by
successful candidates for that purpose [4]. Additionally, immobilized Environmental Scanning Electron Microscope (LEO-EVO 40, Cambridge,
enzymes specially gained attention in dye and phenolic compound England). EDX analysis (Bruker-125 eV, Berlin, Germany) revealed the
treatment due to their effectiveness, low-cost, practical maintenance elemental composition of the cryogel. Surface area analysis was con­
and reusability [12]. ducted with a BET device (Autosorb-6B, Quantachrome). The sample
Acrylamide-alginate combining cryogels are rare in literature. was degassed with N2 at 150 ◦ C for 1 h and mass of sample was recorded
Mourao et al. (2019) synthesized acrylamide-alginate cryogels for IgG before and after degassing. Adsorption and desorption of gas was con­
purification [10]. Herein, fabrication of a support material for immo­ ducted at 77 K and room temperature, respectively. Specific surface area
bilization of laccase enzyme from Trametes versicolor using the combi­ was obtained from the software of the instrument. Swelling degrees of
nation of synthetic polymer (polyacrylamide), and natural polymer the dried cryogel by lyophilization (Freezone 6, Model 77520; Labconco
(alginate) was presented. To the best of our knowledge, this is the first Co. Kansas City, MO) and another dried cryogel at +40 ◦ C were deter­
study about laccase immobilization onto GMA-modified acryl­ mined. For this, dry cryogels were plunged into 50.0 mL of distilled
amide-alginate based monolithic cryogel and further for the treatment of water at 25 ◦ C and the masses of swelling cryogels were recorded until
olive mill wastewater or dye decolorization. Considering the advantages they reached saturation. Using the difference between the masses of
being non-toxic, biocompatible and hydrophilicity, a useful inter­ water-saturated (MS) and dried (MD) cryogel, swelling degree (S) was
penetrating material for several applications was produced without any calculated according to the equation below:
crosslinker for alginate. Utilization of immobilized enzyme for decol­ S = [(MS – MD) / MD] (1)
orization of dyes, dephenolization of olive mill wastewater (OMW) and
degradation of real textile effluent was investigated.

2. Materials and methods 2.2.2. Covalent immobilization of laccase onto polyacrylamide-alginate

2.1. Materials
Acrylamide (AAm), alginic acid sodium salt (low viscosity), glycidyl
methacrylate (GMA), N,N,N’,N’-tetramethylethylenediamine (TEMED),
methanol, citric acid monohydrate, ammonium persulfate (APS), laccase
(from Trametes versicolor, ≥0.5 U/mg), N,N’-methylenebisacrylamide
(MBAAm), KBr, 2,2′ -azinobis-(3-ethylbenzothiazoline-6-sulfonate)
(ABTS), guaiacol (GUA), syringaldazine (SYR), Congo Red, Trypan Blue,
Sunset Yellow FCF, Fast Green FCF, Chlorazol Black, Folin-Ciocalteu
reagent (FCR) and gallic acid were obtained from Sigma (Steinheim,
Germany). Na2HPO4.2H2O and Na2CO3 were purchased from Riedel-de
Haën (Seelze, Germany). Solvents and reagents were of analytical grade
and used without further purification. Ultrapure water was obtained by
cryogels
Laccase immobilization via covalent attachment was performed
using a peristaltic pump (Longerpump BT100-1L-A, China) at 25 ◦ C for 2
h at flow rate of 150 μL/min. Laccase solution [5.0 mL, 5.0 mg/mL in 0.1
M pH 3.0 citrate-phosphate buffer solution (McIlvaine’s buffer)] was
passed through the PAG cryogel column and laccase enzyme was bound
covalently through the epoxy groups of the GMA bearing cryogel. A
control experiment was also employed using a cryogel, prepared
without adding GMA into the polymerization medium, to point out that
immobilization was through the epoxy groups. Effects of pH in range of
2.5–7.5 (0.1 M McIlvaine’s buffer), temperature (5− 55 ◦ C), initial lac­
case concentration (0.5–12.0 mg/mL) and immobilization time (2− 24
h) on enzyme attachment were investigated. Amount of bound laccase
(Q) was determined by calculating the protein concentrations from
laccase standard curve using absorbance data at 280 nm before and after

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R. Yavaşer and A.A. Karagözler Process Biochemistry 101 (2021) 137–146

