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International Journal of Biological Macromolecules 114 (2018) 106–113

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Immobilization of laccase on modified Fe3O4@SiO2@Kit-6 magnetite


nanoparticles for enhanced delignification of olive pomace bio-waste
Reza Amin a, Alireza Khorshidi a,⁎, Abdollah Fallah Shojaei a, Shahla Rezaei b, Mohammad Ali Faramarzi b,⁎
a
Department of Chemistry, Faculty of Sciences, University of Guilan, P.O. Box 41335-1914, Rasht, Guilan, Iran
b
Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research Center, Tehran University of Medical Sciences, P.O. Box 14155–6451, Tehran 1417614411, Iran

a r t i c l e i n f o a b s t r a c t

Article history: Lignocellulose is considered a major source for the production of valuable chemicals. Efficient degradation of lig-
Received 14 October 2017 nin as the natural carrier of the lignocellulosic biomass represents a key limiting factor in biomass digestibility.
Received in revised form 6 December 2017 Recently, biological delignification methods have been promoted as sustainable and environmentally friendly al-
Accepted 18 March 2018
ternatives to the traditional technologies. In this study, porous nanocomposite of Fe3O4@SiO2@KIT-6 with mag-
Available online 19 March 2018
netic properties was synthesized. The immobilized laccase supported on nanocomposite with enhanced
Keywords:
stability in a hydrophobic ionic liquid has been developed for both olive pomace delignification and degradation
Magnetic nanoparticles of phenolic extracts from the pomace. After 6 h incubation, the degradation rate of lignin and phenol by the
KIT-6 immobilized laccase was estimated to be 77.3% and 76.5%, respectively. The immobilized laccase retained 70%
Immobilization of its initial degradation ability after 11 successive batch treatments of olive pomace. Furthermore, the
Laccase immobilized laccase retained N70% of its initial activity after 21 days of storage at room temperature. The ob-
Olive pomace tained results indicated that the immobilized laccase on magnetic mesoporous support together with (1-Butyl-
Delignification 3-methylimidazolium hexafluorophosphate) ([Bmim][PF6]) could potentially provide a promising procedure
for an improved enzymatic degradation of lignin and phenol in the related industries.
© 2018 Elsevier B.V. All rights reserved.

1. Introduction The utilization of ionic liquids (ILs) in delignification, as another eco-


friendly method, has recently been considered. ILs, which have been
Lignocellulosic biomass is a potential major resource on earth with a considered recently as alternative green solvents [9], have shown the
great potential to create a sustainable energy for the future [1]. Lignin capability to dissolve lignocellulosic biomass at high temperatures
removal from biomass helps its recycling, enhances the efficiency of cel- with the production of high amounts of inhibitory chemicals and degra-
lulose extraction and hydrolysis. Therefore, it facilitates the utilization of dation products [7,10]. It seems a combination of two strategies, i.e.,
the carbohydrate portion of biomass in the production of valuable prod- laccase-assisted recycling of lignocellulose in recyclable room-tempera-
ucts, cellulosic ethanol or similar biofuels [2]. Due to recalcitrant nature ture ILs (usable in milder conditions and at a lower cost), is a promising
of the lignin components, delignification has remained as an inefficient method for efficient recycling of these bio-wastes, although the enzyme
process [3]. stability in ILs is challenging [7].
Among various delignification strategies, the enzymatic method The immobilization of laccases can overcome some of the aforemen-
using laccases as an important class of lignolytic enzymes has been tioned limitations by improving the thermo- and pH-stability of the en-
demonstrated as a promising mild and clean process [4]. These environ- zyme and its resistance to extreme conditions and chemical reagents
mentally-friendly treatments have some advantages over other such as ionic liquids [11,12]. In addition, immobilized laccases may be
methods such as mild reaction conditions, higher product yields, easily separated from the reaction products, allowing consecutive use
fewer side reactions, and less energy demand [5]. These treatments of the enzymes and development of a cost-effective reaction system
however, are not efficient and the use of laccases for practical applica- [13].
tions is still limited due to their low stability and high production Many recent studies on enzyme immobilization have focused on
costs [6–8]. laccase [8]. The most widely used method of laccase immobilization
for industrial applications has been covalent binding during the last de-
cade. In covalent immobilization technique, chemical groups on the
⁎ Corresponding authors.
support surface are activated using the short active carriers and react
E-mail addresses: khorshidi@guilan.ac.ir (A. Khorshidi), faramarz@tums.ac.ir with specifically lysine amino groups on the protein surface because
(M.A. Faramarzi). of their frequent presence and high reactivity [14]. The selection of the

