Exploring the structural and catalytic features of lipase enzymes immobilized on g-C3N4: A novel platform for biocatalytic and photocatalytic reactions

Article abstract of DOI:10.1016/j.molliq.2021.116612

The exploration of multifunctional supporting materials for modern enzyme immobilization is an attractive subject for advanced catalytic applications. In the present study, lipase enzyme types, namely palatase 20,000 L, lipozyme TL100L, and lipozyme CALB, were immobilized on graphitic carbon nitride (g-C₃N₄) using physical and covalent immobilization methods in order to obtain biohybrids for utilization in biocatalytic and photocatalytic reactions. Characterization tests confirmed successful immobilization of enzymes without changing the crystal phase of g-C₃N₄. The immobilization yields were calculated as 71.0% and 93.4% for C₃N₄@PLTS; 81.3% and 95.4% for C₃N₄@LPZYM; and 79.4% and 91.7% for C₃N₄@CALB biohybrids using physical adsorption and covalent bonding methods, respectively. The kinetic constant values of K_m and V_max were significantly higher for immobilized lipases than for free forms. The photocatalytic efficiencies of biohybrid catalysts were also greater than raw g-C₃N₄. Among the lipase types, lipozyme TL100L attached on g-C₃N₄ exhibited the best catalytic performance in both biocatalytic and photocatalytic experiments, which was attributed to its open lid structure. As a result, this study opens the door to utilizing g-C₃N₄ as a support material for the immobilization of different enzymes and establishing catalyzed reactions.

Full text of DOI:10.1016/j.molliq.2021.116612

Journal of Molecular Liquids 337 (2021) 116612
Contents lists available at ScienceDirect
Journal of Molecular Liquids
journal homepage: www.elsevier.com/locate/molliq
Exploring the structural and catalytic features of lipase enzymes
immobilized on g-C N : A novel platform for biocatalytic and
3
4
photocatalytic reactions
Esra Bilgin Simsek a, , Didem Saloglu b
⇑
a
Department of Chemical Engineering, Faculty of Engineering, Yalova University, 77100 Yalova, Turkey
Disaster and Emergency Management Department, Disaster Management Institute, Istanbul Technical University, Maslak, 34730 Istanbul, Turkey
b
a r t i c l e i n f o
a b s t r a c t
Article history:
The exploration of multifunctional supporting materials for modern enzyme immobilization is an attrac-
tive subject for advanced catalytic applications. In the present study, lipase enzyme types, namely pala-
tase 20,000 L, lipozyme TL100L, and lipozyme CALB, were immobilized on graphitic carbon nitride (g-
Received 12 January 2021
Revised 16 March 2021
Accepted 27 May 2021
Available online 31 May 2021
C N
3 4
) using physical and covalent immobilization methods in order to obtain biohybrids for utilization
in biocatalytic and photocatalytic reactions. Characterization tests confirmed successful immobilization
of enzymes without changing the crystal phase of g-C . The immobilization yields were calculated
as 71.0% and 93.4% for C @PLTS; 81.3% and 95.4% for C @LPZYM; and 79.4% and 91.7% for
@CALB biohybrids using physical adsorption and covalent bonding methods, respectively. The
kinetic constant values of K and Vmax were significantly higher for immobilized lipases than for free
forms. The photocatalytic efficiencies of biohybrid catalysts were also greater than raw g-C . Among
the lipase types, lipozyme TL100L attached on g-C exhibited the best catalytic performance in both
biocatalytic and photocatalytic experiments, which was attributed to its open lid structure. As a result,
3 4
N
Keywords:
Carbon nitride
Lipase
Immobilization
Biocatalyst
Photocatalysis
N
3 4
3 4
N
3 4
C N
m
3 4
N
3 4
N
this study opens the door to utilizing g-C
N
3 4
as a support material for the immobilization of different
enzymes and establishing catalyzed reactions.
Ó 2021 Elsevier B.V. All rights reserved.
1
. Introduction
of the solid–liquid phases [4]. This situation known as ‘‘interfacial
activation” can be explained by combining the substrate character-
istic and monomolecular film interface in an aqueous medium. In
the absence of interfaces, lipases have some elements of secondary
structures, called a ‘‘lid”, covering their active sites and making
them accessible/inaccessible to substrates [5]. Also, in hydrophobic
interfaces, important conformational changes take place yielding
the ‘‘open structure” of lipases [6]. These changes result in the
exposure of hydrophobic parts, interacting with the hydrophobic
interface, and conferring functionality to the enzyme.
However, free lipase enzymes suffer from low operational
stability and they should be anchored on suitable networks to
enhance the enzyme activity and recyclability as well as ther-
mostability [7]. Therefore, chemical and physical immobilization
methods were developed, namely adsorption, cross-linking, encap-
sulation and covalent binding [8,9]. Among them, immobilization
via adsorption is considered the most useful method since it
involves physical interactions including Van der Waals forces, elec-
trostatic interactions, hydrogen bonding and ionic interactions
Enzymes have received growing attention and been utilized in
extensive biotechnological and industrial sectors due to their high
catalytic activities, high substrate specificity and moderate cat-
alytic conditions in contrast to conventional catalysts [1]. Lipase
enzymes (triacylglycerol ester hydrolase, E.C. 3.1.1.3) were well
studied as catalysts because of their unique physicochemical fea-
tures. Lipases are remarkably effective in versatile reactions, such
as hydrolysis, esterification, transesterification, alcoholysis, and
C–C bond formation [2]. They can catalyze chemical reactions in
both hydrophobic and hydrophilic media and are also stable in
organic or eutectic solvents and supercritical fluids which enables
them to be applied in industrial biocatalysis [3]. Lipases have sim-
ilar molecular structure consisting of a b-sheet and a catalytic triad
of serine, histidine, and carboxylic acid. Their active sites can be
quite different, which affects the substrate specificity and reaction
mechanisms. All enzymes are adapted to operate at the interfaces
[
10]. Although the physical interactions are rather weak, the native
⇑
Corresponding author.
E-mail address: ebilgin.simsek@yalova.edu.tr (E. Bilgin Simsek).
structure as well as the activity of the enzyme can remain stable
https://doi.org/10.1016/j.molliq.2021.116612
167-7322/Ó 2021 Elsevier B.V. All rights reserved.
0

