You are on page 1of 13


Immobilization of L‑Asparaginase on Carrier Materials: A
Comprehensive Review
Ahmet Ulu and Burhan Ates*
Department of Chemistry, Faculty of Science & Arts, Inonu University, Malatya, 44280, Turkey

ABSTRACT: There are two major applications of L-asparaginase (L-ASNase): leukemia
therapy and the food industry. Especially, its chemotherapeutic effect has attracted interest
from the scientific community and individual scientists. Therefore, to protect the intrinsic
activity and half-time of L-ASNase, several carriers and immobilization techniques for
immobilization of L-ASNase have been described in articles. Unfortunately, a
comprehensive review about immobilization of L-ASNase has not been written until
now. In this review, we have thoroughly discussed the carriers for L-ASNase by illustrating
immobilization findings including both past and present applications. In addition, we have
revealed advantages and disadvantages of immobilized enzyme and related it to free form.
We believe that this review will not only provide background information, but also guide
future developments.

L-Asparaginase (L-ASNase, L-asparagine amino hydrolase,
E.C. has been widely used as a chemotherapeutic drug
in the treatment of acute lymphoblastic leukemia (ALL, mainly
in children) and other malignant diseases including Hodgkin’s
disease, acute myelocytic leukemia, acute myelomonocytic
leukemia, chronic lymphocytic leukemia, lymphosarcoma
treatment, reticulosarcoma, and melanosarcoma because of its
high chemotherapeutic effect for years.1 L-ASNase has shown
the following chemotherapeutic effect: L-Asparagine is an
essential amino acid for the growth of both normal and cancer Figure 1. Structural illustration of the of L-ASNase reaction
cells. Normal cells can produce the amino acid by means of L- mechanism.
asparagine synthetase, but if L-asparagine level is insufficient, it
is taken from the blood. However, due to cancer cells not
having L-asparagine synthetase, they must use amino acids in basis of biosensor application is based on L-ASNase activity.
the blood. In the presence of L-ASNase, L-asparagine is Ammonium ions resulting from the hydrolysis of asparagine
hydrolyzed into L-aspartate and ammonia. So, cancer cells are differentiate pH due to the change of color and absorption.7
unable to divide and they die. The hydrolysis reaction of L- As noted above, L-ASNase is a significant enzyme with a
asparagine is similar to the two-step mechanism of serine wide range of applications. It is inclusively used in leukemia
proteases and it is as follows: First, the nucleophilic residue of cancers as chemotherapeutic agent. Yet, there are factors
the enzyme is activated with a strong base and attacks the limiting its chemotherapeutic effect. Although L-ASNase has
amide carbon atom of L-asparagine, resulting in β-acyl-enzyme been produced from a variety of sources such as bacteria, yeast,
as an intermediate product. The second reaction step is an fungi, plant, and actinomycetes, commercial L-ASNase has been
attack on the ester carbon made by a nucleophile activated by a obtained from Escherichia coli (E. coli) and Erwinia
water molecule (Figure 1).2,3 chrysanthemi.3 Because L-ASNase is an enzyme of bacterial
Another important application of the enzyme is in the food origin and high molecular weight, it is exposed to the body’s
industry. Acrylamide is formed in foods cooked at high immune system. Therefore, the half-life of L-ASNase decreases.
temperatures. Since it is a potentially neurotoxic, genotoxic, Another restricting factor is that, when L-ASNase is injected
carcinogenic, and toxic to the reproductive system,4 there are into the patient, it may cause rigorous hypersensitivity reactions
reports indicating adverse effects on human health. Because of such as fever, skin rashes, allergic reactions, and even
its activity to convert L-asparagine to L-aspartate, L-ASNase
significantly reduces the formation of acrylamide.5 Received: April 16, 2017
L-ASNase is also used in biosensor technology to detect Revised: May 10, 2017
asparagine levels in both leukemia and the food industry.6 The Published: May 15, 2017

© 2017 American Chemical Society 1598 DOI: 10.1021/acs.bioconjchem.7b00217
Bioconjugate Chem. 2017, 28, 1598−1610
Bioconjugate Chemistry Review

anaphylactic shock.8,9 Also, it is a very expensive drug, but is
required in therapy, particularly childhood ALL. To overcome
these limitations, enzyme immobilization is one of the most
used strategies. Enzyme immobilization can be considered as
the attachment of native enzymes to different solid carrier
matrixes resulting in decrease or loss of mobility of the
enzyme.10,11 Immobilized versions of L-ASNase have been
improved. Thanks to immobilization, the immobilized enzyme
showed an increased half-life and significantly higher stability to
allergic reactions in comparison with the free enzyme. Until
now, L-ASNase was immobilized using various chemical/
physical methods onto natural or synthetic carrier (from
erythrocytes to poly(ethylene glycol) (PEG)). The carrier Figure 3. Schematic presentation of PEGylated L-ASNase.
matrixes used for L-ASNase immobilization were presented in
Figure 2.
reactions to native L-ASNase were 32%, the hypersensitivity
reaction to PEGylated L-ASNase was only 18%.17 It was
demonstrated that the immobilization really improves the
enzyme’s stability and biotechnological properties.
Carriers for L-ASNase Immobilization. So far, various
carriers such as organic, inorganic, and hybrid materials have
been used for L-ASNase immobilization in the literature.
Almost all carrier matrixes for L-ASNase are catalogued in
Table 1. The aim of this review is to give an overview because
there are no papers on this topic. Therefore, for better
understanding, we classified the carrier type, and the advantages
or disadvantages of immobilized L-ASNase were compared with
that of the free one. At the same time, we thought it necessary
to summarize the available immobilization information. For this
reason, we compiled the most interesting results, optimum pH
and temperature, Michaelis constant (Km), and stability
properties of the immobilized L-ASNase, and these results
are listed in Table 2. These tables will provide important
information to interested researchers for the selection of the
most appropriate carrier/method for L-ASNase immobilization.
L-ASNase Immobilization on Organic Carriers. Organic
carriers are at the forefront of immobilization of L-ASNase.
These organic carriers can be divided into two main classes as
natural carriers derived from renewable sources and petroleum-
Figure 2. Illustration of different carrier matrixes for L-ASNase
immobilization. derived synthetic carriers. Furthermore, organic nanomaterials
are considered an additional class.
Natural sources have been constantly used in enzyme
PEG is a synthetic polymer that is nontoxic, hydrophilic, and immobilization.18 These materials are nontoxic, biocompatible,
water-soluble. Additionally, it enhances biocompatibility, and biodegradable. Besides, their reactive functional groups
increases blood circulation time, and improves resistance to (such as amino and hydroxyl groups) make possible the
nonspecific protein adsorption.12,13 Therefore, it has commonly coupling of enzymes and allows in this way higher enzyme
been used in enzyme immobilization as a carrier matrix. PEG-L- loading.19,20 Therefore, they are prominent materials for
ASNase (polyethylene glycol-L-asparaginase) is the first enzyme immobilization. Carbohydrates (cellulose, dextran,
polymeric formulation approved by the Food and Drug starch, chitosan, and chitin), proteins (albumin, gelatin,
Administration (FDA) for cancer therapy and is still used in collagen, silk fibers, and silk fibroin), and lipids are materials
treatment. PEG-L-ASNase is a modified version of L- most preferred by the researchers. In the literature, natural
asparaginase formed by the covalently linking to PEG in the carriers have been reported for L-ASNase using different
process known as PEGylation.14 The PEGylation process is immobilization techniques. These carriers are chronologically
illustrated in Figure 3.15 PEGylation has been developed to arranged below.
decrease immunogenicity of the enzyme and prolong the Poznansky et al.21 displayed advantages in the use of L-
enzyme’s plasma half-life. It was reported that the half-life of ASNase-albumin as an chemotherapy agent. The L-ASNase-
native L-ASNase (from E. coli) was 1.28 days via intramuscular albumin polymer was more stable to proteolysis than of free
injection, while the half-life of PEGylated L-ASNase was 5.73 enzyme. In terms of antitumor activity, the immobilized
days.16 Besides, after L-ASNase was intravenously injected, the enzyme was ∼20 times more effective than the free L-ASNase.
half-time of PEGylated L-ASNase was approximately 19-fold At the same time, it significantly increased in the inhibition of
higher than that of unmodified L-ASNase. The increase cell growth of human pancreatic tumor cells. Likewise, Uren
resulted from enhanced resistance to trypsin degradation of and co-workers22 covalently attached poly(DL-alanine) peptides
L-ASNase. On the other hand, though hypersensitivity to lysyl residues on the surface of Erwinia carotovora L-ASNase.
1599 DOI: 10.1021/acs.bioconjchem.7b00217
Bioconjugate Chem. 2017, 28, 1598−1610
Bioconjugate Chemistry Review