Table 2
Molecular structures and maximum absorption wavelengths of dyes used in decolorization experiments.
Dye Molecular Structure λmax (nm)

Congo Red 492

Trypan Blue 552

Sunset Yellow FCF 482

Fast Green FCF 427 and 622

Chlorazol Black 540

immobilization measured with a double-beam UV spectrophotometer (3)) equations, measuring initial reaction rates of the catalytic reaction
(Shimadzu UV 1601, Tokyo, Japan). at varying concentrations of ABTS (0− 0.25 mM), SYR (0− 0.02 mM) and
GUA (0− 0.50 mM) in 0.1 M McIlvaine’s buffer at pH 3.0, 5.0, 4.5 and at
2.2.3. Activity measurements of laccase using ABTS, SYR and GUA 50 ◦ C, 60 ◦ C and 70 ◦ C, respectively.
substrates
Vmax [S]
Three substrates, ABTS, SYR and GUA, were used for activity mea­ V= (2)
Km + [S]
surements of free and immobilized laccases to evaluate the behavior of
enzyme towards different substrates. Oxidation of ABTS (ε420 = 36,000 /
Km 1 1
M− 1 cm− 1) to the stable radical, ABTS˙+, was measured according to 1 V= × + (3)
Vmax [S] Vmax
method of Bourbonnais and Paice (1992) and monitored spectrophoto­
metrically at 420 nm [14]. Laccase activity with SYR (ε530 = 65,000 M− 1
2.2.5. Thermal and storage stabilities of free and immobilized laccases
cm− 1) was measured following the increase in absorption at 530 nm
Thermal stabilities of free and immobilized laccase enzymes were
resulting from the oxidation of SYR into the quinone compound [15].
analyzed by incubating free enzyme and laccase immobilized cryogel in
Activity of laccase using GUA (ε470 = 26,600 M− 1 cm− 1) substrate was
0.1 M McIlvaine’s buffer at pH 3.0 for ABTS, pH 5.0 for SYR and pH 4.5
employed by recording the absorbance increments at 470 nm [16].
for GUA in a thermostated water bath (Daihan, Wisebath WB-22, Korea).
Activity of free laccase was followed on the kinetic mode, while
Residual activities of enzymes were measured immediately after defined
discontinuous measurement was applied for immobilized laccase using
incubation periods at 25, 40, 50, 60 and 70 ◦ C by taking samples from
the differences in optical densities between initial and final substrate
the medium at the end of stated time periods.
solutions at corresponding optimum pH and temperature. Maximal
Comparison of the stabilities of free and immobilized laccase en­
value of enzyme activity was expressed as 100 % and relative activities
zymes during their storage at 4 ◦ C were investigated by measuring re­
were calculated for each set of experiment. Average values of triplicate
sidual activities of both forms of laccase within defined time intervals.
experiments were used for calculations.
Cryogels were refreshed with distilled water followed by buffer solution
Optimum pH and optimum temperature were determined by
after each measurement.
measuring activities of free and immobilized laccase enzymes in 0.1 M
McIlvaine’s buffer (pH 2.5–6.0) and 4− 80 ◦ C. One laccase unit was
2.2.6. Decolorization of dye solutions and real textile wastewater with free
defined as the amount of enzyme required to oxidize one micromole of
and immobilized laccases
substrate in a minute under optimum conditions.
Free and immobilized laccase efficiencies to decolorize five
industrially-used dyes. Congo Red, Trypan Blue, Sunset Yellow FCF, Fast
2.2.4. Determination of kinetic parameters
Green FCF and Chlorazol Black (Table 2) were chosen as representatives
Kinetic parameters of free and immobilized laccase enzymes were
of monoazo, disazo, trisazo, and triarylmethane dyes. Aqueous (~pH
calculated using Michaelis-Menten (Eq. (2)) and Lineweaver-Burk (Eq.
7.2) and buffered dye solutions prepared using 0.1 M, pH 3.0