E. Bilgin Simsek and D. Saloglu
Journal of Molecular Liquids 337 (2021) 116612
after immobilization [10,11]. Physical adsorption has also advan-
tage like low-cost and chemical free binding properties [12]. Cova-
lent bonding provides strong strength and overcomes the problem
of leakage of enzymes caused by weak interactions in adsorption.
The covalent attachment enables easy incorporation of crosslinkers
with a variety of functional groups in the support material [11,13].
The lipase molecules with strong nucleophiles can interact with
the electrophilic groups on the support surface and the carriers
can be modified with aldehyde, succinimidyl ester or amino groups
that can react with the amino or thiol groups on the lipase [12].
So far, many types of supporting materials such as activated
carbon, graphene, carbon nanotubes, chitosan, silica, metal organic
framework, and magnetic nanoparticles were successfully applied
to lipase immobilization [12,14–16]. From another point of view,
binding the enzyme to a specific support can result in the combi-
nation of enzyme-catalyzed reactions with chemical oxidation
techniques and enhance the catalytic activities [11,17]. Since the
utilization area of immobilized lipases is synchronized with the
type and structure of the support material, the development of
novel and unique supports plays an important role in the catalytic
reactions. The criteria for choosing suitable support material for
immobilization generally includes cost-effectiveness, availability,
stability and reactivity in specific conditions [9]. In addition, the
physicochemical parameters of the support materials that should
be taken into account are surface area, particle size, pore structure
and type of functional groups present on the surface. Moreover,
there should be affinity between the functional groups of the
enzyme and supporting material to reduce diffusional limitations
and allow the formation of effective binding interactions.
3 4
Therefore, in this work, we investigated the usage of g-C N as a
supporting material for immobilization of different lipase enzymes
in order to promote both photocatalytic and enzymatic activities.
Three lipase enzymes (Palatase 20,000 L, Lipozyme TL100, and
3 4 3
Lipozyme CALB) were attached to the g-C N structure to form C -
N
4
@lipases as biohybrid catalysts via physical adsorption and
covalent binding methods. The physical, structural, and optical
properties of the biohybrids were explored in detail. The photocat-
3 4
alytic performance of C N @lipases was investigated by the degra-
dation of tetracycline (TC) and sulfadiazine (SDZ) as model organic
pollutants, which commonly exist in the environment and are
harmful for living beings.
2
. Materials and methods
2
.1. Materials
p-nitrophenylpalmitate (p-NPP, Aldrich, 99%), Lipozyme TL 100
L (100,000 U per g solid), Palatase 20,000 L (20,000 U per g solid),
and Lipozyme CALB (5,000 U per g solid), absolute ethanol (EtOH),
and glutaraldehyde solution (25% (w/v)) were purchased from
Sigma-Aldrich (St. Louis, USA). Protein Assay Kit was obtained from
3 6 6
Boster Biological Technology (CA, USA). Melamine (C H N , 99%)
was supplied from Merck. Tetracycline and sulfadiazine were
obtained from Sigma-Aldrich. All chemicals were of analytical
reagent grade and used as received without any further
purification.
2
3 4
.2. Preparation of graphitic carbon nitride (g-C N )
3 4
Graphitic carbon nitride (g-C N ), the most stable allotrope of
carbon nitride, offers abundant applications in photo-electro-
catalysis, sensing, bioimaging and energy conversion processes
due to its thermal stability, chemical inertness, electronic struc-
ture, and good mechanical properties [18]. The material looks like
a cross-linked polymer built from s-triazine or s-heptazine units
bonded via secondary or tertiary amino groups [19]. The 2D lay-
The raw graphitic carbon nitride (g-C
3 4
N ) sample was prepared
via thermal decomposition of melamine. The melamine was first
ꢀ
1
heated to 550 ℃ (3 ℃ min ) in a semi-closed crucible under air
and then it was kept at the final temperature for 3 h.
2
.3. Immobilization of lipase enzymes on g-C
3 4
N
3 4
ered structure of g-C N can be easily tuned at the molecular level
without changing the composition, which consists of two compo-
nents abundant on earth (carbon and nitrogen) [20]. The excellent
2
.3.1. Physical immobilization
biocompatibility properties and surface-active sites of g-C
make it an ideal scaffold for successful enzyme immobilization.
The amino groups at the margins of g-C are responsible for
forming more stable hybrids via physical or covalent bonding.
Hence, it can be hypothesized that g-C will work in a harmony
with enzymes. Shen et al. [21] immobilized Candida rugosa lipase
CRL) on mesoporous carbon nitride via physical adsorption and
3 4
N
The immobilization of Palatase 20,000 L, Lipozyme TL100 L and
Lipozyme CALB on g-C was carried out by the physical adsorp-
tion method. 0.15 g Palatase 20000L, 0.03 g Lipozyme TL100, and
.60 g Lipozyme CALB wwere fully dissolved in 50 mL 0.1 M
phosphate-buffered saline (PBS,) at pH 7.0. Then, g-C and
3 4
N
3
N
4
0
3 4
N
3 4
N
enzyme solutions were successively mixed, and the mixture was
incubated at 25 ℃ in a shaker operating at 140 rpm overnight.
(
achieved improved thermal stability of free CRL. Wang et al. [22]
introduced palladium and Candida antarctica lipase B (CalB)
immobilized g-C N as a new biocatalyst for benzyl hexanoate pro-
3 4
duction and demonstrated that the immobilized CalB had superior
performance to the free enzyme. Recently, Li et al. [23] covalently
The immobilized lipases (C
CALB) were collected by refrigerated centrifugation, washed with
.1 M PBS (pH 7.0) 3 times, freeze-dried overnight, and stored in
3 4 3 4 3 4
N @PLTS, C N @LPZYM, and C N @-
0
a refrigerator at 4 ℃.
3 4
attached Candida rugosa lipase on g-C N nanosheets and the
immobilized sample had good enzyme-loading, pH-flexibility,
and thermostability features. According to these statements, the
biocompatibility, low cost, surface-active sites and hydrophobic
2.3.2. Covalent immobilization
The immobilization of Palatase 20,000 L, Lipozyme TL100 L and
3 4
Lipozyme CALB on g-C N was performed by the cross-linking
properties of g-C
immobilization which would further increase the activity of the
enzyme. Additionally, the lid opening structure of lipase might
3
N
4
make it a perfect candidate for lipase
method assisted by glutaraldehyde according to literature reports
[23]. First, 100 mL 0.1 M PBS buffer (pH 7.0) and 30 mL of glu-
taraldehyde solution (25%, w/v) were successively added to
be enhanced upon immobilization on the hydrophobic g-C
N
3 4
. In
3 4
250 mL Erlenmeyer flask including 3.0 g of g-C N . After sonication
the physical immobilization process, hydrogen bonding
interactions can cause propagation into the distal region which
enhances lid opening, while the shift base reactions can improve
the lid structure through covalent attachment. In spite of these
excellent features, the related literature has rarely reported
treatment, the mixture was incubated at 60 ℃ in a shaker operat-
ing at 140 rpm overnight. Subsequently, the activated carrier was
separated and repeatedly washed with distilled water 3 times via
vacuum filtration. Finally, the product was dried in a vacuum oven
3 4
at 25 ℃ overnight, and the material was named G-g-C N .
lipase immobilization on g-C
photocatalysis.
3
N
4
, especially in the field of
Then, 0.15 g Palatase 20,000 L, 0.03 g Lipozyme TL100 L, and
0.60 g Lipozyme CALB were fully dissolved in 50 mL 0.1 M PBS
2

E. Bilgin Simsek and D. Saloglu
Journal of Molecular Liquids 337 (2021) 116612
(
pH 7.0) with mild shaking at 37 ℃. The obtained G-g-C
3
N
4
(1.0 g)
2.9. Photocatalytic activity tests
was dispersed in the lipase solutions which were subsequently
incubated at 37 ℃ in a shaker overnight. The immobilized lipases
(C N @PLTS, C N @LPZYM, and C N @CALB) were collected by
3 4 3 4 3 4
refrigerated centrifugation, washed with 0.1 M PBS (pH 7.0) 3
Photocatalytic performances of prepared samples were carried
out using a square reactor equipped with two visible lamps (k:
400–800 nm). A typical degradation test was conducted in suspen-
times, freeze-dried overnight, and stored in a refrigerator at 4 ℃.
sion including 10 mg of raw g-C
C N
3 4
3
N
4
, C
3
N
4
@PLTS, C
@CALB and 50 mL of 10 mgL pollutant solution at neutral pH
of 6.5. Prior to light illumination, the suspension was stirred for
0 min to ensure the equilibrium of adsorption/desorption. During
3 4
N @LPZYM, and
-
1
2.4. Activity assay and protein determination
3
The activities of the free and immobilized lipases were mea-
sured by the hydrolysis of p-nitrophenol palmitate (p-NPP) in a
buffer solution at 37 ℃ for 5 min at 160 rpm. The protein concen-
3 4 3 4 3 4
trations in C N @PLTS, C N @LPZYM, and C N @CALB were deter-
mined by the Bradford method, using bovine serum albumin as
standard. The amount of immobilized lipases was calculated by
detecting the amount of un-immobilized lipase according to the
method described in Li et al. [23].
the photocatalytic reaction, aliquots (3 mL) were collected at speci-
fic times followed by filtration with a syringe membrane filter. The
residual tetracycline (TC) and sulfadiazine (SDZ) concentrations
were analyzed by UV–vis absorption spectroscopy at wavelengths
of 360 nm and 262 nm, respectively.
The degradation ratio was determined by Eq. (3).
ðC
0
ꢀ C Þ
t
Xð%Þ ¼
ꢁ 100
ð3Þ
C
0
2.5. Immobilization yield
where C0 and C are the concentration before irradiation and
t
after irradiation time t, respectively.
In order to determine the effect of pH, the photo degradation
tests of TC and SDZ were evaluated in the presence of C N @LPZYM
Immobilization yield was calculated using Eq. (1):
ꢀ
ꢁ
3
4
Lipase bound enzyme activity
Immobilization yield ¼
ꢁ 100
ð1Þ
catalyst at different pH values, which were adjusted to pH 3.0, 6.5,
.0, and 13.0. In terms of the scavenger effect, ethylene diamine
Lipase loading actvity
8
tetra acetic acid disodium salt (EDTA-2Na, 1.0 mM), isopropyl alco-
hol (IPA, 1.0 mM), and p-benzoquinone (BQ, 0.1 mM) solutions
The activities obtained from other conditions were defined as
relative activities, and we assumed the maximum activity value
of the immobilized and free lipase under optimal conditions to
be 100%.
+
ꢀ
2
were applied as scavengers for h , ꢃOH, and O
, respectively. More-
over, the effect of co-existing anions was investigated by adding
2
–
2–
2-
CO
the catalyst was examined by increasing ambient temperature to
0 ℃ and 60 ℃. All experiments were performed in duplicate and
3 3 4
, NO and SO anions. Additionally, the thermal stability of
4
2
.6. Effect of pH and temperature on the immobilization yield
the average values were reported.
In order to identify the intermediate products with related
pathways after TC and SDZ degradation, Perkin Elmer Clarus 580
gas chromatograph equipped with mass spectrometer (GC–MS)
was utilized. The solutions were extracted with dichloromethane
before analysis.
The effects of pH on the activities of free and immobilized
lipases were investigated at 37 ℃ at a variety of pHs (5.0, 5.5,
.0, 6.5, 7.0, 7.5, and 8.0) for 1 h. The effect of temperature on
the activities of free and immobilized lipases was investigated at
pH 7.0 at different temperatures (30, 40, 50, 60, 70, and 80 ℃)
for 1 h. Each treatment was performed in triplicate.
6
3
. Results and discussion
2.7. Characterization
3.1. Investigation the effect of immobilization method
FT-IR spectra were recorded on a Bruker Tensor 27 FT-IR
spectrometer with the KBr pellet technique. The crystal phases of
samples were measured using a Bruker D8 Advance X-ray diffrac-
tometer (Bruker, Germany) with a 2theta range from 10 to 80 with
Since the immobilization method for enzymes directly influ-
ences the molecular structure of enzyme and thereby its catalytic
activity [13–15], the effect of physical and covalent attachment
on the immobilization yield were preliminarily investigated in
the present paper. Glutaraldehyde was chosen as cross-linking
agent which can be covalently linked between g-C N and lipase
3 4
structures resulting in efficient immobilization through modifica-
tion of amino groups. It was also reported that the hydrophobicity
Cu-Ka irradiation. The thermal stabilities of g-C
@LPZYM, and C @CALB were studied with a thermogravi-
metric (TGA) analyzer (TAQ50, Netzsch, Germany) at a heating rate
3 4 3 4
N , C N @PLTS,
3
C N
4
3 4
N
ꢀ1
2
of 5 ℃ min in a N atmosphere. The morphologies of obtained
samples were characterized using a scanning electron microscope
operated on 300 kV. The UV–vis absorption spectra were obtained
using a diffuse reflectance UV–Vis absorption spectrophotometer
of g-C
As shown in Table 1, the immobilization yields were found to be
1.0% and 93.4% for C @PLTS; 81.3% and 95.4% for C @LPZYM;
and 79.4% and 91.7% for C @CALB using physical adsorption and
3 4
N could increase by using glutaraldehyde [23].
(
Perkin-Elmer Lambda 750).
7
3
N
4
3 4
N
3 4
N
2
.8. Enzyme kinetics
covalent bonding methods, respectively. In addition, bound
enzyme activities were determined to be 2130 and 2802 U/g g-
C N for C N @PLTS; 2559 and 2862 U/g g-C N for C N @LPZYM;
Hanes Woolf plot is a representation of enzyme kinetics accord-
3
4
3
4
3
4
3
4
ing to the ratio of the initial substrate concentration to the reaction
velocity plotted against initial substrate concentration.
3 4 3 4
and 2382 and 2751 U/g g-C N for C N @CALB using physically and
covalently immobilized enzymes, respectively. Lipase immobiliza-
tion using glutaraldehyde onto g-C showed the highest immo-
bilization yield (95.4%) and the highest activity per gram of carrier
2862 U/g g-C ) using Lipozyme TL100 L.
The strong conjugate bonds [13] and hydrophobic effect of glu-
taraldehyde [24] resulted in an efficient immobilization yield
through covalent attachment on g-C . The surface layers of
3 4
N
½
Sꢂ
1
K
m
¼ V
½Sꢂ þ V
max
ð2Þ
v
max
(
3 4
N
where [S] is the initial pNPP concentration,
v
is the initial
reaction rate, and Vmax is the maximum velocity of free and immo-
bilized lipases.
3 4
N
3