https://doi.org/10.1016/j.ijbiomac.2018.03.086
0141-8130/© 2018 Elsevier B.V. All rights reserved.
R. Amin et al. / International Journal of Biological Macromolecules 114 (2018) 106–113 107

optimum support material is a very crucial part of the immobilization for 6 h. The filtration of the reaction mixture and subsequent treatment
process as it can determine the properties of the supported enzyme of the light brown solid with ethanol yielded Fe3O4@SiO2@Kit-6-NH2.
preparation. The final product dried at 70 °C, overnight.
Magnetic nanoparticles with functionalized surface have been ex-
tensively used in a variety of applications [15]. The iron oxide nanopar- 2.3. Characterization of Fe3O4@SiO2@KIT-6
ticles can be decorated with a thin layer of organic molecules, polymers,
and bio molecules [16]. Inorganic substrates such as silica, metal oxide Fourier transform infrared (FT-IR) analysis was performed using
and metal sulfide [17] could also be utilized. Recent advances in Thermo Nicolet Avatar FT-IR300 BRUKER Germany spectrographs (KBr
nanobiocatalysis have generated promising results, manifesting its po- disks). Powder XRD patterns of synthesized MNPs were determined
tentials in various enzyme applications. Especially, mesoporous mate- on X-PRTPRO (PANalitical, Netherlands) instrument using Cu Ka radia-
rials with a magnetic core have attracted a lot of attention as a host of tion source with 2θ within the range of 0.5–70. Nitrogen adsorption-de-
enzyme immobilization due to their high surface area, controllable sorption experiments were carried out at 77 K on a Belsorpmini II
pore diameter, uniform pore size distributions and strong magnetic re- accelerated surface area and porosimetry system (Bel, Japan). The sur-
sponse. Other notable features of these systems are resistance to physi- face area was calculated by the Brunauer-Emmett-Teller (BET) method
cal and chemical conditions, low toxicity and their ease of synthesis and the pore volume and size distribution were measured using the ad-
[18]. sorption curve and BJH model, respectively. Particle size and geometry
This study aims to show an efficient removal of phenol and were characterized by scanning electron micrographs (SEM) with a
delignification of olive pomace by combining two paradigms into a sin- SIGMA VP-500 FESEM (Carl Zeiss Germany). Sample preparation for
gle system, which integrates an immobilized laccase into a medium transmission electron microscopy (TEM, CM30 Philips 300 kV) was
containing a hydrophobic ionic liquid, i.e., 1-Butyl-3- done by placing a droplet (1 μL) of sample dispersion latex along with
methylimidazolium hexafluorophosphate ([Bmim][PF6]), thereby pro- a droplet of water on a copper grid covered by Formvar foil
ducing enhanced decomposition of the bio-waste. (200 mesh) and analyzed after drying.

2. Materials and methods 2.4. Covalent immobilization of laccase

2.1. Chemicals and the enzyme The enzyme was immobilized onto the Fe3O4@SiO2@KIT-6-NH2
nanomaterials following the method described below.
Cetyltrimethyl ammoniumbromide (CTAB), Folin-Ciocalteus phenol Initially, 0.5 g of amine-functionalized particles was dispersed in
reagent (FCR), tetra ethylorthosilicate (TEOS), and 3,5-dinitrosalicylic 45 mL of a 0.1 M Na2HPO4-NaH2PO4 buffer solution (pH 8.0) through
acid (DNS) were purchased from Merck (Darmstadt, Germany). ultra-sonication. A 50% glutaraldehyde (GA) solution was then added
Aminopropyltriethoxysilane (APTES), laccase was obtained from to the particles dispersed in buffer to reach a final volume of 5% (v/v).
Trametes versicolor (N10 U mg−1), 2,2′-Azino-bis(3-ethylbenzothiazo- The reaction mixture was continuously stirred at a speed of 150 rpm
line-6-sulfonic acid) diammonium salt (ABTS), and pluronic P123 for two hours at room temperature. After magnetic separation, the
(EO20–PO70–EO20) were obtained from Sigma-Aldrich (St. Louis, MO,
USA). All other chemicals, salts, and solvents used as received.