139
R. Yavaşer and A.A. Karagözler
Fig. 1. FTIR spectrum of PAG cryogel.
McIlvaine’s buffer (final pH ~3.4) of 0.01 mg/mL and 0.05 mg/mL dye
concentration were investigated for decolorization. Ten mL of each dye
solution was circulated through the laccase-bound cryogel at 25 ◦ C for 2
h at 100 μL/min flow rate for decolorization with immobilized enzyme.
For free laccase, equivalent mass of laccase bound to cryogel was added
into the dye solution and stirred at 50 rpm/min using a magnetic stirrer
(Daihan Scientific, MSH 20D, Korea) at room temperature. UV–vis
spectra of dye solutions were recorded initially and after passing
through cryogel at defined time intervals. Decolorization percent was
calculated using Eq. (4) and the results were plotted versus time:
A0 − At
Decolorization (%) = × 100
A0
where, A0:initial absorbance of dye solution prior to circulation through
laccase-immobilized cryogel and At:absorbance of dye solution after
circulation through laccase-immobilized cryogel at a definite time.
Real textile wastewater was supplied from a plant in Aydın Province,
Turkey. Decolorization was monitored spectrophotometrically by
recording UV–vis spectra between 200− 500 nm at different time in­
tervals. Experiments were employed at native pH of the wastewater
(11.6) and pH 5.0 adjusted with citric acid at 25 ◦ C.
2.2.7. Dephenolization of OMW by free and immobilized laccases
The amount of phenolic compounds in OMW before and after
treatment with immobilized laccase was determined according to the
spectrophotometric method reported by Singleton et al. [17]. To
determine the initial amount of phenolic compounds, OMW sample
Fig. 2. Images of native (a), freeze-dried (b), swollen in distilled water (c) and squeezed (d) PAG cryogel.
Process Biochemistry 101 (2021) 137–146
(0.25 mL) was diluted to 23.0 mL with distilled water in 100 mL
erlenmeyer flask. Folin-Ciocalteu reagent (0.5 mL) was added and
allowed to react with phenolic compounds for 3 min at 25 ◦ C. Na2CO3
solution (1.5 mL, 2.0 %) was added and the solutions were shaken in a
benchtop shaker (Heidolph, Promax 2020, Germany) for 2 h at 120 rpm
in dark at 25 ◦ C. Then, absorbances of the solutions were measured at
760 nm using distilled water as blank. To evaluate the dephenolization
by immobilized laccase, 10.0 mL of OMW was circulated through the
enzyme-bound cryogel for 8 h. A 0.25 mL aliquot of the eluent was
withdrawn and the experiment was conducted as mentioned above.
Degradation and/or removal of phenolic compounds were estimated by
(4) measuring the initial and final quantities of phenolic compounds present
in olive mill wastewater from the calibration curve plotted using gallic
acid (0− 250 μg) as the reference compound. Dephenolization efficiency
due to pH of the OMW sample and interaction period of OMW with
immobilized laccase (0− 24 h) was also investigated.
2.2.8. Operational stability of immobilized laccase
Operational stability of laccase immobilized on PAG cryogels was
determined by performing ten consecutive operating cycles for decol­
orization of dyes and dephenolization of phenolic compounds in olive
mill wastewater. After each cycle, cryogel was washed with 100 mL
water for three times and then with buffer solutions respectively to
remove any residue and the procedure restarted with a fresh aliquot of
sample.
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R. Yavaşer and A.A. Karagözler
Fig. 3. ESEM micrographs of PAG cryogel with a magnification of 2500 × .
Fig. 4. EDX spectrum of laccase immobilized PAG cryogel.
3. Results and discussion
3.1. Characterization of cryogel
Incorporation of monomers building up the cryogel and GMA, which
functionalize the cryogel with epoxy groups was verified by FTIR. As
seen in Fig. 1, the characteristic C– – O stretching vibrations of AAm
(1672 cm− 1) and alginate (1619 cm− 1) overlapped due to the in­
teractions between carboxyl groups of alginate and amide groups of
AAm. The N–H2 vibration of acrylamide at 3199 cm-1 was widened
with the effect of − OH band of alginate around 3500 cm− 1 confirming
formation of the intramolecular hydrogen bonds. The band belonging to
epoxy group at 906 cm− 1 proved the existence of GMA.
Cryogels can absorb remarkable amounts of water due to their high
porous structure. Swelling of cryogels may occur in two ways: adsorp­
tion of solvent by pore walls via diffusion or pores are filled with solvent
[18]. Swelling degree of lyophilized PAG cryogel was calculated to be
14.0 ± 1.53 g H2O/g cryogel (Fig. 2). The high swelling ability con­
tributes to the flexibility and size recovery of the cryogel after drying
and depicts flow resistance of the cryogel. Cryogels were also dried in
oven at +40 ◦ C in order to investigate the effect of drying method on
swelling behavior of the cryogel. Oven-dried cryogel adsorbed 11.0 g
H2O per gram of cryogel. It was concluded that lyophilization was a
more suitable drying method than drying at high temperatures since
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Process Biochemistry 101 (2021) 137–146
pores are kept fully open during freeze-drying. A similar result was
obtained by Bajpai and Saini (2009) where the swelling ratio of
PVA-haemoglobin cryogels decreased with increasing drying tempera­
ture [19].
Porous properties of the prepared cryogel were examined by ESEM