E. Bilgin Simsek and D. Saloglu
Journal of Molecular Liquids 337 (2021) 116612
Table 1
3 4
Immobilization efficiency of lipase enzymes on g-C N using physical adsorption and covalent attachment.
Immobilization method
Lipase loading on
g-C (U/g)
Bound lipase activity
(U/g g-C N )
3 4
Immobilization
yield (%)
3
N
4
3 4
C N @PLTS
Physical immobilization
Covalent immobilization
3000
3000
2130
2802
71.0
93.4
3 4
C N @LPZYM
Physical immobilization
Covalent immobilization
3000
3000
2559
2862
85.3
95.4
3 4
C N @CALB
Physical immobilization
Covalent immobilization
3000
3000
2382
2751
79.4
91.7
g-C
consists of heterocyclic carbon–nitrogen aromatic rings [19]. Dur-
ing immobilization, the amino groups of g-C interact with C = O
bonds from the aldehyde groups of glutaraldehyde. Then, amide (–
CO-NH-) bonds of lipase enzymes attach to the crosslinked group
resulting in strong covalent immobilization. In the case of physical
3
N
4
are decorated with amino groups, while the internal layer
addition, the photodegradation efficiencies of C
3 4 3
N @CALB and C -
N
4
@PLTS were observed to be 50.1% and 57.5%, respectively. For
3
N
4
both pollutants, the degradation performances of covalently
immobilized biohybrids were lower than physically immobilized
ones. Unless the strong crosslinking groups between g-C N and
3 4
glutaraldehyde created new active sites enhancing immobilization
yield and biocatalytic activity, these sites dramatically decreased
adsorption, hydrogen atoms bind to the amino groups of g-C
3 4
N .
Possible immobilization mechanisms are shown in Fig. 1.
3 4
the photocatalytic efficiency of g-C N owing to the competitive
Among the lipase enzymes, Lipozyme TL100L displayed highest
immobilization yield for both physical and covalent immobiliza-
tion methods. Lipozyme TL100L is a 1,3-regioselective and stable
lipase. The large hydrophobic pocket surrounding the active site
inhibition of end groups. Therefore, further experiments were con-
ducted with physically immobilized biohybrids and their biocat-
alytic and photocatalytic behaviors were investigated in detail.
of Lipozyme TL100L might strongly interact with g-C
3 4
N . More-
over, the adsorption of Lipozyme TL100L on g-C under different
3
N
4
3.2. Characterization of raw g-C N and physically immobilized
3
4
experimental conditions may also affect changes in the shape of
the open active center of the enzyme and different activity-
selectivity properties of the enzyme.
3 4 3 4 3 4
C N @PLTS, C N @LPZYM, and C N @CALB biohybrids
The surface functional groups of raw g-C N and physically
3
4
The influence of immobilization method for Palatase 20000L,
3 4 3 4 3 4
immobilized C N @PLTS, C N @LPZYM, and C N @CALB biohybrids
ꢀ1
Lipozyme TL100L, and Lipozyme CALB enzymes on g-C
also explored for their photocatalytic degradation performances
Fig. 2). In the presence of raw g-C , TC degradation was calcu-
3
N
4
were
were investigated by FT-IR (Fig. 3). The bands near 1000 cm were
2
assigned to graphitic sp and triazine vibrations. The peak around
ꢀ
1
(
3
N
4
800 cm was attributed to out-of-plane bending vibration of the
H atom on the heptazine ring. The three bands at 1250, 1320,
lated as 52% at the end of 180 min. With the enzyme immobiliza-
tion, the TC degradation efficiency was significantly accelerated
and 69.2%, 83.0% and 98% removal percentages were achieved for
ꢀ1
and 1410 cm indicated the aromatic C–N stretching of raw g-
ꢀ
1
C N . The peak at about 1610 cm originated from C = N stretch-
3
4
ꢀ1
physically immobilized C
3
N
4 3
@CALB, C N
4
@PLTS, and C
3
N
4
@LPZYM
ing [25], while the peaks between 3000 and 3500 cm illustrated
biohybrids, respectively. Conversely, lower TC degradation rates
were determined as 39.40%, 49.4% and 79.5% for covalently
attached Lipozyme CALB, Palatase 20,000 L, and Lipozyme TL 100
L enzymes, respectively. Similarly, SDZ degradation performances
increased with enzyme immobilization via physical adsorption
on g-C N ; which resulted from the ability of enzymes to enhance
3 4
the structural and optical properties of the supporting material.
The SDZ degradation ratio was found to increase from 43% to
the stretching of N-H or terminal NH groups in the aromatic ring
2
of raw g-C N [26]. In comparison with raw g-C N , some differ-
3
4
3
4
ences appeared in the FT-IR spectra with the intensities clearly
decreased for C N @PLTS, C N @LPZYM, and C N @CALB biohy-
3
4
3
4
3
4
ꢀ
1
brids. The peaks at about 1250, 1320, 1410, and 1610 cm seen
at lower intensities were assigned to the aromatic C–N and C–N
skeleton. These characteristic absorption peaks of C N @PLTS,
3
4
C N @LPZYM, and C N @CALB highly overlapped with the absorp-
3
4
3
4
6
1% with the physically immobilized Lipozyme enzyme. In
3 4
tion peaks of g-C N due to the physical interaction of lipases with
Fig. 1. Immobilization mechanism of physically and covalently immobilized lipase.
4