Table 1. Several Carriers for L-ASNase Immobilizationa
immobilization immobilization
carrier method refs carrier method refs
Organic Organic
Albumin cross-linking 21 PLGA nanoparticles encapsulation 68, 69
Poly(DL-alanine) peptides covalent binding 22 PHVB nanocapsules encapsulation 71
Dextran covalent binding 23 Hydrogel-magnetic nanoparticles entrapment 72
Dextran sulfate chemical 24 Silk fibroin nanoparticle entrapment 74
modification PANI nanofiber covalent binding 75
Chitosan adsorption 25 PVDMA nanoparticles covalent binding 76
N,O-carboxymethyl chitosan chemical 26 Inorganic
Silica gel adsorption 77, 78, 81
Colominic acid covalent binding 27
Celite adsorption 77, 78
Glyoxyl-agarose covalent binding 28
Tricalcium phosphate adsorption 77, 78
Agarose-glutaraldehyde covalent binding 29
Activated carbon covalent binding 79
Fructose levan covalent binding 30
Inulin chemical 31
modification PEG-albumin chemical 82, 83
Red blood cells encapsulation 33
Calcium alginate-gelatin cross-linking 84
Alginate, gelatin entrapment 34
Poly(dextran/L-arginine)-CaCO3 encapsulation 85
Silk fibroin covalent binding 35, 38
PEG-chitosan and glycol-chitosan conjugation 86
Silk sericin covalent binding 36, 37
Chitosan-TPP nanoparticles encapsulation 87
Fatty acids covalent binding 40
PMMA-S, pHEMA-S, and chemical 88−90
BSA cross-linking 41 P(MAA-co-MMA)-S modification
Phospholipid DPPC adsorption 48 a
Symbols: BSA, bovine serum albumin; DPPC, dipalmitoylphospha-
PEG covalent binding 51
tidylcholine; P(VP-co-AC), poly(N-vinylpyrrolidone-co-acrolein);
Nylon tubing covalent binding 57 P(VP-co-MA), poly(N-vinylpyrrolidone-co-maleic anhydride); PEG,
Polyacrylamide entrapment 58, 59 polyethylene glycol; PEG-g-P(VMP), polyethylene glycol grafted
P(VP-co-AC) covalent binding 60 vinylpyrrolidone−maleic anhydride copolymers; P(GMA-co-EDMA),
P(VP-co-MA) chemical 61 poly(glycidyl methacrylate-co-ethylene dimethacrylate); PLGA: poly-
modification (D,L-lactide-co-glycolide); PHBV, poly(3-hydroxybutyrate-co-3-hydrox-
PEG-g- P(VMP) chemical 62 yvalerate); PANI, polyaniline; PVDMA, poly(2-vinyl-4,4-dimethyla-
modification zlactone); TPP, tripolyphosphate; PMMA, poly(methyl methacrylate);
Epoxy resin covalent binding 63 pHEMA, poly(2-hydroxyethyl methacrylate); P(MAA-co-MMA), poly-
Polyimide entrapment 64 (methacrylic acid-co-methyl methacrylate); S, starch.
Amberlite IR-120 ionic binding 65
P(GMA-co-EDMA) covalent binding 66

The authors observed that immobilized enzyme showed less asparaginase compared to the free L-ASNase. After immobiliza-
immunogenicity in mice and 100-fold longer half-time in rhesus tion the Km values generally decreased and Vmax values
monkey plasma. increased. However, there was no change in Km and Vmax of
Wileman et al.23 prepared soluble dextran/L-ASNase (from the immobilized enzymes. The authors stated that the affinity
Erwinia carotovora) conjugates using covalent binding. The between enzyme and substrate was not affected by chemical
prepared modified L-ASNase maintained 50% of its initial modification. Moreover, the immobilized enzyme was more
activity and exhibited remarkable resistance to proteolysis by stable against trypsin and α-chyrnotrypsin. Therefore, the
trypsin and chymotrypsin and inactivation by L-ASNase- authors suggested that immobilization allowed for a decrease in
specific antibody. In performing in vivo tests, rabbits could its antigenicity and increase in its plasma half-life.
tolerate multiple doses of the L-ASNase conjugate without The L-ASNase from Erwinia carotovora was immobilized to
developing immunity to the enzyme. The work team reported colominic acid as a polysialic acid in the work of Fernandes and
that because the half-time of modified L-ASNase increased and Gregoriadis.27 The attained Km and Vmax values for the
decreased antigen reactivity, it can be used in therapeutic immobilized L-ASNase increased when compared to the free
applications. Similar results were attained by Karsakevich et form. After of incubation 6 h (h) in mouse blood plasma at 37
al.24 for L-ASNase immobilized on biologically active polymer °C, while the free enzyme decreased to 13.5% of its activity, the
dextran sulfate. These results are remarkable in that the immobilized enzyme retained 65−83% of its activity in similar
increased therapeutic properties of L-ASNase and dextran conditions. At this point, polysialic acids such as colominic acid
might be a natural alternative to PEG. might be used as a carrier to enhance its effective use in
Quian et al.25 performed the immobilization of E. coli L- therapeutics of L-ASNase.
ASNase on a chitosan microsphere. The results showed that the Balcao et al.28,29 coimmobilized L-ASNase (from E. coli) and
immobilized enzyme had high enzymatic activity and was more glutamate dehydrogenase onto glyoxyl-agarose and agarose-
stable against trypsin, when compared with the free enzyme. In glutaraldehyde. They deduced that coimmobilization of two
another work of Quian et al.,26 N,O-carboxymethyl chitosan enzymes provided important advantages in comparison to the
was chemically used to L-ASNase for therapeutic application. free form.
As an outcome of immobilization, the enzyme was over 33 Also in 2001, Vina et al.30 bound L-ASNase to biologically
times longer and completely lost its antigenicity toward anti- active fructose polysaccharide levan through covalent binding.
1600 DOI: 10.1021/acs.bioconjchem.7b00217
Bioconjugate Chem. 2017, 28, 1598−1610
Bioconjugate Chemistry Review

Table 2. Obtained Immobilization Results of Immobilized L- over E. coli ASNase for the first remission of ALL.32 Therefore,
ASNasea Kwon et al.33 encapsulated L-ASNase into red blood cells
(RBC) (Figure 4). They hypothesized that RBC overcome
optimum pH kinetic
and parameter thermal storage
carrier matrix temperature Km stability stability refs
inulin 8.0, 50 °C 0.01 mM 65 °C, 30 55 h: 50% 31
m: 66%
silk sericin 6.5−8.0, 60 °C 1.2 mM 60 °C, 30 37 °C, 8 37
m: 30% d: 50%
silk fibroin 6.0−8.0, 60 °C 0.84 mM 60 °C, 30 RT, 30 d: 38
m: 80% 80%
silk sericin 5.0, 50 °C 0.159 mM 60 °C, 30 37 °C, 84 39
m: 90% h: 50%
polyacrylamide 7.0 3.3 mM 37 °C, 15 58
d: 40%
PEG-g-P(VMP) 7.0, 40 °C 60 °C, 30 t0.5:53 h 62
m: 30%
Amberlite IR-120 8.0, 40 °C 0.033 mM 60 °C, 30 65 Figure 4. Schematic for catalytic activity of L-ASNase loading into red
m: 100% blood cells.
silk fibroin 6.0−8.0, 50 °C 2.4195 50 °C, 30 RT, 10 d: 74
mM d: 90% 100%
PANI 8.5, 37 °C 1.809 mM 60 °C, 30 75
toxicity issues related to the enzyme. Because of their abundant
m: supply in blood, erythrocytes would protect L-ASNase from
∼70% inactivation by proteolytic degradation and immune surveil-
activated carbon 7.0, 60 °C 90 °C, 10 79 lance such as destruction by reticuloendothelial system. The
m: 62%
authors also developed a novel method providing RBC
PEG-albumin 37 °C, 50 82
d: 90% incorporation of the L-ASNase using the membrane-trans-
Ca-alginate- 8.5, 50 °C 60 °C, 30 84 locating low molecular weight protamine (LMWP). Thanks to
gelatin m: 66% a novel method, the half-life of RBC/LMWP-L-ASNase was
Chitosan-TPP 9.0, 37 °C 50 °C, 60 37 °C, 6.4 87 prolonged to 4.5 days, whereas RBC-L-ASNase was 2.4 days.
m: 70% d: 50%
The L-ASNase produced by Streptomyces gulbargensis was
PMMA-S 8.5, 45 °C 0.0256 4 °C, 30 88
mM d: 60% immobilized in gelatin and alginate-gelatin fibers by entrap-
pHEMA-S 8.5, 50 °C 0.56 mM 4 °C, 15 89 ment.34 The activity yield obtained with gelatin and alginate-
d: 60% gelatin fibers was 44% and 69%, respectively. Therefore, they
P(MAA-co- 8.5, 35−40 °C 0.0378 RT, 30 d: 90 chosen alginate-gelatin entrapped L-ASNase for further
MMA)-S mM 80% characterizations. The free and immobilized enzyme displayed
the same optimum temperature (40 °C). However, the
Time units: m (minutes), d (days), h (hours). Symbols: Km
(Michaelis−Menten constant), % (residual activity), t0.5: half-life immobilization shifted the optimal pH of the both enzyme.
time, RT: room temperature. Silk fibroin (SF) is a fibrous protein and it has been produced
from Bombyx mori. Although it generally has been used in the
The polysaccharide levan was produced by periodate oxidation textile industry, it has also been used as a biomaterial in human
of levan following reductive alkylation. The covalent binding tissue engineering materials, cell culture matrixes, and carriers
was a result of the direct reaction of dialdehyde groups of for the modification of enzymes or drugs due to its high
oxidized levan with ε-amino groups of lysine and N-terminal mechanical strength, thermal stability, biocompatibility, and
amino groups of L-ASNase. The residual enzyme activity biodegradability, as well as being inexpensive and easy to find.
(≥55%) of L-ASNase/polysaccharide levan conjugates was Because SF has a high concentration of hydrophobic amino
observed at ≤24% oxidation degree. It was found that the acid residues, it is insoluble in water. Silk sericin, silk fibroin,
immobilized L-ASNase demonstrated enhanced biocatalytic and silk fibroin nanoparticles were commonly used for
activity and stability over free enzymes in similar assay immobilization of L-ASNase in the literature. For instance,
conditions. silk fibroin-L-ASNase bioconjugates were reported by Wang
Inulin is a natural polysaccharide produced by many types of and Cao.35 They prepared the modified L-ASNase by direct
plants. To enhance the pharmacokinetic and immunological reaction of the dialdehyde groups of a cross-linker with the ε-
properties of L-ASNase, Tabandeh and Aminlari31 synthesized amino groups of lysine and the N-terminal amino groups of the
oxidized inulin-L-ASNase bioconjugates and examined their enzyme and examined optimum parameters of the modified
physicochemical and immunological properties. Immobilized enzyme. Whereas the relative activity of free L-ASNase
enzyme had good thermal, pH, and trypsin stability. In decreased in pH below 6.0 and above 8.0, modified L-ASNase
addition, the Km value decreased about five times. The results maintained its stability from pH 4.0 to 9.0. Thermal stability of
indicated that the affinity between immobilized enzyme with its both was determined from 30 to 80 °C. According to the
substrate was increased following the immobilization process. obtained results, below 50 °C, both L-ASNases showed ∼90%
The half-time of free and immobilized enzyme was 20 and 55 h relative activity. Yet, free L-ASNase almost lost its activity
in human serum, respectively. Moreover, the oxidized inulin-L- relative to modified L-ASNase at 80 °C; modified L-ASNase
ASNase bioconjugates displayed a decrease in antibody titer demonstrated ∼30% relative activity. Modified L-ASNase was
and immunogenecity after repeated injection in rabbits. more stable than free L-ASNase against proteolysis.
Immunogenicity is a major problem due to the bacterial In the literature, there are many works similar to the above
origin of L-ASNase in medical application. Unfortunately, it was article. Silk sericin powder,36 silk sericin protein,37 silk fibroin,38
reported that pegaspargase has not demonstrated superiority and silk sericin peptides39 were used for L-ASNase immobiliza-
1601 DOI: 10.1021/acs.bioconjchem.7b00217
Bioconjugate Chem. 2017, 28, 1598−1610
Bioconjugate Chemistry Review