technique. Pore size and morphology of the cryogel were demonstrated
in Fig. 3 with a magnification ratio of x2500 and x1000. The cryogel had
macropores with diameters between 10− 100 μm and smaller pores (1− 5
μm) on the walls. BET data revealed that the surface area of PAG cryogel
was 6.574 m2/g.
The existence of Cu and S elements in EDX spectrum (Fig. 4) proved
that laccase molecule was bound to the cryogel since neither the starting
materials in cryogel formation nor the solutions used in immobilization
experiments contain Cu and S elements. They were inserted into the
cryogel structure via laccase molecule.
3.2. Covalent immobilization of laccase onto polyacrylamide-alginate
cryogels
Since only 2.53 mg/g laccase protein was bound to PA cryogel, it
ensured that laccase enzyme was immobilized via GMA molecule.
Determination of the pH at which maximum immobilization occurs is a
critical point for evaluation of the interactions between support material
and the enzyme molecule. Epoxy-bearing support materials may bind
R. Yavaşer and A.A. Karagözler
Fig. 5. Effect of pH (A: initial laccase concentration: 5.0 mg/mL; flow rate: 150 μL/min, temperature: 25 ◦ C) and temperature (B: in 0.1 M pH 3.0 citrate-phosphate
buffer; laccase concentration: 5.0 mg/mL; flow rate: 150 μL/min) on laccase immobilization onto PAG cryogel.
Fig. 6. Effect of initial laccase concentration on the amount of immobilized
protein onto PAG cryogel (in 0.1 M pH 3.0 citrate-phosphate buffer, flow rate:
150 μL/min; temperature: 25 ◦ C).
enzyme molecules at pH range of 1.0− 12.0. At acidic pH, enzyme may
be bound from carboxyl and thiol groups whereas at neutral and alkaline
pH it can be attached via amino groups [20]. In this study, immobili­
zation experiments were carried out at pH range of 2.5–7.5 and
maximum immobilization occurred at pH 3.0 (Fig. 5a). Some covalent
immobilization studies of T. versicolor laccase on epoxy supports re­
ported maximum binding at pH 5.0 [21] and pH 4.5 [22]. Temperature
had a slight effect on immobilization of laccase between 4− 45 ◦ C but at
55 ◦ C (Fig. 5b). At elevated temperatures, interaction between cryogel
and laccase molecule was weak due to the chemical nature of the
polymer and the enzyme molecule.
PAG cryogel was able to attach high amount of laccase as demon­
strated in Fig. 6. However, in immobilization experiments 5.0 mg/mL
laccase concentration was chosen both to reduce the amount of
consumed enzyme and to avoid extreme loading of the support with
protein molecules. At high protein concentrations, binding sites may be
crowded and steric hindrances may occur inhibiting the interaction of
enzyme with the support material. Also, active site of enzyme may be
blocked or narrowed therefore substrate cannot fit.
Effect of immobilization time on immobilization was investigated for
optimization of the process. A 120 min circulation of laccase solution
through the cryogel was sufficient for binding 68.7 ± 1.45 mg laccase
per gram of cryogel. It was seen that amount of immobilized laccase
slightly changed after 5 h and 24 h.
3.3. Determination of activities of free and immobilized laccases
Enzyme activity strongly depends on the pH of the reaction medium.
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Process Biochemistry 101 (2021) 137–146
Fig. 7. Effect of pH on laccase activity in 0.1 M citrate-phosphate buffer (solid
lines: free laccase, dotted lines: immobilized laccase).
Laccase activity was measured in 0.1 M McIlvaine’s buffer at different
pH values (Fig. 7). Maximum substrate oxidation was observed at pH 3.0
for ABTS, pH 5.0 for SYR and 4.5 for GUA with free laccase. No
displacement of optimum pH values was detected post-immobilization
of the enzyme. ABTS is an intensively studied substrate for activity
assay of T. versicolor laccase and optimum pH values were reported to be
between 3.0–5.0 by several researchers [23–25]. Syringaldazine was
oxidized to a quinone compound at pH 5.0 maximally and that result
was consistent with the work of Manole et al. [26], Bayramoğlu et al.
[21], and Rotkova et al. [4]. It was also stated that upon immobilization,
optimum pH of laccase varied only about half or one pH unit. Optimum
pH of the laccase catalyzed oxidation of GUA was determined to be 4.5.
Optimum temperature of laccase enzymes differs in a wide range of
25− 80 ◦ C [27,28]. Additionally, depending on the source, laccase ac­
tivity may depend both on the conformation of enzyme and interaction
of the substrate with the active site at the studied temperature [29].
Effect of temperature on activities of free and immobilized laccases,
enzyme activities were measured at 5.0–80.0 ◦ C with ABTS, SYR and
GUA (Fig. 8). Maximum activity was observed at 50 ◦ C with ABTS
substrate and it was consistent with the results of Han et al. [30] and
Doğan et al. [31]. When SYR and GUA were used, optimum tempera­
tures were 60 and 70 ◦ C for free laccase and 50 and 70 ◦ C for immobi­
lized laccase.
3.4. Determination of kinetic parameters
Substrate specifity of laccases varies according to the geometry and
chemical environment of the substrate binding site as well as the mo­
lecular structure of the substrate [32]. Thus, kinetic behavior of the
R. Yavaşer and A.A. Karagözler Process Biochemistry 101 (2021) 137–146