2.2. Preparation of Fe3O4@SiO2@KIT-6

Fe3O4 nanoparticles were prepared with a slight modification of a


previously reported procedure [19]. Briefly, FeCl3·6H2O and FeCl2·4H2O
were dissolved in double distilled water (DDW) with molar proportion
of 1:2. Under constant stirring of the mixture at 80 °C with a mechanical
stirrer (1200 rpm), pH of the solution was increased to 10.0 by dropwise
addition of 3.0 M ammonia solution. After 30 min, magnetite nanopar-
ticles were separated by an external magnet and washed with deionized
water to reach pH 7.0. To enhance tolerance to acidic conditions re-
quired for the synthesis of mesoporous KIT-6, magnetite nanoparticles
were first covered by an amorphous silica shell. For this purpose,
500 mg of the Fe3O4 nanoparticles were dispersed in 200 mL of ethanol
at 60 °C using of a mechanical stirrer (1200 rpm). After the addition of
12 mL of an ammonia solution (25%), TEOS (5.5 mL) was added
dropwise and the SiO2 coated magnetite nanoparticles were separated
and washed with deionized water until pH 7.0 was reached. Extension
of mesoporous KIT-6 over the surface of the obtained Fe3O4@SiO2 was
achieved by the method of Kleitz et al. [20]. Briefly, Pluronic P123
(1.25 g) was dissolved in 50 mL of DDW. Then, Fe3O4@SiO2 (1.0 g)
and concentrated HCl solution (37%, 2.4 mL) were added under me-
chanical stirring (at 1200 rpm). Following the addition of n-butyl alco-
hol (2 mL), TEOS (2.7 mL) was added and after 24 h, the reaction
mixture was transferred to a Teflon lined stainless steel autoclave and
incubated at 100 °C for 12 h. After treatment of the resulting solid
with ethanolic solution of HCl, the product was calcinated at 550 °C
for 6 h. NH2 functionalization of the core-shell Fe3O4@SiO2@Kit-6 nano-
particles was achieved by post synthesis grafting method [21] using Fig. 1. a) The FTIR spectrum of the Fe3O4@SiO2@Kit-6-NH2 particles (bottom curve) and
APTES in dry toluene. A solution of 1 g of Fe3O4@SiO2@Kit-6 in 50 mL the glutaraldehyde modified Fe3O4@SiO2@Kit-6-NH2 particles (top curve) and b) the
of dry toluene was refluxed at 110 °C in the presence of 4 mL of APTES XRD pattern of the Fe3O4@SiO2@Kit-6-NH2 nanoparticles.
108 R. Amin et al. / International Journal of Biological Macromolecules 114 (2018) 106–113

GA-modified particles were collected, washed with the phosphate


buffer and utilized to react with the amino groups of the enzyme. The
enzyme solution was prepared by dissolving 1 mg of laccase (10 U) in
1 mL of a 100 mM citrate buffer solution (pH 4.5). Finally, 5 mg of the
GA-modified Fe3O4@SiO2@KIT-6-NH2 particles was re-dispersed in the
enzyme solution by ultra-sonication. The reaction mixture was then
stirred at 25 °C for 120 min. and the enzyme-bound magnetic particles
were collected with a magnet. The unbound enzymes were removed
by rinsing with a 100 mM citrate buffer solution. The enzyme-bound
particles were applied to determine the enzyme loading and the en-
zyme activity. Incubation time (10–120 min), pH (2.5–10.5), and tem-
perature (25–65 °C) were independent variables selected in this study
to optimize conditions for maximum enzyme immobilization.
After optimizing the conditions, the amount of laccase immobilized
on the nanoparticles was estimated by Bradford assay at 595 nm [22].
Immobilization efficiency was calculated using following equation:

Immobilization efficiency ð%Þ ¼ ðA0 −A1 Þ=A0  100 ð1Þ

where A0 and A1 are the amounts of total protein introduced for immo-
bilization and the amount of protein present in the filtrate solutions
after immobilization, respectively.
The yield of immobilization was also calculated via following equa-
tion:

Immobilization yield ð%Þ ¼ ðB0 −B1 Þ=B0  100 ð2Þ

where B0 and B1 are the enzyme specific activity in the solution before
and after immobilization, respectively.

2.5. Enzyme activity assay and protein determination

Laccase activity was determined spectrophotometrically (Pharmacia


LKB ultraspect III, Sweden), using ABTS as the substrate. The oxidation of
0.2 mM ABTS in citrate buffer (100 mM, pH 4.5) was determined by
measuring the increase in absorbance at 420 nm after 15 min incubation
at 40 °C [23]. Enzyme activity was expressed as international units (IU),
where 1 IU represents the amount of enzyme that forms 1 μmol of prod- Fig. 3. Effect of a) incubation time and b) pH-temperature on the relative activity profile of
uct per minute under standard assay conditions. The absorbance of the immobilized laccase.
blank samples was obtained using similar samples excluding enzyme.
Protein was also determined using the method of Bradford with bovine implementing the typical oxidizing reaction of ABTS as described in
serum albumin as the protein standard. Section 2.4. All enzyme activities were assayed in triplicate.

2.6. Immobilized enzyme stability studies 2.6.2. Stability with [Bmim][PF6]


Initial and residual activities of free and immobilized laccase were
2.6.1. Thermal and pH stability measured by the standard method described above. Stability assays
The thermal and pH stability was investigated by incubating the free against [Bmim][PF6] were carried out by incubation in citrate buffer
and immobilized laccase solutions (pH 2.0–10.0) at temperatures rang- (pH 4.5, 100 mM) at 25 °C with three concentrations of [Bmim][PF6]
ing from 25 °C to 65 °C for 6 h. The pH was adjusted using 0.1 M citrate (0, 25, and 50% (v/v)). The initial activity was measured after the addi-
buffer (pH 2.0–6.0), 0.1 M phosphate buffer (pH 6.5–8.0) and 0.1 M Tris- tion of the immobilized laccase into the incubation media. Residual ac-
HCl buffers (pH 8.5–10.0). The residual activity was then examined by tivities were determined after 12 h, using the standard procedure

Fig. 2. a) BET analysis, b) Scanning electron microscopy (SEM) image, and c) Transmission electron microscopy (TEM) image of the Fe3O4@SiO2@Kit-6-NH2 nanoparticles.
R. Amin et al. / International Journal of Biological Macromolecules 114 (2018) 106–113 109