tion. In all of them, L-ASNase was improved against denaturing may preserve the catalytic activity after weeks.43,44 The lipid
factors after modification. Langmuir−Blodgett technique has advantages such as accurate
Ashrafi et al.40 synthesized lipid/L-ASNase conjugates in control of thickness, composition, surface elasticity, and
order to cope with activity limitations of free L-ASNase using molecular density; therefore, it has been widely utilized for
short (lauric acid, C12), medium (palmitic acid C16), and long enzyme immobilization on ultrathin films particularly in sensor
(behenic acid, C22) fatty acids. They hoped that lipid/L- applications.45−47 Rocha Junior and Caseli48 revealed that lipid
ASNase conjugates can improve stability, biological half-life, Langmuir and Langmuir−Blodgett ultrathin films have been
and biological activity of the L-ASNase. Covalent binding was used for immobilization of L-ASNase via adsorption. In the
preferred as the immobilization method. The amine group of study, L-ASNase was immobilized on Langmuir monolayers of
lysine in L-ASNase was conjugated with the carboxylic group of the phospholipid dipalmitoylphosphatidylcholine (DPPC)
fatty acids. In this work, they first performed necessary which was chosen due to abundance in several cells. The
optimizations such as lipid:L-ASNase ratio, reaction time, and enzyme immobilized in the ultrathin lipid films preserved more
reaction medium. Then, both free enzyme and the synthesized than 78% of the enzyme activity after 30 days, while
lipid/L-ASNase conjugates were characterized by conjugation homogeneous medium preserved less than 13%. Therefore,
degree, particle size, size distribution, zeta potential, and the lipid films are a suitable carrier matrix to protect the activity
enzyme activity. Free L-ASNase was compared with lipid/L- of L-ASNase and they can also be considered for sensing
ASNase conjugates by pH effect, in vitro half-time (phosphate- asparagine.
buffered saline (abbreviated PBS) and plasma), Km, and trypsin The second type of organic sources used for L-ASNase
digestion. According to the results, the optimum pH range of immobilization is synthetic carriers. The main advantages of
immobilized L-ASNase was larger than that of the free enzyme. synthetic carriers are their physical properties, nature of reactive
The optimum pH range of free enzyme was 7.5−8.5, while that groups, and micro and macro-environment.49 Nevertheless,
of modified enzyme was 6.5−9.0. The active site of modified L- they are nonrenewable, nondegradable, and cause serious
ASNase was protected against high/low values of pH and complications in the body when they were used. Therefore,
improved pH stability via chemical modification. The reduction biocompatibility synthetic carriers were preferred for enzymes,
in the Km value was an indicative increasing affinity for the which are used as drugs in the body. Because it is a nonionic
substrate of the modified L-ASNase. The Km value of all polymer with high aqueous solubility and biocompatibility, the
modified L-ASnase was lower free enzyme, but behenic acid/L- most important synthetic carrier is PEG for L-ASNase.50 PEG-
ASNase had the lowest Km values. The half-life of free enzyme L-ASNase (pegaspargase) is a commercial bioconjugated L-
in PBS and plasma was determined as 19 and 21 h, respectively. ASNase form that has been used in therapy since 1994.31 There
Lipid bioconjugates improved in vitro half-time of L-ASNase in are many studies about this carrier. For instance, in the work of
both PBS and plasma. For instance, the half-time of modified Soares et al.,51 they aimed to investigate PEG-L-ASNase
enzyme was increased to 46 h in plasma when used palmitic stability and activity changes in different pH and temperature.
acid/L-ASNase. Besides, the stability of modified L-ASNase was Similar workers have reported increased stability of the
investigated against trypsin digestion. The resistance of the immobilized enzyme with PEG-modification.52−56 The work
modified L-ASNase to trypsin digestion was distinctively demonstrated that the activity of immobilized L-ASNase was
greater than that of the free enzyme. Even though free enzyme remarkably improved as compared to the free form. Since the
maintained 13.8% of its initial activity after 30 min, the effect of PEGylation is a proven process, we did not need to
modified enzyme (Behenic acid/L-ASNase) maintained ∼80% give a description of this information. We focused on synthetic
of its initial activity. carriers aside from PEG for L-ASNase.
The L-asparagine biosensor having high sensitivity, low cost, In order to compare the immobilization parameters and
along with good stability and selectivity can considerably kinetic properties of the free and immobilized L-ASNase, it was
contribute to applications toward food and clinical chemistry. immobilized on nylon tubing via covalent binding by Allison et
In a recent study, a novel biosensor was designed using a new al.57 Following immobilization, the optimum pH of the enzyme
form of L-ASNase from a strain of Erwinia carotovora (EcaL- did not shift, but the range of optimal activity was limited.
ASNase) and was used for the determination of L-asparagine in Similarly, the immobilization led to a great increase in thermal
biological samples (potato and human serum). The enzyme stability of the enzyme. In addition, a decrease in Km was
was immobilized by cross-linking with glutaraldehyde and monitored as compared to the free enzyme.
bovine serum albumin (BSA). While the free enzyme totally Mori et al.58 prepared L-ASNase entrapped in the lattice of
lost its activity after 8 days of incubation at 4 °C, the polyacrylamide gel. The immobilized L-ASNase showed the
immobilized enzyme maintained at about 50% of its intrinsic highest catalytic activity when L-glutamine (23.7 μmol/min)
activity after about 30 days under the same conditions. The and L-asparagine (22.6 μmol/min) were used. The maximum
biosensor showed high sensitivity for the detection of L- activity of free L-ASNase was at pH 8.0, whereas immobilized
asparagine with a linear range of 10−200 μM and a detection L-ASNase exhibited maximum activity at pH 7.0. Generally, the
limit of 10 μM. Moreover, the method’s reproducibility and L- Km value decreases after immobilization. However, the Km of
asparagine mean recovery were 3−6% and 101.5%, respectively. immobilized L-ASNase increased 200 times in this work. The
The designed microplate biosensor provided a novel approach authors asserted that a semipermeable polyacrylamide gel
for specific, convenient, cost-effective, and rapid measurement lattice prevented the rate of permeabilization of the substrate.
and analysis of L-asparagine.41 The immobilized L-ASNase retained 40% of its original activity
Enzyme immobilization in Langmuir and Langmuir− along 15 days in storage conditions (pH 7.0, 37 °C) and
Blodgett films of lipids is a convenient strategy to control showed more resistance than free L-ASNase against proteolytic
catalytic activity at the molecular level.42 Since the conservation enzymes. Moreover, in order to examine the effectiveness of the
of activity relies on the molecular-level interactions, the entrapped L-ASNase in the blood, a work team examined
molecular environment provided by the lipid−enzyme system asparagine, glutamine, and ammonia concentration levels in the
1602 DOI: 10.1021/acs.bioconjchem.7b00217
Bioconjugate Chem. 2017, 28, 1598−1610
Bioconjugate Chemistry Review