E. Bilgin Simsek and D. Saloglu
Journal of Molecular Liquids 337 (2021) 116612
3 4 3 4 3 4 3 4
Fig. 2. Effect of immobilization method on the photocatalytic performances of raw g-C N , C N @PLTS, C N @LPZYM, and C N @CALB.
3 4, 3 4 3 4 3 4
Fig. 3. FT-IR spectrum of raw g-C N C N @PLTS, C N @LPZYM, and C N @CALB biohybrids.
the support matrix. The findings revealed that the immobilization
process affected the basic surface structure of g-C
Additionally, the most significant difference was determined in
the spectra of C @LPZYM sample, demonstrating the efficient
interaction between Lipozyme TL100L and g-C . Comparing the
curves of raw g-C and C @LPZYM, the N–H and H vibration
peaks of g-C at around 3000–3500 and 800 cm weakened
after immobilization, which can be explained by the successful for-
mation of N–H and H bonds between Lipozyme TL100L and g-C
istic peak at 27.7° was assigned to the graphitic (002) plane of g-
. Also, two obvious additional peaks at 2h of 34.8° and 47.8°
were detected in the pattern of C @LPZYM. These crystal peaks
correspond to the crystalline and/or stereo complex crystals of
aspartate, histidine, and serine of all lipases. These features illus-
trated the typical amino acid/protein phases of enzyme biohybrid
3
N
4
.
3 4
C N
3 4
N
3
N
4
3
N
4
3
N
4
3 4
N
ꢀ
1
3
N
4
catalysts. Furthermore, the XRD pattern of C
a minor peak at 13.2° ascribed to the (100) planes in-planar tris-
s-triazine of g-C . On the other hand, high crystallinity of g-
was observed in the biohybrid sample due to the strong inter-
action between g-C and Lipozyme TL100L enzyme. This interac-
3 4
N @LPZYM showed
N .
3 4
3 4
N
In other words, the amino and carboxylic groups, which are active
sites of enzymes, bonded with the surface N–H and H groups
resulting in changes in the microenvironment.
3 4
C N
3 4
N
tion proved the close packaging of g-C
the formation of regular crystallites.
3 4
N molecules required for
The thermal stabilities of raw g-C
and C @CALB were characterized by TGA (Fig. 4(a)). The char
yields of C @PLTS, C @LPZYM, and C @CALB were nearly
same as that of raw g-C . All biohybrid catalysts were stable
up to about 750 °C, while obvious weight losses of about 4.5% were
observed from TGA curves of C @PLTS, C @LPZYM, and C
@CALB, respectively. The weight loss beginning from 200 °C
3 4 3 4 3 4
N , C N @PLTS, C N @LPZYM,
3
N
4
Scanning electron microscopy (SEM) was carried out to verify
the surface morphology of g-C and C @PLTS, C @LPZYM,
and C @CALB (coded as C @Lipases in the figure) (Fig. 5).
The raw g-C had a lamellar, non-homogenous, and porous sur-
face structure with wrinkles. The particle size of synthesized g-
were below 100 mm in diameter. Also, non-homogenous
internal structure of raw g-C was obvious in the high magnifi-
cation SEM image. Meanwhile, C @Lipases displayed smooth
3
N
4
3
N
4
3 4
N
N
4
N
4
3 4
N
3
3
3 4
N
3
N
4
3 4
N
3
N
4
3
N
4
3
N
4
3
-
N
4
3 4
C N
was derived from the decomposition of immobilized lipases. In
addition, it can be clearly seen from Fig. 4 that lower degradation
rates were obtained for C N @PLTS, C N @LPZYM, and C N @CALB
3 4 3 4 3 4
in the temperature range of 25 ℃ and 750 ℃. Higher thermal sta-
3 4
N
3 4
N
layers which were attributed to the immobilized lipase layer.
These observations demonstrated successful immobilization of
bility with lower degradation rate for biohybrid catalysts could
lipases onto the g-C
In Fig. 6, the UV–vis absorption curves showed that both raw g-
and lipase immobilized g-C biohybrids absorbed a consid-
3 4
N structure.
be attributed to the extensive interaction between g-C
lipases.
3 4
N and
3
C N
4
3 4
N
In Fig. 4(b), the XRD patterns of g-C
3 4 3 4
N and C N @LPZYM biohy-
erable amount of visible light and in turn have potential for use as
visible-light-driven photocatalysts. The corresponding band gap
brid catalysts were quite different from each other. The character-
5

E. Bilgin Simsek and D. Saloglu
Journal of Molecular Liquids 337 (2021) 116612
3.3. Biocatalytic activities of biohybrids
To investigate the biocatalytic applications of C
3 4 3
N @PLTS, C -
N
4
@LPZYM, and C @CALB samples, the enzyme activities were
3 4
N
determined using the hydrolysis reaction of p-NPP. It was foreseen
that the interactions between lipase enzymes and triazine rings of
g-C N might increase the catalytic activity of lipases, thus enhanc-
3 4
ing the enzymatic activity of the hydrolysis of substrate molecules
at active sites.
Since pH of solution and temperature seriously affect the rela-
tive activities of free and immobilized lipases when hydrolyzing
p-NPP, the effects of these parameters were investigated, and the
results are presented in Fig. 7(a). Compared to the free Palatase
2
5
0000L, Lipozyme TL100 L, and Lipozyme CALB (pH 7.0, 7.0, and
.0, respectively), the optimum pH of C @PLTS, C @LPZYM
@CALB shifted toward the basic region (pH 7.5, 7.5, and
.5, respectively). This shift could be associated with the conforma-
3
N
4
3 4
N
3 4
and C N
6
tional variation of enzyme on the support and protonation of the
micro-environment. When the free lipase enzymes directly
accessed the substrate in soluble form, the –NH
g-C might stabilize the pH of the microenvironment surround-
ing the enzyme, thereby affecting the protonation of the protein
molecule [27]. Meanwhile, the enzyme activity of C @PLTS, C
@LPZYM and C @CALB biohybrids showed better performance
2
groups on the
3
N
4
3
N
4
3
-
N
4
3 4
N
when the pH was changed from 5.0 to 8.0, which demonstrated
improved stability in comparison to the free form. It could be spec-
ulated that the integrity of Palatase 20000L, Lipozyme TL100 L, and
Lipozyme CALB enzymes was preserved over a wider pH range by
3 4
immobilization on the g-C N matrix. A similar approach was sta-
ted about Candida antarctica lipase B which displayed an enhanced
catalytic rate when it was adsorbed on a hydrophobic interface and
it was demonstrated that the increased activity was due to the con-
formational changes in open active sites [2].
Fig. 4. (a) TGA and (b) XRD results for samples.
3 4 3 4 3 4
As shown in Fig. 7(b), C N @PLTS, C N @LPZYM and C N @CALB
biohybrid catalysts exhibited better relative enzyme activities than
their free forms in the range from 30 ℃ to 40 ℃. Due to denatura-
tion, the relative activity of the free lipases decreased rapidly when
there was an increase in temperature (0% of initial activity at 70 ℃
and 80 ℃). In contrast, the immobilized lipases were more heat-
resistant than the free lipases at higher temperatures. This might
3 4 3 4 3 4 3 4
energies of g-C N , C N @PLTS, C N @LPZYM, and C N @CALB
were 2.75 eV, 2.70 eV, 2.6 eV and 2.6 eV, respectively (Fig. 5(b)).
Of note, such a decrease in band gap energy value suggests
enhanced potential for visible light activation, enabling these bio-
hybrids to be promising candidates for photocatalytic applications.
3 4 3 4 3 4 3 4 3 4
Fig. 5. SEM images of g-C N , C N @PLTS, C N @LPZYM, and C N @CALB (coded as C N @Lipases in the figure) (x100 and x5000).
6