Fig. 8. Effect of temperature on activities of free (left) and immobilized laccase (right) with ABTS, SYR and GUA (in 0.1 M of citrate phosphate buffer of pH 3.0 for
ABTS, pH 5.0 for SYR and pH 4.5 for GUA).

Table 3 3.5. Thermal and storage stabilities of free and immobilized laccases
Kinetic parameters of free and immobilized laccase with different substrates
(ABTS, SYR and GUA). High thermal stability enhances practical usage of enzymes in an
Substrate ABTS SYR GUA economic way. Immobilization is an effective strategy that protect/in­
Kinetic Km Vmax Km Vmax Km Vmax
crease the conformational stability of enzyme molecule therefore pro­
parameter (mM) (U/mg) (mM) (U/mg) (mM) (U/ vides stability against thermal denaturation [35]. In current study,
mg) thermal stabilities of free and immobilized laccases were tested at 25,
Free laccase 0.0071 3.546 0.00643 1.128 0.343 0.423 40, 50, 60 and 70 ◦ C (data not shown). For practical uses, it is important
Immobilized 0.234 0.0072 0.0383 0.0011 3.66 0.007 to determine the stability of laccase at room temperature as well as high
laccase temperatures. Free and immobilized laccases showed lower activity at
ABTS: 2,2ˈ-azinobis-(3-ethylbenzothiazoline-6-sulfonate; SYR: syringaldazine; higher temperatures (60− 70 ◦ C). Additionally, both forms of the
GUA: guaiacol. enzyme were similar in thermal stability at corresponding temperatures.
However, activity loss occurred in a longer period for immobilized
enzyme may differ when different substrates are employed. Kinetic pa­ laccase with ABTS and GUA compared to free laccase whereas it was
rameters point that the affinity of laccase enzyme towards its substrates similar when SYR was used. Therefore, it may be concluded that the
decreased upon immobilization (Table 3). Maximum reaction rate was structural changes of laccase occurred during heat treatment resulted in
also lower compared to free enzyme. This phenomenon may result due different interactions depending on the type of the substrate.
to limited accessibility of the substrate to enzyme molecule after Storage stability data revealed immobilization provided improved
immobilization. Diffusional limitation occurred during passage of sub­ stability to laccase (Fig. 9). Free laccase lost its activity within 150 days
strate through the pores of cryogel can be considered as another reason. when activity was measured using ABTS and GUA substrates whereas
Similar situations were reported by Li et al. [33] and Vera and Rivas [34] within 60 days with SYR. Immobilized laccase was ~30 % more stable
where T. versicolor laccase was covalently attached onto bacterial cel­ compared to its free counterpart. This was an expected feature since one
lulose/TiO2 membrane and poly(GMA) membrane, respectively. of the advantages of enzyme immobilization is to provide stability of the

Fig. 9. Storage stabilities of free and immobilized laccases.