fresh blood. They reported that both substrates were scavenged
by the L-ASNase entrapped in polyacrylamide gel. Galaev et
al.59 also immobilized L-ASNase on polyacrylamide gel. As
compared to the free form, immobilized L-ASNase displayed
more stability to pH, denaturation, and proteolytic enzyme, a
200-fold increase in Km. Furthermore, immobilized L-ASNase
hydrolyzed both L- and D-substrate isomers, but the free L-
ASNase was highly stereospecific.
Karsakevich et al.60 prepared vinylpyrrolidone and acrolein
copolymers for E. coli L-ASNase immobilization. Owing to the
interactions between enzyme and the carrier, the immobilized
L-ASNase exhibited pH and thermal stability. A rather similar
study was reported by Qian and co-workers61 that E. coli L-
ASNase was immobilized on poly(N-vinylpyrrolidone-co-maleic Figure 5. Schematic diagram of L-ASNase/polyimide/Pt electrode.
anhydride) (P(VP-co-MA)). After immobilization, L-ASNase
showed more stability at various pH, temperature, and storage
stability when compared to the intrinsic enzyme. The optimum El-Refai et al.65 reported a successful immobilization and
pH of immobilized enzyme shifted ∼0.8 pH unit more than improved stabilization of L-ASNase onto Amberlite IR-120 by
that of free enzyme due to the polyanionic microenvironment ionic binding. Immobilization did not change optimum pH
surrounding the enzyme. The optimum temperature of the (8.0) and temperature (40 °C) compared to free enzyme, but it
immobilized L-ASNase was ∼10 °C higher than that of the free showed enhanced thermostability as a result of immobilization.
form. In addition, the immobilized L-ASNase was markedly The immobilized enzyme retained 100% activity when it was
resistant to trypsin digestion; free L-ASNase almost lost all of exposed at 60 °C and it also retained 70% of its relative activity
its initial activity. at pH 9.0 for 50 min. Km was 0.0259 mM and 0.033 mM for
A similar work was reported for immobilized L-ASNase on free and immobilized L-ASNase, respectively. Vmax for the
polyethylene glycol grafted vinylpyrrolidone−maleic anhydride soluble and the immobilized enzyme was 757.6 U/mg protein
(PEG-g-P(VMP)) copolymer by Qian et al.62 The goal of the and 581 U/mg protein, respectively.
study was to decrease the antigenicity and to improve the half- Qiao et al. prepared monolithic reactors for L-ASNase using
time of the L-ASNase in serum. First, PEG-g-P(VMP) poly(glycidyl methacrylate-co-ethylene dimethacrylate) (P-
copolymer was synthesized via radical copolymerization. (GMA-co-EDMA)) materials.66,67 Afterward, L-ASNase was
Then, E. coli L-ASNase was chemically modified with these covalently immobilized to the polymeric monolith and coating
copolymers. To investigate the immobilization parameters, in the capillary. To determine these enzymatic microreactors,
PEG-g-P(VMP)-4 modified L-ASNase was selected as carrier kinetic constants were calculated. The kinetics data were Km =
because it gave the best results. Immobilized enzyme was more 3.8 μM, Vmax = 106.2 μM min−1 for monolith and Km = 7.7 μM,
stable than free enzyme, especially in alkaline medium. Vmax = 157.4 μM min−1 for coating enzymatic reactors,
Nonetheless, immobilized enzyme responded more sensitively respectively. Furthermore, the amount of L-aspartate in the
than free enzyme to temperature. For that reason, the writers reactor outlet was determined periodically using the microchip
stated that the thermal motion of polymer chains in the electrophoresis−laser-induced fluorescence method. After 21
immobilized enzyme would affect the conformation of the days, the decrease in L-aspartate values was not detected, but a
enzyme much more vigorously at higher temperature. The half decrease was observed at 28 days.
immobilized enzyme also showed an enhancement to trypsin In addition to the previously mentioned materials, some new
digestion. Additionally, in vivo half-time of free and immobilized types of organic nanomaterials (such as nanoparticle, nanofiber,
enzyme was 1.2 and 53 h in plasma of rabbits, respectively. nanocomposite, and nanosphere) have been attractive due to
The L-ASNase was covalently immobilized to an epoxy resin tunable pore size, high surface area, and effective enzyme
by Almendral et al.63 The study was based on spectrophoto- loading. Therefore, they have shown enormous promise in the
metric determination of L-asparagine by flow-injection analysis. preparation of immobilized enzymes in recent years. In the
The reactor was used for more than 5 months and retained literature, many organic-based nanocarriers were reported for
∼88% of its origin activity after 700 determinations. L-ASNase immobilization.
Consequently, the authors stated that this enzymatic For example, L-ASNase was immobilized on poly(lactide-co-
immobilization procedure was more simple, stable, economical, glycolide) (PLGA) nanospheres and was determined to
and operational than other methods. influence polymer properties such as enzyme loading, activity,
One of the first attempts at developing L-ASNase-based and in vitro release by Gaspar and co-workers.68 Nanospheres
biosensors was made by Erdogan et al.64 They developed were fabricated by water-in-oil-in-water solvent evaporation.
polyimide based amperometric biosensor for determination of Then L-ASNase was immobilized by encapsulation and its
L-asparagine in serum samples. Polyimides are attractive binding confirmed by transmission electron microscopy
because of their excellent thermal, chemical, electrical, and (TEM). In another similar work,69 PLGA nanospheres were
mechanical properties. So, a polyimide membrane was exploited for the immobilization of L-ASNase via encapsulation.
evaluated as carrier for L-asparagine biosensor. The schematic Also, Vasudev et al.70 mainly showed the effect of PEGylation
diagram of this process was illustrated in Figure 5. Experimental on enzyme loading, activity, and in vitro release. So, they
data demonstrated that proposed biosensors showed a wide formulated PEGylated L-ASNase loaded PLGA nanoparticles
linear detection range (15−113 μM), acceptable reproduci- and compared them to the unmodified counterpart. It was
bility, high sensitivity, long-term stability, and low detection found that PEGylation protected the enzyme against
limit (1 × 10−6 M). denaturation during encapsulation.
1603 DOI: 10.1021/acs.bioconjchem.7b00217
Bioconjugate Chem. 2017, 28, 1598−1610
Bioconjugate Chemistry Review