E. Bilgin Simsek and D. Saloglu
Journal of Molecular Liquids 337 (2021) 116612
3 4 3 4 3 4 3 4
Fig. 6. (a) UV–vis absorption spectra (b) Kubelka-Munk function for raw g-C N , C N @PLTS, C N @LPZYM, and C N @CALB biohybrids.
Fig. 7. Effects of (a) pH and (b) temperature on the activities of free and physically immobilized lipases.
ꢀ
1
be explained by the formation of chemical bonds during the phys-
ical bonding process, which could effectively restrict the conforma-
tion distortion or damage during periods of elevated temperature.
Analogous results were obtained by other researchers for lipase
immobilization, as summarized in Table S1.
to free enzyme values of 3.73
ally, Vmax and K values of immobilized Lipozyme CALB were
found to be 2.13
values of 3.42 Mmin and 19.83 mM respectively. K
values of immobilized Lipozyme TL100L were calculated to be
lMmin and 55.23 mM. Addition-
m
ꢀ1
l
Mmin and 36.6 mM compared to free enzyme
ꢀ
1
l
m
and Vmax
ꢀ1
7
4
1.5 mM and 1.77
.2 mM and 1.75 Mmin . As is known, K
represent higher affinity of the
enzyme [28]. In other words, high K values indicate how effi-
ciently an enzyme selects its substrate and converts to product.
In this study, the increment in all K values after the immobiliza-
tion process indicates the increase in enzyme affinity. K values of
Lipozyme TL 100 L increased 17 fold after immobilization. The K
value of the immobilized Lipozyme TL 100 L, 71.5 mM, was higher
than the K of the free one, 4.2 mM; revealing that the immobi-
lMmin compared to free enzyme values of
ꢀ1
l
m
represents the enzyme
3.3.1. Kinetic studies
affinity; higher values of K
m
m
Kinetic parameters (K and Vmax) for free and physically immo-
bilized lipases were determined using Hanes Woolf model at opti-
mum pH and room temperature. The immobilization process
m
changed the K
0,000 L, and Lipozyme CALB enzymes (Table 2).
max and K values of immobilized Palatase 20,000 were deter-
m
and Vmax values of free Lipozyme TL 100 L, Palatase
m
2
m
V
m
m
ꢀ1
mined to be 5.81 lMmin and 81.39 mM, respectively, compared
m
lized enzyme needs a higher concentration of substrate. This situ-
ation represents higher mass transfer resistance, steric hindrance,
enzyme conformational changes, and limited accessibility to the
enzyme active sites [29]. These findings confirmed the successful
Table 2
Kinetic parameters of free and physically immobilized Lipozyme TL 100 L, Palatase
2
0,000 L, and Lipozyme CALB enzymes.
M.min 1
ꢀ
)
Enzyme form
K
m
(mM)
V
max
(l
3 4
attachment of all enzymes to the g-C N surface.
C
3
N
4
@PLTS
The maximum reaction velocity (Vmax) values for the immobi-
lized Lipozyme TL 100 L, Palatase 20,000 L, and Lipozyme CALB
Free
Physical immobilization
@LPZYM
55.23
81.9
3.73
5.81
were determined. It was found that Vmax values decreased from
C
N
3 4
ꢀ1
1
.77 to 1.75
l
mol min
for Lypozyme TL 100 L and 5.81 to
Free
Physical immobilization
@CALB
4.20
71.5
1.75
1.77
ꢀ
1
3
.73 mol min
l
for Palatase 20,000 L, respectively. These
C
N
3 4
data showing higher K
cally immobilized lipases agree with the findings of Kashefi et al.
29].
m
value with lower Vmax value for physi-
Free
Physical immobilization
19.83
36.60
3.42
2.13
[
7

E. Bilgin Simsek and D. Saloglu
Journal of Molecular Liquids 337 (2021) 116612
3.4. Photocatalytic degradation studies
the TC degradation performance with removal percentages
increasing from 52% to 98%, while the SDZ degradation ratio
Photocatalytic performance of raw and enzyme immobilized g-
C N was also investigated towards pharmaceuticals, namely tetra-
3 4
cycline (TC) and sulfadiazine (SDZ), under visible light irradiation
and the results are illustrated in Fig. 8. In order to examine the role
of adsorption in the removal performance, the preliminary control
experiments were conducted in the dark and the results are pre-
increased from 42.5% to 60.5% for the C
the immobilized lipase enzyme types, the Lipozyme TL100L
enzyme attached to g-C exhibited the best photocatalytic per-
3 4
N @LPZYM sample. Among
3 4
N
formance for both antibiotics. The difference in the photocatalytic
activities could be ascribed to the structural and functional differ-
ences between the studied lipase enzyme types. Researchers
demonstrated that the amino acid sequence and lid configurations
of lipases play important roles in the catalytic activity and speci-
ficity [15]. The lid of the lipase enzyme reflects to its amphipathic
structure, in which the hydrophilic side faces the solvent in the
closed form, while the hydrophobic side is exposed to the catalytic
site [2]. When the lid is closed, it enables the active site to face
towards substrate and the lipase will be inactive; while in open
conformation, substrates can reach active sites [30]. It can be sta-
ted that lipases with similar lid configuration or flexibility might
have similar immobilization efficiency. Lipozyme TL100L is a speci-
fic lipase and shows high substrate selectivity to large side chains/
large groups. Additionally, Lipozyme TL100L has the highest open
lid and large hydrophobic pocket surrounding the active site
among the other selected enzymes; thus, the highest photocat-
sented in the inset of Fig. 8. The raw g-C
of TC in the dark adsorption period. The introduction of enzymes
into the g-C structure favors a slight improvement in the TC
adsorption performance and about 29%, 27% and 32% adsorptive
removal rates were observed for C @CALB, C @PLTS, and C
@LPZYM samples, respectively. This reveals the fact that adsorp-
3 4
N itself adsorbed 21%
3
N
4
3
N
4
3
N
4
3
-
N
4
tion might play a minor role in the degradation efficiency of TC. On
the other hand, different from TC removal, the SDZ adsorption effi-
ciency of raw g-C
3 4
N was very low, calculated as 3.0% at the end of
3
h. After the immobilization of Lipozyme CALB, Palatase 20000L,
and Lipozyme TL100L enzymes, 9.4%, 14.3% and 14.4% of SDZ
adsorption rates were calculated indicating that SDZ degradation
could be mainly enhanced due to the catalytic degradation. In gen-
eral, the immobilized enzymes cannot significantly oxidize or
adsorb the tested antibiotics demonstrating that enhanced visible
light absorption properties of biohybrid catalysts might be more
dominant for photocatalytic performance.
alytic activity could be obtained with C
alyst. Considering these results, subsequent experiments were
conducted with C @LPZYM as the best catalyst.
3 4
N @LPZYM biohybrid cat-
3
N
4
The photodegradation experiments revealed that the enzyme
immobilization on the g-C N structure significantly improved
3 4
The degradation performances of the enzyme immobilized cat-
alysts were further evaluated based on the Langmuir-Hinshelwood
Fig. 8. Photocatalytic degradation performances of raw g-C
3
N
, C
4 3
4
N @PLTS, C
3
N
4 3 4
@LPZYM, and C N @CALB (Inset shows the dark adsorption performances of samples).
8