described above. All activity measurements were performed in 2.8. Delignification potential of the immobilized enzyme with olive pomace
triplicate.
The almond shell, which was provided from a local store in Astakol
2.6.3. Reusability and storage stability village (36°56′N 48°53′E), Zanjan province, Iran, was used to evaluate
The operational stability of the immobilized laccase was assessed by the delignification potential of free and immobilized laccase in the pres-
repeated oxidation reaction of ABTS in the citrate buffer system over ence or absence of 25% (v/v) ionic liquid [Bmim][PF6] by the method de-
several consecutive cycles. At the end of each oxidation cycle, the scribed below. The olive pomace was first washed with distilled water,
immobilized laccase was separated by magnet, washed three times dried at 65 °C in an oven overnight, grinded in a knife mill (or ball
with the same buffer and the procedure was repeated with a fresh ali- mill), and sieved using the sieve mesh NO.10 ASTEM-E11 [25]. Then
quot of substrate at the same reaction conditions. The activity of the the delignification pre-treatment of the prepared powder was per-
immobilized enzyme was considered to be 100% in the initial cycle. Ac- formed by laccase in a reaction mixture containing sodium citrate buffer
tivity in each cycle was defined as the residual activity of the enzyme in (100 mM, pH 4.5), the free and immobilized enzyme (~50 U), and olive
the reaction mixture. All assays were performed in triplicate. pomace powder (final concentration 5% w/v) at 40 °C with orbital shak-
For testing the storage stability of enzymes, free and immobilized ing at 150 rpm for 48 h. Control samples without the enzyme were proc-
laccases in 0.1 M sodium citrate buffer (pH 4.5) were stored at 25 °C essed in parallel with the test samples. After the enzymatic treatment,
for several days. Then the remaining activity of the enzyme was mea- delignification activity on olive pomace was determined by measuring
sured under standard conditions. the increase in released sugar and the decrease in the total phenol con-
tent before and after the enzymatic treatment.
2.7. Kinetics study The kappa number of olive pomace powder was analyzed at the end
of delignification process according to the Tappi Test Method of T 236
The kinetic parameters, such as Michaelis-Menten constant (Km) om-99 for kappa number measurement. Delignification efficiency was
and maximum reaction rate (Vmax) of free and immobilized laccases determined as bellow:
were estimated using the SigmaPlot software (SigmaPlot version 12.0,
Systat Software, Inc., San Jose, CA) on a wide range of substrate (ABTS) %Delignification efficiency ¼ ½ðK i −K f Þ=K i   100 ð5Þ
concentrations (ranged from 0.01 to 1.0 mM) in 100 mM citrate buffer
(pH 4.5). The Km and Vmax values were calculated according to the
where Ki is the initial kappa number and Kf is the final kappa number
Lineweaver-Burk double reciprocal models as mentioned below in
[26].
Eqs. (3) and (4), respectively [24]:
To measure the released sugar, a modified DNS method was used
and glucose standard curve was drawn under the same conditions [27].
V ¼ ðV max ½SÞ=ð½S þ K m Þ ð3Þ
In the modified DNS method, the supernatant of the reaction mix-
ture was mixed with DNS reagent in a 1:1 ratio and it was heated in boil-
1=V ¼ ½ðK m =V max Þ ð1=½SÞ þ ð1=V max Þ ð4Þ ing water for 10 min. After cooling, absorbance was recorded at 540 nm

Fig. 4. Influence of a, b) pH-temperature and c, d) ionic liquid [Bmim][PF6] on the free and immobilized enzyme stability.
110 R. Amin et al. / International Journal of Biological Macromolecules 114 (2018) 106–113

against the relevant blank in a spectrophotometer. In addition, a modi- 3.2. Laccase immobilization
fied Folin-Ciocalteu method was applied for total polyphenol content
determination [28]. Briefly, 800 μL FCR 10% was added to 200 μL super- The constructed nanocomposite was applied as a support for the im-
natant and equilibrated at room temperature within 5 min. After adding mobilization of laccase, and the optimal conditions for maximal laccase
2 mL NaHCO3 7.5%, the mixture was diluted to 7 mL with distilled water, immobilization onto Fe3O4@SiO2@Kit-6-NH2 were determined indi-
incubated at 45 °C for 15 min with shaking at 150 rpm. Finally, the mix- rectly by measuring the relative activity of the enzyme. The optimal im-
ture was left in a dark place at room temperature for 2 h and the absor- mobilization conditions observed included 30 min incubation time, pH
bance was recorded at 765 nm against the appropriate blank. of 4.5 and the temperature of 35 °C (Fig. 3). After the optimization of
the attachment time and pH-temperature, the percentages of immobili-
zation yield and immobilization efficiency were recorded as 76.2% and
2.9. Statistical analysis 84.4%, respectively. Several reports published in the literature have de-
scribed new supports for laccase immobilization under different condi-
To show the significant difference between the groups, One-way and tions and different organisms were used as the laccase source.
Two-way ANOVA tests were applied (SigmaPlot version 12.0, Systat Therefore, it is almost impossible to compare these immobilization
Software, Inc., San Jose, CA). A probability level of p b 0.05 was consid- results.
ered statistically significant.
3.3. Thermal- and pH-stability of the free and immobilized laccase