In another survey, Baran et al.71 investigated the in vivo half-
time of immobilized L-ASNase into heparin coated poly(3-
hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanocapsules.
The aim of their study was increased in vivo half-time of L-
ASNase and decreased immunogenicity as well as toxicity.
Based on the enzyme activity results in blood, in vivo half-time
of encapsulated L-ASNase was ∼6.5 h, while that of the
unmodified L-ASNase was between 3 and 4 h. In addition, the
authors reported that encapsulated L-ASNase demonstrated no
adverse effects and anaphylaxis symptoms after injection to
Figure 6. Schematic representation of immobilizing L-ASNase enzyme
mice whereas unmodified L-ASNase led to hypersensitive on polyaniline nanofiber.
Teodor et al.72 prepared hydrogel magnetic nanoparticles
using magnetic nanoparticles and successive layers of chitosan environmental factors. The base of pH results, optimum pH
and hyaluronic acid. They then immobilized L-ASNase in the was 8.5 for both free and immobilized L-ASNase. Yet, free L-
hydrogel magnetic nanoparticles by entrapment and they ASNase exhibited ∼30% activity, while immobilized L-ASNase
unfolded swelling behavior, degradation behavior, and the exhibited approximately 70% activity. In addition, immobilized
release of L-ASNase from 7.0 to pH 4.5. Delivery of L-ASNase enzyme was more stable than the free one for all pH values
was determined using protein and L-ASNase activity assay. The (from 7.5 to 9.5). The optimum temperature was found to be
release of L-ASNase is greatly long-lasting compare with free 37 °C for both forms. Furthermore, Km value of immobilized L-
form. The residual activity of the immobilized L-ASNase ASNase was significantly smaller than that of the free L-
remained the same after 6 months from immobilization during ASNase, after immobilization.75
storage at 4 °C. Consequently, prepared biocompatible Several workers designed an enzyme reactor for L-ASNase.
nanocarrier could be utilized for immobilization of L-ASNase. They prepared magnetic nanoparticles functionalized by
The same research group also reported a biocompatible poly(2-vinyl-4,4-dimethylazlactone) (PVDMA) using a rever-
polymer for biologic active agents such as L-ASNase in another sible addition−fragmentation chain transfer polymerization
study.73 technique.76 L-ASNase was immobilized onto a fabricated
Zhang et al.74 preferred silk fibroin nanoparticle (SFN) as enzyme reactor (Figure 7). The prepared L-ASNase reactor
natural carrier for L-ASNase immobilization. SFN/L-ASNase showed high loading capacity of 318.0 μg mg−1 magnetic
bioconjugates were briefly obtained as follows. The liquid silk nanoparticles. Immobilized enzyme retained 95.7% of its
was mixed with L-ASNase and was introduced rapidly into original activity after its 10th repeated use and more than
excess acetone. The obtained SFN/L-ASNase bioconjugates 72.6% activity after 10 weeks of storage. The work indicated
were easily prepared by isolating from acetone solution by that the stability and activity of the enzyme could be enhanced
centrifugation. Afterwards, modified enzyme was used to though immobilization to the magnetic nanoparticles and the
comprehensively determine routine immobilization parameters designed carrier could be used in several biological and clinical
(pH, thermal stability, kinetic and storage stability, etc.) of approaches.
modified L-ASNase and was compared to free enzyme. The L-ASNase Immobilization on Inorganic Carriers. Even
optimum pH was similar for the both and was between 6.0 and though inorganic carriers are inexpensive and easily accessible,
8.0. Although the optimum pH did not change, the pH stability they have not been used for L-ASNase immobilization. Because
of modified L-ASNase improved over that of free L-ASNase. biocompatible carriers have been preferred for L-ASNase
The same was also true for optimum temperature. The immobilization, there are not many articles about this topic
optimum temperature was 50 °C for free and modified enzyme. in the literature. Typically, these carriers were used for other
When L-ASNase was entrapped in silk fibroin, the affinity applications outside of medicine.
between L-ASNase and L-asparagine increased because the Silica gel, activated carbon, Celite, and tricalcium phosphate
active site of L-ASNase was not affected. Therefore, the Km were used as support for immobilization of L-ASNase by
value of modified L-ASNase was lower by 2.5-fold than the free Sundaramoorthi et al.77 and El-Sayed et al.78 Sundaramoorthi
one. While free L-ASNase showed 60% relative enzyme activity and co-workers produced L-ASNase from fungal isolates which
for 10 d at 4 °C, SFN/L-ASNase retained 100% of its original were isolated from soil samples collected from different regions
activity for 10 d at room temperature. Also, the immobilized of the Arabian Sea. They immobilized L-ASNase on different
enzyme is more resistant to heat and to digestion by trypsin. carriers (i.e., silica gel, agarose, activated carbon, Celite,
Polyaniline nanofiber (PANI) is another nanocarrier for the carboxymethyl cellulose, egg shell, tricalcium phosphate) via
immobilization of L-ASNase by covalent binding. Polyaniline covalent binding and obtained the highest immobilization yield
has great importance in energy storage devices, sensors, and with silica gel carrier. On the other hand, El-sayed et al.
biological materials due to biocompatibility, high resistance to assessed immobilization, properties, and antitumor activity of L-
environmental factors, high yield, and easy preparation. First, ASNase of bean (Vicia faba). They used simple adsorption as
PANI was synthesized and characterized. L-ASNase was then the L-ASNase immobilization method and the best carrier for
covalently immobilized with polyaniline nanofibers (Figure 6). L-ASNase was selected as silica gel with a specific activity of
To evaluate the presence of enzyme binding, they used FTIR, 2.75 U/mg protein. The immobilized enzyme retained activity
SEM, TEM, X-ray photoelectron spectroscopy (XPS), and in the alkaline pH range compared to the free enzyme, which
XRD. All results indicated the electronic interaction of L- reported that the stability of the immobilized L-ASNase was
ASNase and PANI. Also, the presence of L-ASNase was verified higher than that of the free L-ASNase at the alkaline pH range.
in terms of catalytic activity. Immobilized enzyme showed more The immobilized enzyme was much more stable than its free
activity than the free enzyme and was less affected by counterpart at higher temperature. Besides, the cytotoxicity
1604 DOI: 10.1021/acs.bioconjchem.7b00217
Bioconjugate Chem. 2017, 28, 1598−1610
Bioconjugate Chemistry Review

Figure 7. Schematic illustration of L-ASNase immobilization onto PVDMA-modified magnetic nanoparticles. (Reprinted with permission from Mu
et al., 2014.76)

effect in vitro of bean L-ASNase on the growth of hepatocellular benefit from using it as a food additive in, e.g., bread, white and
tumor cell lines (Hep-G2) was studied using MTT assay. L- whole wheat flour, and baked goods.
ASNase caused a gradual inhibition in the cell growth and L-ASNase Immobilization on Hybrid Carriers. In this
calculated IC50 value was 217.71 μg/mL. review, hybrid carriers were described as materials derived from
Moharam and co-workers79 studied antitumor and antiox- both synthetic and natural structures. Hybrid carriers have
idant activities as well as immobilization parameters (pH, useful properties. Because the carriers contained natural
thermal, and storage stability) of immobilized L-ASNase in structures, they are both biocompatible and biodegradable.
activated carbon. In this study, L-ASNase was isolated from Moreover, they exhibited advantages such as functional groups,
Bacillus sp. R36 and was efficiently immobilized by covalent various physical properties, and desired modification of
binding on activated carbon with immobilization yield of 73.6%. synthetic structures. The new material having superior
Free and immobilized L-ASNase exhibited the same optimum properties was produced by the combination of these two
pH at pH 7.0, while the optimum temperature was increased materials. Hybrid carriers have been increasingly used in recent
from 50 to 60 °C for the immobilized enzyme. Furthermore, times for enzyme immobilization. Such works are present
immobilized enzyme retained 100% of its activity up to 80 °C. relating to L-ASNase immobilization.
In addition to other work, this study also investigated IC50 value Francois and Fortier82 developed an approach to enhance the
and antioxidant properties (scavenging of DPPH) of L-ASNase. stability of L-ASNase. They prepared a hydrogel matrix made of
The results showed that the enzyme possessed low scavenging PEG-albumin and investigated the feasibility of the immobiliza-
activity with high SC50 (half maximal scavenging concentration) tion of E. coli L-ASNase. In this study, albumin was used to
values of 325.4 μg/mL compared to the scavenging activity of improve biocompatibility of PEG. Thus, this matrix can be very
ascorbic acid. The IC50 values of the enzyme were calculated as useful for L-ASNase due to its good biocompatibility and it will
112.19 μg/mL and 218.7 μg/mL for Hep-G2 and colon also provide a functional bioreactor for use in vivo. The authors
carcinoma (HCT-116) cell line, respectively. observed that the immobilized L-ASNase had optimal activity
In the same study, L-ASNase from Bacillus licheniformis compared to free enzyme at different pH. After 50 days of
STRAIN HSA3−1a was immobilized by activated glutaralde- storage at 37 °C, the immobilized L-ASNase maintained its
hyde-carbon carrier using covalent coupling.80 They evaluated initial activity (more than 90%). The free enzyme had 43%
various characteristics of immobilized enzyme such as optimum activity, while immobilized enzyme retained 90% of its activity.
temperature, pH, and reusability compared with the free Also, Francois and co-workers83 evaluated the performance of
enzyme. The authors observed that the optimum pH changed biocompatible PEG-albumin hydrogel carriers in rats. Immo-
after immobilization. They reported the capacity of enzyme bilized L-ASNase on the carrier showed great in vivo stability
protein and its matrix carrier as the reason for this change. after 4 months of implantation.
Besides, the immobilized L-ASNase retained its initial activity The efficiency of immobilized L-ASNase on calcium
by 84.79% after 2 times repeated use. alginate−gelatin composite was studied by Youssef and Al-
We considered that L-ASNase has applications in the food Omair.84 They reported that the L-ASNase II gene was cloned
industry as well as medicine. Immobilization techniques have from E. coli W3110, was purified utilizing several chromatog-
also been used for these studies. For instance, Muslim et al.81 raphy technique, and was purified with SDS-PAGE. Finally, the
investigated enhancement of the activity and stability of L- purified enzyme was immobilized on calcium alginate−gelatin
ASNase. They isolated L-ASNase from Acinetobacter baumannii composite. According to the results obtained, immobilized and
and purified it by chromatography. L-ASNase was then free L-ASNase showed optimal activity at pH 8.5 and 7.5 and
immobilized on silica gel (adsorption), and Sephadex G-50 optimal temperature at 50 and 37 °C, respectively. The
(entrapment) was used to retain the activity and increase the immobilized L-ASNase completely maintained its origin activity
stability of the enzyme. The authors stated that adsorption was for 30 min storage at 60 °C.
more efficient than entrapment for immobilization of L- Glutaminase-free Saccharomyces cerevisiae cytoplasmic L-
ASNase. They point out that the immobilized L-ASNase was ASNase I was encapsulated in biocompatible multilayered
found to be stable during continuous operation for one month polyelectrolyte microcapsules by Karamitros et al.85 They used
and to be resistant to attack by proteolytic enzymes, which led calcium carbonate (CaCO3) particles as carrier matrix for
to enhanced activity and stability of L-ASNase and increased enzyme immobilization. Afterward, the matrix was coated with
1605 DOI: 10.1021/acs.bioconjchem.7b00217
Bioconjugate Chem. 2017, 28, 1598−1610
Bioconjugate Chemistry Review