E. Bilgin Simsek and D. Saloglu
Journal of Molecular Liquids 337 (2021) 116612
2ꢀ
2
–, NO
ꢀ
3
kinetic model. According to the linear plots of the pseudo-first-
order kinetic model (Fig. 8), both TC and SDZ degradation by the
synthesized biohybrids satisfied the requirements for pseudo-
were investigated by adding SO
4
and CO
3
(0.1 M) into the
suspension and the results are depicted in Fig. 9(b). The addition
of sulfate caused a slight depression in the TC degradation (86.5%
removal), while the presence of carbonate significantly decreased
the TC removal from 98% to 58.3%.
first-order kinetics (ln(C/C
0
) = kappt). The apparent reaction rate
-2
ꢀ1
constants (kapp) of raw g-C
3
N
4
were calculated as 0.35x10 min
-
2
ꢀ1
and 0.27x10 min for TC and SDZ degradations, respectively. The
Unlike TC degradation, the presence of sulfate anions had no
inhibitory effect on SDZ degradation. Luna et al. [37] underlined
the fact that the sulfate anions could react with photo induced
holes producing oxidizing radicals and promoting ꢃOH formation
Lipozyme TL100 immobilized g-C
3
N
4
catalyst possessed higher
degradation rate constants for TC (kapp = 3.73x10-2 min ) and
ꢀ1
SDZ degradation (kapp = 0.44x10-2 min ) than for raw g-C
ꢀ1
3
N
4
.
Compared with tetracycline antibiotic, the sulfadiazine degra-
dation performances of both raw g-C and lipase immobilized
2
by reacting with H O molecules resulting in enhanced photocat-
3
N
4
alytic degradation. On the other hand, SDZ removal was inhibited
in the presence of nitrate (49.6% removal) and carbonate (38.3%
removal) as the anions served as radical scavengers and prevented
radical formation (Eqs. 4–6).
The decrease may also be related to the coverage of active sur-
face sites on the catalysts by anions, reducing the number of
surface-active sites [36].
catalysts were lower, indicating that it is a more recalcitrant pollu-
tant. It is known that the sulfonamide groups exhibit strong chem-
ical stability and solubility in aqueous media; thereby, the rapid
and complete degradation of these groups are an important issue
and effective techniques are needed [31]. The decomposition path-
way of SDZ could begin by breaking the pyrimidine ring that is a
feature of high chemical stability; while the TC degradation mech-
anism is generally proposed as open-ring reactions and further oxi-
dation of byproducts into small organic acids [32]. These
phenomena might explain the lower photodegradation of SDZ than
TC. Similar phenomena were observed by Garcia-Delgado et al.
2
ꢀ
ꢃ
ꢀ
+ OH—
CO
3
+ HOꢃ!CO
3
(4)
(5)
(6)
ꢃ
ꢀ + H2
—
+ OH—
2CO
3
O ! 2CO
2
+ HO
2
[
33] who explored the biocatalytic removal of tetracycline and sul-
fonamide via mediator-assisted laccase oxidation. Ding et al. [34]
demonstrated that the K values were important for the removal
of antibiotics. They underlined that tetracycline could be easily
adsorbed due to high K values, whereas the K of sulfonamides
3ꢀ
3
ꢃ
+ OH—
NO
+ HOꢃ!NO
d
d
d
were relatively low. Accordingly, these assumptions can explain
the difference in the adsorption and photocatalytic removal rates
for TC and SDZ antibiotics.
3
.4.3. Effect of reactive oxidative species
The trapping experiments were carried out in order to explore
the involvement of reactive oxidative species in the TC and SDZ
photodegradation with the C @LPZYM biohybrid catalyst. For
3
N
4
3.4.1. Effect of solution pH
this purpose; isopropyl alcohol (IPA), benzoquinone (BQ) and
EDTA-2Na were added to trap the generated hydroxyl radicals
Since the solution pH affects the generation of hydroxyl radicals
and also influences the surface charge and interface potential prop-
erties of the catalyst, it is one of the important factors in the pho-
tocatalytic degradation. Therefore, the effect of solution pH on the
–
+
(ꢃOH), superoxide radicals (ꢃO
2
) and holes (h ), respectively. As
seen in Fig. 9(c), TC degradation decreased from 98% in the absence
of scavenger to 79% upon addition of IPA, indicating the minor role
of hydroxyl radicals. Additionally, with the addition of EDTA-2Na
and BQ, the degradation efficiencies significantly reduced to
3 4
performance of C N @LPZYM catalyst for the TC and SDZ degrada-
tion were evaluated in the pH range of 3.0 and 13.0. When the
solution pH was adjusted to pH 3.0, almost complete TC degrada-
tion was observed within 120 min, while 98% and 80% of TC were
degraded at pH 6.5 and 8.0, respectively. This indicates that the
catalysts can exhibit ideal performance in a range of pH from 3.0
to 8.0 (Fig. 9(a)). On the other hand, almost no catalytic degrada-
tion was observed at pH 13. These findings may be related with
3
8.3% and 16.8%, respectively. Similarly, the SDZ photodegradation
was sharply decreased upon the addition of BQ, implying that the
superoxide radicals were the predominant reactive species in the
degradation process for both antibiotics.
both enzyme properties and pK
The pK
ferent chemical states at these pH values as: (i) H
a
characteristics of tetracycline.
3.4.4. Effect of temperature on the photodegradation performance of
biocatalysts
Since the catalytic properties of the enzymes can be influenced
3 4
by temperature, the photodegradation performances of C N @-
a
values of TC are 3.3, 7.7, 9.7 and 12.0, which results in dif-
+
4
TC (pH < 3.3),
ꢀ
2ꢀ
(
ii) H
3
TC (3.3 < pH < 7.7), (iii) H
2
TC (7.7 < pH < 9.7) and (iv) HTC
(
pH > 12) [35]. Accordingly, at higher pH values, the repulsion
LPZYM catalyst for tetracycline and sulfadiazine removal were
investigated in a range of temperatures from 25 °C to 60 °C. From
the results shown in Fig. 10, for both antibiotics, the highest
removal efficiencies were observed at 25 °C; while the slight
decrease with increasing temperature revealed the thermal stabil-
2
ꢀ
forces might be dominant between HTC
and the negatively-
charged catalyst surface, limiting the photocatalytic removal.
Besides, SDZ degradation reached maximum photocatalytic effi-
ciency at pH 8.0 (75.1% degradation), while it reduced at pH 3.0
(
31.3% degradation) and 13.0 (40.1% degradation). According to
the pK values of SDZ (1.8 and 5.7) [34], at lower pHs than 5.7, elec-
trostatic repulsion forces might occur between the protonated
3 4
ity of the C N @LPZYM enzyme.
a
With the increase in temperature from 25 °C to 60 °C, the TC
degradation slightly decreased from 98% to 75% at the end of 3 h.
Similarly, the SDZ degradation rates were calculated as 60%, 57%
and 54% at temperatures of 25 °C, 40 °C and 60 °C, respectively.
+
CNH
2 2 3 4
and C NH groups on the g-C N surface and SDZ molecules;
thereby, lower degradation efficiency was observed.
3 4
This decreasing trend could be ascribed to the properties of C N @-
3
.4.2. Effect of co-existing anions
It is well-known that the presence of co-existing anions
LPZYM, which exhibits maximum activity in the temperature range
30–60 °C. Since free enzymes can be unstable above ambient tem-
peratures, the slight decrease in degradation could be the result of
the formation of stable and strong interaction of lipase on the car-
bon nitride surface. Similar phenomena were reported by Zdarta
et al. [38], who examined the biocatalytic degradation of tetracy-
cline on laccase immobilized electrospun nanofibers.
strongly affects the removal efficiency of pollutants in industrial
wastewater treatment. Especially, in catalytic removal processes,
the anions act as free radical scavengers and produce anion reac-
tive species [36]. Therefore, the effects of co-existing inorganic
3 4
anions on TC and SDZ degradation performance of C N @LPZYM
9

E. Bilgin Simsek and D. Saloglu
Journal of Molecular Liquids 337 (2021) 116612
Fig. 9. Effects of solution pH (a), co-existing anions (b) and reactive oxidative species (c) on the photocatalytic degradation of TC and SDZ.
3
.5. Photodegradation pathways of target molecules
singlet oxygen followed by partial breakage of the double C–C
bond [39]. The intermediate with m/z of 392 was attributed to
the breaking of amido and amino groups which subsequently
degraded to a product with m/z of 306 via oxidation and ring open-
ing reactions [40]. After losing methyl groups on the carbon atom
ring, the intermediate product with m/z of 278 was formed while
alcohols and acids were broken on the carbocycle resulting in the
formation of a product with m/z of 230. In case of Pathway II, the
The degradation products of TC and SDZ were identified by gas
chromatography coupled with mass spectrometer, and degrada-
tion pathways are shown in Fig. 11. The EI mass spectra of the
intermediate products are indicated on Figs. S1-S2. The tetracy-
cline degradation might have occurred via two possible degrada-
tion pathways. In Pathway I, a product with m/z of 464 could be
formed through radical attack which breaks C = C bonds of tetracy-
3
product with m/z of 355 could form via breakage of -N-CH group
ꢀ
1
cline (m/z = 445). It was suggested that the electrophilic ꢃO
2
/ O
2
followed by loss of amino and acylamino groups [41]. Further
degradation led to its demethylation and formation of a corre-
sponding product with m/z of 281. Afterwards, the aromatic com-
radicals firstly attacked the TC molecule leading to the formation
of intermediates with m/z ~ 464 accompanied with epoxide and
10

E. Bilgin Simsek and D. Saloglu
Journal of Molecular Liquids 337 (2021) 116612
Fig. 10. Effect of temperature on the photocatalytic degradation of TC and SDZ.
3 4
Fig. 11. Proposed degradation pathway for selected molecules with C N @LPZYM biohybrid catalyst.
11