3. Results and discussion The effect of pH and temperature on the stability of the free and
immobilized laccase on Fe3O4@SiO2@Kit-6-NH2 was investigated at dif-
3.1. Characterization of the support ferent pH and temperature values varying from 2.5–10.5 and 25–65 °C,
respectively (Fig. 4a and Fig. 4b). Both free and immobilized laccases ex-
In the FTIR spectrum of Fe3O4@SiO2@Kit-6-NH2 (Fig. 1a, bottom hibited maximal activity at a pH of 4.5 and a temperature of 35 °C. The
curve), the following characteristic bands were observed: Fe\\O covalently immobilized laccase showed broadening in the pH and
stretching at 558 cm−1, Si\\O\\Si symmetric stretching, asymmetric
stretching and binding vibrations at 1076, 793 and 459 cm−1, C\\H
stretching vibrations of propyl chains at 2926 cm−1 and N\\H
stretching vibrations of \\NH2 functional groups at 3413 cm−1. In the
FTIR spectrum of glutaraldehyde modified Fe3O4@SiO2@Kit-6-NH2 par-
ticles (Fig. 1a, top curve) in addition to the above vibrations, the charac-
teristic peaks of aldehyde moiety was observed at 1727 cm−1,
attributable to C_O stretching and 2854 cm−1, attributable to aldehyde
C\\H stretching. Moreover, the intensity of N\\H stretching was signif-
icantly decreased and slightly shifted to higher wavenumbers. The FT-IR
results provided evidence that glutaraldehyde, as a cross-linker for pro-
tein binding, was attached successfully to the carrier through amine
groups.
In the XRD pattern of Fe3O4@SiO2@Kit-6-NH2 nanoparticles (Fig.
1b), corresponding reflections of pure magnetite were observed at 2θ
of 29.72, 35.57, 43.17, 57.15, and 62.77, and matched well with the
XRD pattern of the standard Fe3O4 (JCPDS no.19-692) [29]. Inset of Fig.
1b compares the low-angle pattern of pure KIT-6 with Fe3O4@SiO2@
Kit-6-NH2. It is clear that well resolved reflections corresponding to
211 and 332 planes of KIT-6 were shifted to smaller angles with simul-
taneous decrease of intensity.
BET analysis of the Fe3O4@SiO2@Kit-6-NH2 nanoparticles showed
that mesoporous structure of KIT-6 was preserved in the obtained
hybrid material. From Fig. 2a it is obvious that nitrogen adsorption-
desorption isotherm of Fe3 O 4@SiO2 @Kit-6-NH 2 corresponds to a
type IV curve with its hysteresis loop in the p/p0 range of 0.6 to 0.9.
Inset of Fig. 2a shows the BJH pore size distribution curve. Based on
these analyses, physicochemical properties of the Fe3O4@SiO2@Kit-
6-NH2 nanoparticles determined from N2 adsorption-desorption iso-
therms are a BET surface area of 238.2 m2 g−1, mean BJH pore diam-
eter of 5.8 nm, total pore volume of 0.48 cm3 g −1, and mean pore
volume of 0.501 cm3 g−1.
Scanning electron microscopy (SEM) and transmission electron mi-
croscopy (TEM) were also used to illustrate the surface morphology and
the core-shell structure of the obtained nanoparticles. From Fig. 2b it is
clear that the nanoparticles were aggregated due to their magnetic na-
ture. TEM imaging, however, revealed the core-shell structure of nano-
particles. In the Fig. 2c, it is notable that the mesoporous shell has been
extended over the surface of dark Fe3O4 magnetic cores. Similarly, the
extension of shell over the core (dependent on mesoporous type and
layer thickness) was also discussed in previous studies by Wang et al. Fig. 5. Residual activity of free and immobilized laccase with a) storage time and b)
[30] and Guo et al. [31]. repeated use.
R. Amin et al. / International Journal of Biological Macromolecules 114 (2018) 106–113 111