cationic (poly(L-arginine)) and anionic (dextran sulfate) layers. temperatures. Moreover, in vitro half-time of free L-ASNase was
Then CaCO3 was removed by treating it with ethylenediamine- about 33 h when it was stored in PBS, while immobilized L-
tetraacetic acid (EDTA) disodium salt, and polyelectrolyte ASNase was about 6.4 days in similar storage conditions. The
capsules were prepared (Figure 8). A slight increase was obtained results revealed that the stability and activity of the L-
ASNase were considerably improved against pH, thermal
stability, and storage stability in comparison to that of the free
In the literature, some materials may be used as carrier for L-
ASNase immobilization following functionalization or derivati-
zation. Recently, novel techniques have been studied for L-
Figure 8. Schematic illustration of the preparation of L-ASNase I-filled ASNase immobilization. For instance, Ulu and co-workers88−90
polyelectrolyte capsules. (Reprinted with permission from Karamitros recently used poly(methyl methacrylate) (PMMA)-starch
et al., 2013.86)
(Figure 9), poly(2-hydroxyethyl methacrylate) (pHEMA)-
starch, and poly(methacrylic acid-co-methyl methacrylate)
observed in thermal stability after immobilization. On the other (P(MAA-co-MMA)-starch (Figure 10) composites to immobi-
hand, although free enzyme almost lost all of its initial activity, lize L-ASNase. First, they prepared both polymers and
the immobilized enzyme lost ∼50% of its activity during storage functionalized them using starch. The prepared composites
for 3 months at 4 °C. were characterized structurally, thermally, and morphologically
The L-ASNase from Erwinia carotovora was conjugated with by various techniques. Then, an enzyme solution was then
PEG-chitosan and glycol-chitosan by Sukhoverkov and directly mixed with functionalized carriers for immobilization.
Kudryashova.86 They aimed to upgrade the therapeutic effect Following the immobilization procedure, the immobilized
of L-ASNase and evaluated some parameters. The optimum pH enzyme exhibited both pH stability and thermostability relative
of immobilized L-ASNase was displaced toward more acidic to the non-immobilized enzyme. The immobilized enzyme had
values because of the polycationic nature of the copolymer, and a lower Km value than the former form. Also, the authors noted
the thermostability of the immobilized enzyme was higher than that immobilization of L-ASNase using this technique resulted
that of the free form. Moreover, it was observed that glycol- in excellent storage stability and enabled the reuse of the
enzyme relative to the non-immobilized enzyme.

chitosan conjugates demonstrated the best results than present
There are many candidate biocompatible synthetic materials FINAL REMARKS
(mostly polymers) as carriers for L-ASNase immobilization. Herein, we review the use of various materials as carrier matrix
However, they do not have chemical groups to interact with the for immobilizing L-ASNase, pointing out the obtained results in
enzyme’s active site. Thus, these carriers must be functionalized different immobilization techniques. Based on reported results,
with a material containing a functional group to achieve enzyme we can interpret advantages and drawbacks of the previously
immobilization. The natural polymers containing hydroxyl or mentioned carrier matrix. An ideal carrier matrix should be
amino group such as cellulose, chitin, chitosan, and starch were biocompatible, easily accessible, cheap, stable, and suitable for
generally used functionalized. Hereby, a new chemical function regeneration.91 Natural carriers can be found widely in
is introduced onto the carrier. Generally, while synthetic renewable sources and they are biocompatible, biodegradable,
polymers were used as the main matrix, natural structures were and inexpensive supports. This is the underlying reason for the
preferred as reinforcement. most commonly used materials in enzyme immobilization
Bahreini et al.87 reported production, purification, and systems. With respect to synthetic carriers, they are suitable for
immobilization of L-ASNase by nanoencapsulation in chito- immobilization because of their many functional groups. Their
san-tripolyphosphate (TPP) nanoparticles produced by the further advantage is the possibility to regulate their structure at
ionotropic gelation method. The goal of this study was the macromolecular level. Furthermore, shape, form, porosity,
increased in vitro efficiency of L-ASNase-entrapped chitosan pore diameter, polarity, and hydrophobicity of the carriers may
nanoparticles cross-linked with TPP. They examined immobi- be controlled during synthesis. However, the synthetic
lization parameters of free and immobilized L-ASNase such as polymers are made from nonrenewable petroleum resources;
optimum pH, thermal stability, release, and in vitro half-time. this has been criticized due to the fossil fuel depletion
The optimum pH for both forms was between 8.5 and 9.0. Yet, problem.91,92 On the other hand, inorganic carriers have a
immobilized enzyme was more stable at alkaline pH and at high high chemical, physical, and biological resistance. Nevertheless,

Figure 9. Schematic image of preparation and effect mechanism of PMMA-Starch-L-ASNase. (Reprinted with permission from Ulu et al., 2016.88)

1606 DOI: 10.1021/acs.bioconjchem.7b00217
Bioconjugate Chem. 2017, 28, 1598−1610
Bioconjugate Chemistry Review

Figure 10. Schematic diagrams of preparation of P(MAA:7-co-MMA:3) copolymer (A), P(MAA:7-co-MMA:3)-starch composite and L-ASNase
immobilization (B). (Reprinted with permission from Ulu et al., 2016.90)

these carriers include the following drawback: they have ultimate aim of this review. In conclusion, we tried to list and
sufficient functional groups, which limits bonding of the explain almost all carriers for L-ASNase. This review pointed
enzyme.91 Compared with other carrier matrixes, since out promising carriers for L-ASNase immobilization. In this
nanocarriers have larger surface areas and high porosity, they way, this review also described how to produce an improve-
can provide relatively high enzyme loading and facilitate the ment in enzyme activity, stability, and reusability. Perhaps,
accessibility of active sites and substrate. Therefore, the enzyme future research can contribute to development of more suitable
displays high catalytic efficiency. For that reason, we think that formulations than the present L-ASNase formulations. We
hybrid carriers are more worthwhile because they combining anticipate that more novel carriers and applications will be
properties of natural and synthetic carriers. Recently, ongoing reported in the near future, including medicine, food, and
research in advanced delivery of protein-based drug formula- biosensor.
tions focused on micro/nanoencapsulation methods and
stimuli-sensitive or smart molecular designs such as liposomes,
micelles, carbon nanotubes, dendrimers, and magnetic nano-
Corresponding Author
particles and pH, thermal, or dual response hydrogels. These *E-mail: Phone: 90-422 377 3888.
novel techniques and smart materials can benefit from L- Fax: 90-422 341 0037.
ASNase immobilization. For instance, in a recent work, L- ORCID
ASNase was immobilized onto Ca-alginate beads via micro- Burhan Ates: 0000-0001-6080-229X
encapsulation by Bahraman and Alemzadeh.93 In addition, we
hope that nanoencapsulation of L-ASNase in nanocarriers can
The authors declare no competing financial interest.

be very promising approach for future applications. On the
other hand, the red blood cell (RBC)-encapsulated L-ASNase ACKNOWLEDGMENTS
has been used as a technique in bioreactors. Artificial biomimic
The author would like to thank Inönü University.

materials have been made in recent years to replace natural
biomolecules. We highlight that these materials will be very ABBREVIATIONS
important in applications of L-ASNase by using micro/
nanoencapsulation. These and similar innovative ideas can L-ASNase, L-asparaginase; ALL, acute lymphoblastic leukemia;
further advance the use of L-ASNase. E. coli, Escherichia coli; PEG, polyethylene glycol; FDA, Food

and Drug Administration; PEG-L-ASNase, polyethylene glycol-
L-asparaginase; RBC, red blood cells; LMWP, low molecular
CONCLUSIONS weight protamine; SF, silk fibroin; PBS, phosphate-buffered
Immobilized enzymes have been increasingly used in a variety saline; BSA, bovine serum albumin; P(VP-co-MA), poly(N-
of fields such as medicine, industry, and food. L-ASNase is one vinylpyrrolidone-co-maleic anhydride); PEG-g-P(VMP), poly-
of the most important agents used as a drug−enzyme conjugate ethylene glycol grafted vinylpyrrolidone−maleic anhydride;
in the treatment of cancer. Hence, studies to increase the half- TEM, transmission electron microscopy; PLGA, poly(lactide-
life and stability of this enzyme are important as innovative co-glycolide); PHBV, poly(3-hydroxybutyrate-co-3-hydroxyval-
carriers and immobilization methods must be developed to erate); SFN, silk fibroin nanoparticle; FTIR, Fourier transform
achieve this phenomenon. At this point, all information and infrared spectroscopy; SEM, scanning electron microscopy;
experience from past to present are required. That is the SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electro-
1607 DOI: 10.1021/acs.bioconjchem.7b00217
Bioconjugate Chem. 2017, 28, 1598−1610
Bioconjugate Chemistry Review

phoresis; XRD, X-ray diffraction; PANI, polyaniline nanofiber; (18) Bezerra, C. S., de Farias Lemos, C. M. G., de Sousa, M., and
XPS, X-ray photoelectron spectroscopy; TPP, tripolyphos- Gonçalves, L. R. B. (2015) Enzyme immobilization onto renewable
phate; PVDMA, poly(2-vinyl-4,4-dimethylazlactone); TGA, polymeric matrixes: Past, present, and future trends. J. Appl. Polym. Sci.
thermogravimetric analysis; EDTA, ethylenediamine-tetraacetic 132, n/a.
acid; PMMA, poly(methyl methacrylate); pHEMA, poly(2- (19) Datta, S., Christena, L. R., and Rajaram, Y. R. S. (2013) 9.
Enzyme immobilization: an overview on techniques and support
hydroxyethyl methacrylate); (P(MAA-co-MMA), poly-
materials. 3 Biotech 3, 1−9.
(methacrylic acid-co-methyl methacrylate); GMA, glycidyl (20) Guzik, U., Hupert-Kocurek, K., and Wojcieszyńska, D. (2014)
methacrylate; EDMA, ethylene dimethacrylate; DPPC, dipal- Immobilization as a strategy for improving enzyme properties-