E. Bilgin Simsek and D. Saloglu
Journal of Molecular Liquids 337 (2021) 116612
pounds with smaller m/z values of 221, 207, and 149 were released
through oxidation reactions [40,42]. Eventually, all products were
further mineralized into CO2, H2O and other small molecules.
For the photodegradation of SDZ, intermediate products with
m/z of 95, 174, 266 were detected and the corresponding MS spec-
tra are presented in Fig. S2. According to the detected products,
three pathways were proposed. In Pathway I, the sulfadiazine
molecule was destroyed through the cleavage of the S–N bond
resulting in formation of and intermediate with m/z of 174 (p-
aminobenzene sulfonic acid) [43]. In terms of Pathway II, the for-
mation of the product (m/z 266) could be attributed to the ꢃOH
radicals which caused hydroxylation reactions on the benzene ring
of SDZ [43,44]. Then, C–N breakage might occur generating a pro-
duct with m/z of 217. After opening of heterocyclic rings and the
cleavage of S–N and C–N bonds, the products might be decom-
posed to the intermediate with m/z 174. In the third photodegrada-
tion pathway, the sulfadiazine molecule might degrade to the
intermediate with m/z of 125 (2-nitropyrimidin) by breaking the
pyrimidine ring which subsequently transformed to pyrimidine
molecules with m/z 96 (1H-pyrimidin-2-one) and m/z 95 (2-
aminopyrimidine) [45].
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.molliq.2021.116612.
References
[
1] S. Aggarwal, A. Chakravarty, S. Ikram, A comprehensive review on incredible
renewable carriers as promising platforms for enzyme immobilization
&
thereof strategies, Int. J. Biol. Macromol. In press, https://doi.org/10.1016/j.
ijbiomac.2020.11.052.
[2] F.I. Khan, D. Lan, R. Durrani, W. Huan, Z. Zhao, Y. Wang, The Lid Domain in
Lipases: Structural and Functional Determinant of Enzymatic Properties, Front.
Bioeng. Biotechnol. 5 (2017) 16, https://doi.org/10.3389/fbioe.2017.00016.
[
3] C. Ortiz, M.L. Ferreira, O. Barbosa, J.C.S. dos Santos, R.C. Rodrigues, A.
Berenguer-Murcia, L.E. Briand, R. Fernandez-Lafuente, Novozym 435: the
‘
‘perfect” lipase immobilized biocatalyst, Catal. Sci. Technol. 9 (2019) 2380,
https://doi.org/10.1039/c9cy00415g.
[
[
4] M. Lotti, L. Alberghina, Chapter 16 Lipases: Molecular Structure and Function, J.
Polaina and A.P. MacCabe (eds.), Industrial Enzymes, pp. 263–281. Springer
(
2007) https://doi.org/10.1007/1-4020-5377-0_16.
5] L. Polgár, The catalytic triad of serine peptidases, CMLS 62 (2005) 2161–2172,
https://doi.org/10.1007/s00018-005-5160-x.
[6] R. Fernandez-Lafuente, Lipase from Thermomyces lanuginosus: Uses and
prospects as an industrial biocatalyst, J. Mol. Catal. B Enzym. 62 (2010) 197–
212. https://doi:10.1016/j.molcatb.2009.11.010.
[
7] M.A. Maria-Solano, E. Serrano-Hervás, A. Romero-Rivera, J. Iglesias-Fernández,
S. Osuna, Role of conformational dynamics in the evolution of novel enzyme
function, Chem. Commun. 54 (2018) 6622–6634, https://doi.org/10.1039/
C8CC02426J.
4
. Conclusion
In this work, a green approach was developed for enhanced bio-
[
[
8] W. Zhou, W. Zhang, Y. Cai, Laccase immobilization for water purification: A
comprehensive review, Chem. Eng. J. 403 (2021), https://doi.org/10.1016/j.
cej.2020.126272 126272.
9] J. Zdarta, A.S. Meyer, T. Jesionowski, M. Pinelo, Review : A General Overview of
Support Materials for Enzyme Immobilization: Characteristics, Properties,
Practical Utility, Catalysts 8 (2018) 92, https://doi.org/10.3390/catal8020092.
catalytic and photocatalytic performance by utilizing graphitic car-
bon nitride as a new platform for the immobilization of lipase
enzymes. The synthesized biocatalysts showed high enzyme activ-
ity as well as thermal stability and pH tolerance. The enzyme activ-
ity of immobilized lipase species showed better performance when
the pH was changed from 5.0 to 8.0, when compared to the free
form. The photocatalytic activities were investigated for the degra-
dation of two antibiotics and the results showed that the biohy-
brids yielded higher photodegradation rates which was ascribed
to the narrow band gap energy and hydrophilic properties of g-
[10] T. Jesionowski, J. Zdarta, B. Krajewska, Enzyme immobilization by adsorption:
a review, Adsorption 20 (2014) 801–821, https://doi.org/10.1007/s10450-014-
9623-y.
[
[
[
[
[
11] C. Ding, J. Liang, Z. Zhou, Y. Li, Wenchao Peng, Guoliang Zhang, Fengbao Zhang,
Xiaobin Fan Photothermal enhanced enzymatic activity of lipase covalently
immobilized on functionalized Ti C T nanosheets, Chem. Eng. J. 378 (2019),
3 2 X
https://doi.org/10.1016/j.cej.2019.122205 122205.
12] A.R. Ismail, K.H. Baek, Lipase immobilization with support materials,
preparation techniques, and applications: Present and future aspects, Int. J.
Biol. Macromol. 163 (2020) 1624–1639, https://doi.org/10.1016/j.
ijbiomac.2020.09.021.
13] L. Zhong, Y. Feng, G. Wang, Z. Wang, M. Bilal, H. Lv, S. Jia, J. Cui, Production and
use of immobilized lipases in/on nanomaterials: A review from the waste to
biodiesel production, Int. J. Biol. Macromol. 152 (2020) 207–222, https://doi.
org/10.1016/j.ijbiomac.2020.02.258.
14] S.E. Collins, V. Lassalle, M.L. Ferreira, FTIR-ATR characterization of free
Rhizomucor meihei lipase (RML), Lipozyme RM IM and chitosan-
immobilized RML, J. Mol. Catal. B Enzym. 72 (2011) 220–228, https://doi.
org/10.1016/j.molcatb.2011.06.009.
15] J. Mulinari, J.V. Oliveira, D. Hotza, Lipase immobilization on ceramic supports:
An overview on techniques and Materials, Biotechnol. Adv. 42 (2020), https://
doi.org/10.1016/j.biotechadv.2020.107581 107581.
C
3
N
4
. The trapping experiments of active species demonstrated
that the hydroxyl radicals and holes were the main active species
–
accounted for the TC degradation, while ꢃO
2
radicals were domi-
@lipases are
nant for SDZ removal. This study revealed that C
3
N
4
a promising biomimetic photocatalytic material for the degrada-
tion of environmental pollutants in an ecofriendly way. Also, g-
C N can be considered for immobilizing more types of enzymes
3 4
which will be important in guiding the development of multifunc-
tional catalysts both in enzymatic and photocatalytic processes.
CRediT authorship contribution statement
[16] Z. Ashkan, R. Hemmati, A. Homaei, et al., Immobilization of enzymes on
nanoinorganic support materials: An update, Int. J. Biol. Macromol. xxx (xxxx)
xxx. https://doi.org/10.1016/j.ijbiomac.2020.11.127.
Esra Bilgin Simsek: Conceptualization, Methodology, Investiga-
tion, Data curation, Visualization, Writing - original draft, Writing -
review & editing. Didem Saloglu: Conceptualization, Methodology,
Investigation, Data curation, Writing - original draft, Writing -
review & editing.
[
17] M. Bilal, S.S. Ashraf, D. Barceló, H.M.N. Iqbal, Biocatalytic degradation/
redefining ‘‘removal” fate of pharmaceutically active compounds and
antibiotics in the aquatic environment, Sci. Total Environ. 691 (2019) 1190–
1211, https://doi.org/10.1016/j.scitotenv.2019.07.224.
[
[
[
18] T.O. Ajiboye, A.T. Kuvarega, D.C. Onwudiwe, Graphitic carbon nitride-based
catalysts and their applications: A Review, Nano-Struct. Nano-Objects 24
(
2020), https://doi.org/10.1016/j.nanoso.2020.100577 100577.
19] M. Fronczak, Adsorption performance of graphitic carbon nitride-based
materials: Current state of the art, J. Environ. Chem. Eng. 8 (2020), https://
doi.org/10.1016/j.jece.2020.104411 104411.
Declaration of Competing Interest
20] K.R. Reddy, C.H.V. Reddy, M.N. Nadagouda, N.P. Shetti, S. Jaesool, T.M.
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Aminabhavi,
Polymeric
graphitic
carbon
nitride
3 4
(g-C N )-based
semiconducting nanostructured materials: Synthesis methods, properties
and photocatalytic applications, J. Environ. Manage. 238 (2019) 25–40,
https://doi.org/10.1016/j.jenvman.2019.02.075.
[
21] W. Shen, L. Ren, H. Zhou, S. Zhang, W. Fan, Facile one-pot synthesis of bimodal
mesoporous carbon nitride and its function as a lipase immobilization support,
J. Mater. Chem. 21 (2011) 3890, https://doi.org/10.1039/C0JM03666H.
22] Y. Wang, N. Zhang, R. Hübner, D. Tan, M. Löffler, S. Facsko, E. Zhang, Y. Ge, Z. Qi,
Acknowledgement
[
3 4
C. Wu, Enzymes Immobilized on Carbon Nitride (C N ) Cooperating with Metal
Nanoparticles for Cascade Catalysis, Adv. Mater. Interfaces (2019) 1801664,
https://doi.org/10.1002/admi.201801664.
The authors thank Ozlem Tuna for kind help with carbon nitride
synthesis.
12