temperature stability profile as compared to the free enzyme. This ob- shell of the protein [7]. The results suggest that the support might trap
servation means laccase immobilization led to a significant stabilizing and prevent the stripping off enzyme-bound water, essential to main-
effect towards heat denaturation and induced an enhanced activity tain the native conformation of the enzyme for catalysis [39]. In addi-
within the tested temperature and pH ranges. However, temperatures tion, the immobilized enzymes result in an increased stability towards
above 65 °C resulted in a clear decrease in activity due to the enzyme de- ILs due to the limited conformational changes in enzyme structure,
naturation [32]. The great adaptability at a broader pH and temperature thus preventing the formation of protein aggregation [40]. Similar re-
ranges should depend on the immobilization method, as well as on the sults have also been observed by other researchers, which mean that
carrier charge and structure [32,33]. Patel et al. [23] immobilized laccase the immobilization methods preserve the enzyme activity.
from Trametes versicolor on SiO2 nanoparticles and showed that the op-
timal pH for free and immobilized laccase was about 3.0 and 3.5, respec- 3.5. Reuse and storage stability of immobilized laccase
tively. However, the activity of immobilized enzyme in the pH range of
3.5–7.0 was retained significantly, as compared to the free enzyme. The Reusability and storage time are two important factors to evaluate
catalytic activity of the majority of fungal laccases is optimal in the tem- the immobilized enzyme properties, which can reduce the total cost of
perature range between 30 and 55 °C and under mild acidic conditions the industrial application of biocatalysts [11]. The results obtained
(pH 4.0–6.0) [34,35]. The thermal and pH-stability are the most impor- from the reusability experiment in this study, showed a mild decrease
tant factors for the applicability of the biocatalyst. The improvement of in the activity of immobilized laccase after 11 cycles (Fig. 5a).
the thermal and pH-stability of the enzyme can be explained by the re- Immobilized laccase retained about 71% of the initial activity after 11 cy-
duced conformational mobility of the immobilized enzyme due to the cles. Reusability of immobilized enzymes is an important parameter for
covalent binding with an aldehyde group [36]. Indeed, the covalent im- industrial application view points, because immobilized enzymes re-
mobilization of an enzyme on a support often limits its movements and duce the cost of production due to their repeatability, or larger scale
conformational changes, resulting in an increased stability towards uses. A decrease in the enzyme activity upon repeated usage is ex-
thermal denaturation [37,38]. pected. The denaturation of the laccase and the leakage of enzyme
from the support could be an explanation for the inactivation of the en-
3.4. Stability with [Bmim][PF6] zyme after each cycle. In addition, upon repeated uses, pore blockage of
support by substrate or product may take place.
Initial and residual activities against free and immobilized laccase Generally, enzymes in solution are not stable and their activities de-
were measured after 12 h incubation in [Bmim][PF6] (0, 25, and 50% cline with time, thus the storage of free enzymes is particularly difficult.
(v/v)). The effect of [Bmim][PF6] on the stability of the both laccases is One of the best methods to address this problem is immobilization. Fig.
shown in Fig. 4c and Fig. 4d. The immobilized enzyme showed better 5b shows the storage stabilities of free and immobilized laccase at 25 °C.
stability in the absence or presence of [Bmim][PF6] than the free laccase With increased storage time, a remarkable difference was observed be-
after 12h incubation at 25 °C. Similar to the denaturing effect of salts, ILs tween the activity variations of free and immobilized laccase. Free
inhibit enzymatic activity mainly by disrupting hydrogen bonding, hy- laccase completely lost its initial activity, whereas immobilized laccase
drophobic interactions and removing the internally water hydration retained about 70% of initial activity after 20 days. One possible