application to oxidoreductases. Molecules 19, 8995−9018.
(21) Poznansky, M. J., Shandling, M., Salkie, M. A., Elliott, J., and
REFERENCES Lau, E. (1982) Advantages in the use of L-asparaginase albumin
(1) Nadumane, V. K., Venkatachalam, P., and Gajaraj, B. (2016) polymer as an antitumor agent. Cancer Res. 42, 1020−1025.
Aspergillus applications in cancer research. In New and Future (22) Uren, J. R., Hargis, B. J., and Beardsley, P. (1982)
Developments in Microbial Biotechnology and Bioengineering, Aspergillus Immunological and pharmacological characterization of poly-DL-
System Properties and Applications (Gupta, V. K., Ed.) pp 235−247, alanyl modified Erwinia carofoyora L-Asparaginase. Cancer Res. 42,
Elsevier, New York. 4068−4071.
(2) Cachumba, J. J. M., Antunes, F. A. F, Dias Peres, G. F., Brumano, (23) Wileman, T. E., Foster, R. L., and Elliott, P. N. C. (1986)
L. P., Dos Santos, J. C., and Da Silva, S. S. (2016) Current applications Soluble asparaginase-dextran conjugates show increased circulatory
and different approaches for microbial L-asparaginase production. persistence and lowered antigen reactivity. J. Pharm. Pharmacol. 38,
Braz. J. Microbiol. 47, 77−85. 264−271.
(3) Verma, N., Kumar, K., Kaur, G., and Anand, S. (2007) L- (24) Karsakevich, A. S., Dauvarte, A. Z., Zvirgzda, I. K., Lebedeva, L.
Asparaginase: A promising chemotherapeutic agent. Crit. Rev. V., and Vina, I. A. (1986) Effective complexes of the antileukemic
Biotechnol. 27, 45−62. enzyme L-asparaginase with dextran sulfate. Vopr. Med. Khim. 32, 47−
(4) Kumar, N. S. M., Shimray, C. A., Indrani, D., and Manonmani, H. 51.
K. (2014) Reduction of acrylamide formation in sweet bread with L- (25) Qian, G., Zhou, J., Ma, J., Wang, D., and He, B. (1996)
Asparaginase treatment. Food Bioprocess Technol. 7, 741−748. Immobilization of E-coli L-asparaginase on chitosan microsphere.
(5) Xu, F., Oruna-Concha, M. J., and Elmore, J. S. (2016) The use of Chem. J. Chinese U 17, 1147−1150.
asparaginase to reduce acrylamide levels in cooked food. Food Chem. (26) Qian, G., Zhou, J., Ma, J., He, B., and Wang, D. (1997)
210, 163−171. Chemical modification of L-asparaginase with N, O carboxymethyl
(6) Batool, T., Makky, E. A., Jalal, M., and Yusoff, M. M. (2016) A chitosan and its effects on plasma half-life and other properties. Sci.
comprehensive review on L-asparaginase and its applications. Appl. China, Ser. B: Chem. 40, 337−341.
Biochem. Biotechnol. 178, 900−923. (27) Fernandes, A. I., and Gregoriadis, G. (1997) Polysialylated
(7) Kumar, K., Kataria, M., and Verma, N. (2013) Plant asparaginase- asparaginase: preparation, activity and pharmacokinetics. Biochim.
based asparagine biosensor for leukemia. Artif. Cells, Nanomed., Biophys. Acta, Protein Struct. Mol. Enzymol. 1341, 26−34.
Biotechnol. 41, 184−188. (28) Balcao, V. M., Mateo, C., Fernandez-Lafuente, R., Malcata, F. X.,
(8) Ali, U., Naveed, M., Ullah, A., Ali, K., Shah, S. A., Fahad, S., and and Guisan, J. M. (2001) Coimmobilization of L-asparaginase and
Mumtaz, A. S. (2016) L-asparaginase as a critical component to glutamate dehydrogenase onto highly activated supports. Enzyme
combat Acute Lymphoblastic Leukaemia (ALL): A novel approach to Microb. Technol. 28, 696−704.
target ALL. Eur. J. Pharmacol. 771, 199−210. (29) Balcao, V. M., Mateo, C., Fernandez-Lafuente, R., Malcata, F. X.,
(9) Jackson, J. A., Halvorson, H. R., Furlong, J. W., Lucast, K. D., and and Guisan, J. M. (2001) Structural and functional stabilization of L-
Shore, J. D. (1979) A new extracorporeal reactor-dialyzer for enzyme asparaginase via multi-subunit immobilization onto highly activated
therapy using immobilized L-asparaginase. J. Pharmacol. Exp. Ther. supports. Biotechnol. Prog. 17, 537−542.
209, 271−274. (30) Vina, I., Karsakevich, A., and Bekers, M. (2001) Stabilization of
(10) DiCosimo, R., McAuliffe, J., Poulose, A. J., and Bohlmann, G.
anti-leukemic enzyme L-asparaginase by immobilization on poly-
(2013) Industrial use of immobilized enzymes. Chem. Soc. Rev. 42,
saccharide levan. J. Mol. Catal. B: Enzym. 11, 551−558.
(31) Tabandeh, M. R., and Aminlari, M. (2009) Synthesis,
(11) Meryam Sardar, R. A. (2015) Enzyme immobilization: an
physicochemical and immunological properties of oxidized inulin−L-
overview on nanoparticles as immobilization matrix. Biochem. Anal.
Biochem. 4, 178. asparaginase bioconjugate. J. Biotechnol. 141, 189−195.
(12) Popat, K. C., Mor, G., Grimes, C., and Desai, T. A. (2004) Poly (32) Graham, M. (2003) Pegaspargase: a review of clinical studies.
(ethylene glycol) grafted nanoporous alumina membranes. J. Membr. Adv. Drug Delivery Rev. 55, 1293−1302.
Sci. 243, 97−106. (33) Kwon, Y. M., Chung, H. S., Moon, C., Yockman, J., Park, Y. J.,
(13) Kumagai, M., Imai, Y., Nakamura, T., Yamasaki, Y., Sekino, M., Gitlin, S. D., David, A. E., and Yang, V. C. (2009) L-Asparaginase
Ueno, S., Hanaoka, K., Kikuchi, K., Nagano, T., Kaneko, E., et al. encapsulated intact erythrocytes for treatment of acute lymphoblastic
(2007) Iron hydroxide nanoparticles coated with poly(ethylene leukemia (ALL). J. Controlled Release 139, 182−189.
glycol)-poly(aspartic acid) block copolymer as novel magnetic (34) Amena, S., Lingappa, K., Vishalakshi, N., Prabhakar, M., and
resonance contrast agents for in vivo cancer imaging. Colloids Surf., Dayanand, A. (2010) Immobilization and Characterization of L-
B 56, 174−181. Asparaginase from Streptomyces gulbargensis. J. Pure Appl. Microbiol. 4,
(14) Fu, C. H., and Sakamoto, K. M. (2007) PEG-asparaginase. 623−628.
Expert Opin. Pharmacother. 8, 1977−1984. (35) Wang, B., and Cao, Y. (2011) Purified silk fibroin-L-
(15) Rytting, M. (2010) Peg-asparaginase for acute lymphoblastic asparaginase bioconjugates show increased thermostability and
leukemia. Expert Opin. Biol. Ther. 10, 833−839. resistance to trypsin digestion. Eng. Life Sci. 11, 44−51.
(16) Asselin, B. L., Whitin, J. C., Coppola, D. J., Rupp, I. P., Sallan, S. (36) Tao, M. L., Zhang, Y. Q., Shen, W. D., Wang, X. B., Zhang, H.
E., and Cohen, H. J. (1993) Comparative pharmacokinetic studies of P., Zhou, W. L., and Ma, Y. (2002) Preparation of silk sericin powder
three asparaginase preparations. J. Clin. Oncol. 11, 1780−1786. and its application as a support for the immobilization of L-
(17) Ettinger, J. L., Ettinger, A. G., Avramis, V. I., and Gaynon, P. S. asparaginase. Prog. Biochem. Biophys. 29, 961−965.
(1997) Acute lymphoblastic leukemia a guide to asparaginase and (37) Zhang, Y. Q., Tao, M. L., Shen, W. D., Zhou, Y. Z., Ding, Y., Ma,
pegaspargase therapy. BioDrugs 7, 30−39. Y., and Zhou, W. L. (2004) Immobilization of L-asparaginase on the