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Table 4
Effect of pH and dye concentration on decolorization with immobilized laccase.
Dye pH Decolorization with Decolorization with immobilized laccase pH Decolorization with Decolorization with immobilized laccase
immobilized laccase (0.05 mg/mL, aqueous) immobilized laccase (0.05 mg/mL, buffered)
(0.01 mg/mL, aqueous) (0.01 mg/mL, buffered)

Congo Red 7.58 99.1 75.4 3.44 100 90.5

Fast Green FCF 7.13 94.1 94.2 3.40 59.0 47.2
Sunset Yellow FCF 7.20 97.9 91.9 3.45 46.4 52.3
Trypan Blue 7.63 95.7 98.4 3.40 100 100
Chlorazol Black 7.51 93.3 73.5 3.40 100 97.2

Temperature:25 ◦ C; decolorization time: 300 min; buffer: 0.1 M pH 3.0 citrate-phosphate buffer.

Fig. 10. UV–vis spectra of real textile wastewater (insert) and gradual decrease after treatment with immobilized laccase (pH 5.0, 25 ◦ C).

enzyme and prolong the life time of immobilized enzyme. Soluble pre-treatments such as clarification or precipitation. Different decolor­
enzyme may be destabilized by the distortion effect of aqueous media on ization percentages can be explained by the structural varieties affecting
the active site of the enzyme. However, for immobilized laccase, sub­ the electron-donating properties of dye molecules (Table 4). Degrada­
strate binding site is usually conserved and it results in enhancement of tion of aromatic part of azo dyes appears as a decline of absorbance in
storage stability [36]. UV–vis spectrum at 200− 400 nm. The gradual decrease between
400− 800 nm is due to decolorization [39].
Fig. 10 depicts the spectrophotometric scans of textile wastewater
3.6. Treatment of dye solutions and real textile effluent and before and after treatment with immobilized laccase. The effluent used
dephenolization of OMW with free and immobilized laccases in this study was a complex mixture of different dyes, salts and several
auxiliary chemicals. However, it was light grey and it was not possible to
Decolorization of dyes by enzymatic catalysis offer new opportu­
nities in environmental biotechnology. Dye decolorization capacities of
various free and immobilized laccase enzymes have been reported [37,
38]. In current study, laccase was more active at pH 3.4 for decolor­
ization of Congo Red (90.5 %) and Chlorazol Black (97.2 %) and at pH
7.5 for Sunset Yellow FCF (91.9 %), Fast Green FCF (94.2 %) and Trypan
Blue (98.4 %). Control cryogel was responsible for 10 % of decoloriza­
tion as it adsorbed some of the dye molecules at 0.05 mg/mL dye con­
centration. Color removal by immobilized laccase was rapid and
pH-dependent. In a similar study performed by Bayraktaroğlu et al.
[38], Congo Red and Sunset Yellow FCF decolorization via immobilized
laccase onto lectin-modified poly(2-hydroxyethyl methacrylate-glycidyl
methacrylate) cryogel was investigated and 34.71 % and 52.08 % effi­
ciency was observed at 0.1 mg/mL dye concentration, respectively.
Besides the efficiency of immobilized laccase on decolorization, the
structural properties of PAG cryogel are also advantageous in the success
of the process. Monolithic and interconnected macroporous morphology Fig. 11. Time-dependent dephenolization of OMW by plain cryogel and laccase
of the cryogel allowed the process to be utilized without additional immobilized cryogel.