Fig. 6. a, b) The classic Michaelis-Menten curves and c, d) the Lineweaver-Burk plots for the free and immobilized laccases activity on ABTS.
112 R. Amin et al. / International Journal of Biological Macromolecules 114 (2018) 106–113

explanation for this increased storage time may be the covalent bonding components for hydrogen bonding, thus breaking up the overall struc-
between the enzyme and support, which improved the enzyme's stabil- ture of the biomass and as a result, IL increases the accessibility of the
ity against structural denaturation; although the mechanisms proposed enzyme to the substrate. So, the combination of these two methods
for the storage stability of immobilized enzymes are complex and un- yields high efficiency delignification. Many Studies have already re-
clear [37,38,41]. ported similar results where lignin was degraded by laccases [49]. In a
similar study, Sondhi et al. [50] reported 27% reduction in kappa num-
3.6. Kinetics study ber using a laccase from Bacillus tequilensis in the absence of a mediator.
Furthermore, Call and Mücke [51] have reviewed the laccase-assisted
Kinetic Parameters were estimated from the Lineweaver-Burk plot delignification of pulp from several origins with 30–60% delignification
using ABTS as substrate (Fig. 6). Immobilization decreased at Vmax yield in the presence of different concentrations of HBT.
value from 121.25 μmol mg−1 min−1 to 39.59 μmol mg−1 min−1. The The total polyphenol content of sample was measured using FCR
decreasing trend of Vmax may have occurred due to the inactivation of [52]. The results of FCR method in Table 1 show a reduction in total pro-
the enzyme, caused by structural changes, which reduces accessibility duced phenol, indicating enzymatic degradation of the phenols.
of the substrate to the active site of the immobilized enzyme. This The results of DNS method also revealed that enzymatic treatment
could also reduce the production of intermediate compounds during reduced the degree of lignocellulose polymerization and released the
degradation of the substrate [42]. The Km value of immobilized laccase saccharides linked to lignin. Lignin is the complex matrix of aromatic
(345.37 μM) was 1.6 times higher than that of free laccase (211.13 compounds that are linked to hemicellulose polysaccharides via ester,
μM), which means the immobilized laccase had lower affinity towards phenyl, and covalent bonds [53]. Also, lignin degradation causes release
the substrate. The most important factors affecting the immobilized en- of reducing sugars [12]. Some studies have shown similar results for re-
zyme affinity towards the substrate are the loss of enzyme flexibility leasing the reducing sugar after delignification of different substrates
necessary for substrate binding, the steric hindrance of the active site [54].
by the support and diffusional resistance to substrate and product mol-
ecules transport near the particles of the support [43]. 4. Conclusion
These results are in agreement with reports over significant reduc-
tion in affinity in immobilized catalysts [44,45]. Immobilization of Laccase was immobilized by covalent attachment on the prepared
laccase on Amberlite IR-120 H by covalent bonding was explored by magnetic nanocomposite Fe3O4@SiO2@KIT-6-NH2 by glutaraldehyde
Spinelli et al. [46], and the Km value increased 92-fold and the Vmax crosslinking. The immobilized enzyme exhibited an improved thermal,
value decreased 5-fold for the kinetic study. Çetinus and Öztop [47] re- pH and storage stability with a great (potential) performance for reus-
ported the Km values were increased after enzyme was immobilized on ability. The catalytic performance of laccase in the degradation of lignin
glyoxal-crosslinked chitosan beads, whereas Vmax value was decreased. and phenol from olive pomace in [Bmim][PF6] was improved by immo-
bilization on the magnetic nanoparticle. Regarding the immobilization
3.7. Delignification potential of immobilized laccase for olive pomace process, our results demonstrated that the immobilized laccase devel-
oped in this study was effective and provides a useful technique to in-
Due to the ability of the enzyme to oxidize a wide range of sub- crease enzymatic stability and practical applications in an industrial-
strates, delignification of olive pomace by immobilized laccase was ex- scale setting.
amined and the proceeding was followed by changing in total
phenolic content, sugar released and kappa number. Acknowledgement
Application of the immobilized system in lignin removal was inves-
tigated in a batch system, and a decrease in kappa number value of Partial support of this study by the research council of the University
about 77.3% was obtained after 6 h at pH 4.5 and at 35 °C without any of Guilan is gratefully acknowledged.
mediator (Table 1). Table 1 exhibits the kappa number changing along
the different treatment compared with control, which had an initial References
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