E. Bilgin Simsek and D. Saloglu
Journal of Molecular Liquids 337 (2021) 116612
[
23] Y. Li, Z. Ruan, M. Zheng, Q. Deng, S. Zhang, C. Zheng, H. Tang, F. Huang, J. Shi,
Candida rugosa lipase covalently immobilized on facilely-synthesized carbon
nitride nanosheets as a novel biocatalyst, RSC Adv. 8 (2018) 14229, https://doi.
org/10.1039/c8ra00536b.
[35] S. Jiao, S. Zheng, D. Yin, L. Wang, L. Chen, Aqueous photolysis of tetracycline
and toxicity of photolytic products to luminescent bacteria, Chemosphere 73
(3) (2008) 377–382, https://doi.org/10.1016/j.chemosphere.2008.05.042.
[36] F. Hayati, A.A. Isari, B. Anvaripour, M. Fattahi, B. Kakavandi, Ultrasound-
assisted photocatalytic degradation of sulfadiazine using MgO@CNT
heterojunction composite: Effective factors, pathway and biodegradability
studies, Chem. Eng. J. 381 (2020), https://doi.org/10.1016/j.cej.2019.122636
122636.
[
24] A.A. Mendes, R.C. Giordano, R. de LC Giordano, H.F. de Castro, Immobilization
and stabilization of microbial lipases by multipoint covalent attachment on
aldehyderesin affinity: application of the biocatalysts in biodiesel synthesis, J.
Mol. Catal.
B Enzym. 68 (1) (2011) 109–115. https://doi.org/10.1016/
j.molcatb.2010.10.002.
[37] M.D.G. de Luna, L.K.B. Paragas, R.-A. Doong, Insights into the rapid elimination
[
25] P. Yan, D. Li, X. Ma, J. Xue, Y. Zhang, M. Liu, Hydrothermal synthesis of Bi2WO6
with a new tungsten source and enhanced photocatalytic activity of Bi2WO6
hybridized with C3N4, Photochem. Photobiol. Sci. 17 (8) (2018), https://doi.
org/10.1039/x0xx00000x.
3 4
of antibiotics from aqueous media by tunable C N photocatalysts: Effects of
dopant amount, co-existing ions and reactive oxygen species, Sci. Total
Environ. 669 (2019) 1053–1061, https://doi.org/10.1016/j.scitotenv.2019.03.003.
[38] J. Zdarta, K. Jankowska, K. Bachosz, E. Kije n´ ska-Gawro n´ ska, A. Zgoła-
[
26] X. Gao, J. Feng, D. Su, Y. Ma, G. Wang, H. Ma, J. Zhang, In-situ exfoliation of
porous carbon nitride nanosheets for enhanced hydrogen evolution, Nano
Energy 59 (2019) 598–609, https://doi.org/10.1016/j.nanoen.2019.03.016.
27] K. Wang, S. Sun, B. Ma, F. Dong, T. Huo, X. Li, Y. Zhao, H. Yu, Y. Huang,
Construction and characterization of a nanostructured biocatalyst consisting
of immobilized lipase on aminopropyl-functionalized montmorillonite, Appl.
Clay Sci. 183 (2019), https://doi.org/10.1016/j.clay.2019.105329 105329.
28] N. Carrillo, E.A. Ceccarelli, O.A. Roveri Choi, Usefulness of Kinetic Enzyme
Parameters in Biotechnological Practice, Sci. Rep. 7 (2017) 17018, https://doi.
org/10.1080/02648725.2010.10648157.
29] S. Kashefi, S.M. Borghei, N.M. Mahmoodi, Covalently immobilized laccase onto
graphene oxide nanosheets: Preparation, characterization, and biodegradation
of azo dyes in colored wastewater, J. Mol. Liq. 276 (2019) 153–162, https://doi.
org/10.1016/j.molliq.2018.11.156.
30] S.H. Albayati, M. Masomian, S. Nor Hasmah, I.M.S. bin Mohamad Ali, A.L.
Thean, F. M. Shariff, N.D. Muhd Noor, R.N.Z. Raja Abd Rahman, Main Structural
Targets for Engineering Lipase Substrate Specificity, Catal 10 (2020)747.
https://doi:10.3390/catal10070747.
Grze s´ kowiak, E. Kaczorek, T. Jesionowski,
A
promising laccase
immobilization using electrospun materials for biocatalytic degradation of
tetracycline: Effect of process conditions and catalytic pathways, Catal. Today
348 (2020) 127–136, https://doi.org/10.1016/j.cattod.2019.08.042.
[39] X. Yuan, S. Qu, X. Huang, X. Xue, C. Yuan, S. Wang, L. Wei, P. Cai, Design of Core-
shelled g-C3N4@ZIF-8 Photocatalyst with Enhanced Tetracycline Adsorption
for Boosting Photocatalytic Degradation, Chem. Eng. J. https://doi.org/10.1016/
j.cej.2021.129148.
[40] Y. Wu, X. Zhao, S. Huang, Y. Li, X.n Zhang, G. Zeng, L. Niu, Y. Ling, Y. Zhang,
Facile construction of 2D g-C3N4 supported nanoflower-like NaBiO3 with
direct Z-scheme heterojunctions and insight into its photocatalytic
degradation of tetracycline, J. Hazard. Mater., https://doi.org/10.1016/j.
jhazmat.2021.125547
[41] M. Li, C. Lai, H. Yi, D. Huang, L. Qin, X. Liu, B. Li, S. Liu, M. Zhang, Y. Fu, L. Li, J. He,
Y. Zhang, L. Chen, Multiple charge-carrier transfer channels of Z-scheme
bismuth tungstate-based photocatalyst for tetracycline degradation:
transformation pathways and mechanism, J. Colloid Interface Sci. 555 (2019)
770–782. https://doi.org/10.1016/j.jcis.2019.08.035
[42] T. Pan, D.D. Chen, W.C. Xu, J.Z. Fang, S.X. Wu, Z. Liu, K. Wu, Z.Q. Fang, Anionic
polyacrylamide-assisted construction of thin 2D–2D WO3/g-C3N4 Step-
scheme heterojunction for enhanced tetracycline degradation under visible
light irradiation, J. Hazard. Mater. 393 (2020), https://doi.org/10.1016/j.
jhazmat.2020.122366 122366.
[
[
[
[
[
[
[
31] Z. Guan, Q. Li, B. Shen, S. Bao, J. Zhang, B. Tian, Fabrication of Co
3
O
4
and Au co-
modified BiOBr flower-like microspheres, with high photocatalytic efficiency
for sulfadiazine degradation, Sep. Purif. Technol. 234 (2020), https://doi.org/
1
0.1016/j.seppur.2019.116100 116100.
32] A. Vázquez, D. Hernández-Uresti, S. Obregón, Electrophoretic deposition of CdS
coatings and their photocatalytic activities in the degradation of tetracycline
antibiotic, Appl. Surf. Sci. 386 (2016) 412–417, https://doi.org/10.1016/j.
apsusc.2016.06.034.
33] C. García-Delgado, E. Eymar, R. Camacho-Arévalo, M. Petruccioli, S. Crognalec,
A. D’Annibale, Degradation of tetracyclines and sulfonamides by stevensite-
and biochar-immobilized laccase systems and impact on residual antibiotic
activity, J. Chem. Technol. Biotechnol. 93 (2018) 3394–3409, https://doi.org/
[43] Y. Song, J. Tian, S. Gao, P. Shao, J. Qi, F. Cui, Photodegradation of sulfonamides
3 4
by g-C N under visible light irradiation: Effectiveness, mechanism and
pathways, App. Catal. B. 210 (2017) 88–96, https://doi.org/10.1016/j.
apcatb.2017.03.059.
[44] H. Zenga, H. Zhanga, L. Deng, Z. Shi, Peroxymonosulfate-assisted
photocatalytic degradation of sulfadiazine using self-assembled multi-
3 4
layered CoAl-LDH/g-C N heterostructures: Performance, mechanism and
1
0.1002/jctb.5697.
eco-toxicity evaluation, J. Water Process Eng. 33 (2020), https://doi.org/
10.1016/j.jwpe.2019.101084 101084.
[45] P. Gholamia, A. Khataee, B. Vahide, A. Karimi, M. Golizadeh, M. Ritala,
Sonophotocatalytic degradation of sulfadiazine by integration of
microfibrillated carboxymethyl cellulose with Zn-Cu-Mg mixed metal
[
34] H. Ding, Y. Wu, B. Zou, Q. Lou, W. Zhang, J. Zhong, L. Lu, G. Dai, Simultaneous
removal and degradation characteristics of sulfonamide, tetracycline, and
quinolone antibiotics by laccase-mediated oxidation coupled with soil
adsorption, J. Hazard. Mater. 307 (2016) 350–358, https://doi.org/10.1016/j.
jhazmat.2015.12.062.
3 4
hydroxide/g-C N composite, Sep. Purif. Technol. 245 (2020), https://doi.org/
10.1016/j.seppur.2020.116866 116866.
13