144
R. Yavaşer and A.A. Karagözler
Fig. 12. Reusability of immobilized laccase for decolorization of dye solutions (pH:7.5; time: 1 h; dye solution volume: 10.0 mL) and for dephenolization of OMW
(pH:5.6; time: 8 h; OMW volume: 10.0 mL).
observe color removal by naked eye. The complex dye solution causes
absorption in a range from 250 to 600 nm in UV–vis region. Textile
effluent actually may consist of hundreds of different shades or hues.
They absorb light in the wavelength range of 350–500 nm [40]. The
changes in spectra (Fig. 10) may be due to the reduction of dye con­
centration via both decolorization and degradation. After 24 h, at pH
5.0, absorption decreased about 55.6 ± 0.72 % and at pH 11.6 it was
about 17 % whereas cryogel without laccase was effective to be 2.3 ±
0.5 %. Immobilized enzyme retained about half of its initial activity after
five reuses.
Free and immobilized laccases are reported to be effective on
dephenolization. The work of Minussi et al. revealed that 55 % of
phenolic removal was succeed in 24 h of treatment with laccase from
T. versicolor CCT4521 [41]. A study performed by D’Annibale et al. re­
ported 67 % of polyphenol abatement in OMW with laccase from Len­
tinula edodes immobilized on chitosan [42]. In this study, laccase
immobilized cryogel system was successful (70.0 ± 1.2 %) on dephe­
nolization of OMW. Control cryogel provided 25 % of this removal
whereas 45 % of it was in charge of the laccase enzyme. The process was
continued for 24 h to investigate the effect of interaction time of OMW
with immobilized laccase and it was observed that 8 h was sufficient to
reach a plateau (Fig. 11).
Operational stability, in other words reusability, is an important
criterion in enzyme immobilization studies and it is one of the superi­
ority of immobilized enzymes over free enzymes. Effect of repeated use
on activity of immobilized laccase was investigated for both dye
decolorization and dephenolization of olive mill wastewater. Fig. 12
indicates that immobilized laccase retained approximately 50 % of its
initial activity after 7 reuses for each dye. Reduction of enzyme activity
after each use could be attributed to the distortion of enzyme active site
due to frequent interaction with dye molecule as the substrate and also
stereochemical changes of enzyme molecule [43]. No leakage of laccase
from cryogel was determined since laccase was covalently bound.
Operational stability of immobilized laccase for OMW dephenoliza­
tion was investigated for 13 cycles (Fig. 12). Laccase immobilized cry­
ogel was usable for the consecutive treatment of OMW. Half of the initial
activity was conserved up to sixth cycle but decreased gradually after­
wards. Activity loss could result from the accessibility limitation of
phenolic compounds to active site since the pores of the cryogel might be
blocked by this complex pollutant. Denaturation of laccase caused by
OMW ingredients (i.e. salts) may be responsible for the decline in
enzymatic activity.
4. Conclusion
Immobilization of enzymes is a promising method for confining these
valuable and sensitive biocatalysts on a support material. Cryogels are
promising solid supports for enzyme immobilization. In current study,
145
Process Biochemistry 101 (2021) 137–146
GMA-functionalized polyacrylamide-alginate cryogels were fabricated
and laccase enzyme from Trametes versicolor was covalently attached via
epoxy groups of GMA. The attachment of enzyme was characterized
using FTIR, ESEM, EDX and BET instrumental techniques. FTIR spectra
verified the formation of cryogel by monomer polymerization and
addition of epoxy functional groups via GMA. ESEM technique verified
that synthesized cryogel had large (10− 100 μm diameter) and small
(1− 5 μm diameter) pores. BET technique revealed that the surface area
of the cryogel was 6.574 m2/g. Presence of Cu and S atoms in EDX
spectrum verified that laccase enzyme molecules were immobilized onto
the cryogel. It is important that enzyme is still active and characteristics
don’t change after immobilization procedure. Activity measurements
using free and immobilized laccase was conducted using three different
substrates (ABTS, SYR, and GUA) to demonstrate that immobilization do
not corrupt enzyme specificity towards three different substrates.
Additionally, it was also important that the enzyme do not lose its ac­
tivity after immobilization. Therefore, optimum working conditions and
kinetic parameters of free and cryogel immobilized laccase were
compared. Efficient decolorization of selected dyes and dephenolization
of OMW by immobilized laccase was demonstrated. Moreover, it was
verified that cryogel immobilized laccase enzyme exhibited high effi­
ciency with higher stability and repeated use compared to the free form.
Finally, this work has unveiling results in both synthesizing a new cry­
ogel to immobilize laccase enzyme and to use this cryogel to eliminate
some dyes form wastewaters and some phenolics from OMW.
CRediT authorship contribution statement
Rukiye Yavaşer: Conceptualization, Methodology, Data curation,
Writing - original draft, Visualization, Investigation. Arife Alev Kar­
agözler: Supervision, Conceptualization, Methodology, Data curation,
Writing - original draft, Visualization, Investigation.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgments
Authors thank to Aydın Adnan Menderes University Research Fund
for the financial support to this research with the project number FEF-
15024.
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