1608 DOI: 10.1021/acs.bioconjchem.7b00217
Bioconjugate Chem. 2017, 28, 1598−1610
Bioconjugate Chemistry Review

microparticles of the natural silk sericin protein and its characters. (55) Pinheiro, J. P. V., Wenner, K., Escherich, G., Lanvers-Kaminsky,
Biomaterials 25, 3751−3759. C., Würthwein, G., Janka-Schaub, G., and Boos, J. (2006) Serum
(38) Zhang, Y. Q., Zhou, W. L., Shen, W. D., Chen, Y. H., Zha, X. M., asparaginase activities and asparagine concentrations in the cere-
Shirai, K., and Kiguchi, K. (2005) Synthesis, characterization and brospinal fluid after a single infusion of 2,500 IU/m(2) PEG
immunogenicity of silk fibroin-L-asparaginase bioconjugates. J. asparaginase in children with ALL treated according to protocol
Biotechnol. 120, 315−326. COALL-06−97. Pediatr. Blood Cancer 46, 18−25.
(39) Zhang, Y. Q., Tao, M. L., Shen, W. D., Mao, J. P., and Chen, Y. (56) Rizzari, C., Citterio, M., Zucchetti, M., Conter, V., Chiesa, R.,
H. (2006) Synthesis of silk sericin peptides−Lasparaginase bio- Colombini, A., Malguzzi, S., Silvestri, D., and D’Incalci, M. (2006) A
conjugates and their characterization. J. Chem. Technol. Biotechnol. 81, pharmacological study on pegylated asparaginase used in front-line
136−145. treatment of children with acute lymphoblastic leukemia. Haemato-
(40) Ashrafi, H., Amini, M., Mohammadi-Samani, S., Ghasemi, Y., logica 91, 24−31.
Azadi, A., Tabandeh, M. R., Kamali-Sarvestani, E., and Daneshamouz, (57) Allison, J. P., Davidson, L., Gutierrez-Hartman, A., and Kitto, G.
S. (2013) Nanostructure L-asparaginase-fatty acid bioconjugate: B. (1972) Insolubilization of L-asparaginase by covalent attachment to
synthesis, preformulation study and biological assessment. Int. J. Biol.
nylon tubing. Biochem. Biophys. Res. Commun. 47, 66−73.
Macromol. 62, 180−187. (58) Mori, T., Tosa, T., and Chibata, I. (1974) Preparation and
(41) Labrou, N. E., and Muharram, M. M. (2016) Biochemical
properties of asparaginase entrapped in the lattice of polyacrylamide
characterization and immobilization of Erwinia carotovora L-
gel. Cancer Res. 34, 3066−3068.
asparaginase in a microplate for high-throughput biosensing of L-
(59) Galaev IuV, C. E. G., and Klementeva, T. A. (1981)
asparagine. Enzyme Microb. Technol. 92, 86−93.
(42) Nobre, T. M., Pavinatto, F. J., Caseli, L., Barros-Timmons, A., Immobilization of citrobacter L-asparaginase in polyacrylamide gel.
Dynarowicz-Latka, P., and Oliveira, O. N., Jr. (2015) Interactions of Vopr. Med. Khim. 27, 534−537.
bioactive molecules & nanomaterials with Langmuir monolayers as cell (60) Karsakevich, A. S., Vina, I. A., and Liduma, G. Y. (1988)
membrane model. Thin Solid Films 593, 158−188. Immobilization of the enzyme E. coli L-asparaginase on a water-soluble
(43) Rocha, J. M., Pavinatto, A., Nobre, T. M., and Caseli, L. (2016) copolymer of vinylpypurolidone and acrolein. Translated from Khimiya
Acylated carrageenans changes the physicochemical properties of Prirodnykh Soedinenii 4, 562−565.
mixed enzyme-lipid ultrathin films and enhances the catalytic (61) Qian, G., Ma, J., Zhou, J., He, B., and Wang, D. (1997)
properties of sucrose phosphorylase nanostructured as smart surfaces. Chemical modification of E. coli L-asparaginase with poly (N-
J. Phys. Chem. B 120, 5359−5366. vinylpyrrolidone-co-maleic anhydride). React. Funct. Polym. 32, 117−
(44) de Araújo, F. T., and Caseli, L. (2016) Rhodanese incorporated 121.
in Langmuir and Langmuir- Blodgett films of dimyristoylphosphatidic (62) Qian, G., Ma, J., Zhou, J., He, B., and Wang, D. (1997)
acid: physical chemical properties and improvement of the enzyme Chemical modification of E. coli L-Asparaginase with polyethylene
activity. Colloids Surf., B 141, 59−64. glycol grafted vinylpyrrolidone−maleic anhydride copolymer. Polym.
(45) Girard-Egrot, A. P., Godoy, S., and Blum, L. J. (2005) Enzyme Adv. Technol. 8, 581−586.
association with lipidic Langmuir−Blodgett films: Interests and (63) Almendral, M. J., Porras, M. J., Alonso, A., Baez, M. D., and
applications in nanobioscience. Adv. Colloid Interface Sci. 116, 205− Alonso, C. (1995) Spectrophotometric determination of L-asparagine
225. by flow-injection analysis using L-asparaginase immobilized on an
(46) Siqueira, J. R., Jr., Caseli, L., Crespilho, F. N., Zucolotto, V., and epoxy resin. Anal. Chim. Acta 308, 170−177.
Oliveira, O. N., Jr. (2010) Immobilization of biomolecules on (64) Erdogan, A., Koytepe, S., Ates, B., Yilmaz, I., and Seckin, T.
nanostructured films for biosensing. Biosens. Bioelectron. 25, 1254− (2014) Preparation of the L-asparaginase-based biosensor with
1263. polyimide membrane electrode for monitoring L-asparagine levels in
(47) Zanon, N. C. M., Oliveira, O. N., Jr., and Caseli, L. (2012) leukemia. Int. J. Polym. Mater. 63, 909−917.
Immbolization of uricase enzyme in Langmuir and Langmuir-Blodgett (65) El-Refai, H. A., Shafei, M. S., Mostafa, H., El-Refai, A. M. H.,
films of fatty acids: Possible use as a uric acid sensor. J. Colloid Interface Araby, E. M., El-Beih, F. M., Easa, S. M., and Gomaa, S. K. (2016)
Sci. 373, 69−74. Comparison of free and immobilized L-asparaginase synthesized by
(48) da Rocha Junior, C., and Caseli, L. (2017) Adsorption and gamma-irradiated Penicillium cyclopium. Pol. J. Microbiol. 65, 43−50.
enzyme activity of asparaginase at lipid Langmuir and Langmuir- (66) Qiao, J., Qi, L., Mu, X., and Chen, Y. (2011) Monolith and
Blodgett films. Mater. Sci. Eng., C 73, 579−584. coating enzymatic microreactors of L-asparaginase: kinetics study by
(49) Torchilin, V. P. (1991) Progress in Clinical Biochemistry and MCE−LIF for potential application in acute lymphoblastic leukemia
Medicine. In Immobilized Enzymes in Medicine (Kellen, J. A., Ed.) pp (ALL) treatment. Analyst 136, 2077−2083.
13−28, Springer, New York. (67) Qiao, J., Qi, L., Ma, H., Chen, Y., Wang, M., and Wang, D.
(50) Mishra, P., Nayak, B., and Dey, R. K. (2016) PEGylation in anti-
(2010) Study on amino amides and enzyme kinetics of L-asparaginase
cancer therapy: An overview. Asian J. Pharm. Sci. 11, 337−348.
by MCE. Electrophoresis 31, 1565−1571.
(51) Soares, A. L., Guimaraes, G. M., Polakiewicz, B., de Moraes
(68) Gaspar, M. M., Blanco, D., Cruz, M. E. M., and Alonso, M. J.
Pitombo, R. N., and Abrahao-Neto, J. (2002) Effects of polyethylene
glycol attachment on physicochemical and biological stability of E. coli (1998) Formulation of L-asparaginase-loaded poly(lactide-co-glyco-
L-asparaginase. Int. J. Pharm. 237, 163−170. lide) nanoparticles: influence of polymer properties on enzyme
(52) Ashihara, Y., Kono, T., and Yamazaki, S. (1978) Modification of loading, activity and in vitro release. J. Controlled Release 52, 53−62.
E. coli L -asparaginase with polyethylene glycol dispearance of binding (69) Wolf, M., Wirth, M., Pittner, F., and Gabor, F. (2003)
ability to anti-L-asparaginase serum. Biochem. Biophys. Res. Commun. Stabilisation and determination of the biological activity of L-
83, 385−391. asparaginase in poly(D,L-lactide-co-glycolide) nanospheres. Int. J.
(53) Taylor, C. W., Dorr, R. T., Fanta, P., Hersh, E. M., and Salmon, Pharm. 256, 141−152.
S. E. (2001) A phase I and pharmacodynamic evaluation of (70) Vasudev, S. S., Ahmad, S., Parveen, R., Ahmad, F. J., Anish, C.
polyethylene glycol-conjugated L-asparaginae in patients with K., Ali, M., and Panda, A. K. (2011) Formulation of PEG-ylated L-
advanced solid tumors. Cancer Chemother. Pharmacol. 47, 83−88. asparaginase loaded poly (lactide-co-glycolide) nanoparticles: influ-
(54) Hawkins, D. S., Park, J. R., Thomson, B. G., Felgenhauer, J. L., ence of PEGylation on enzyme loading, activity and in vitro release.
Holcenberg, J. S., Panosyan, E. H., and Avramis, V. I. (2004) Pharmazie 66, 956−960.
Asparaginase pharmacokinetics after intensive polyethylene glycol- (71) Baran, E. T., Ozer, N., and Hasırcı, V. (2002) In vivo half life of
conjugated L-asparaginase therapy for children with relapsed acute nanoencapsulated L-asparaginase. J. Mater. Sci.: Mater. Med. 13, 1113−
lymphoblastic leukemia. Clin. Cancer Res. 10, 5335−5341. 1121.

1609 DOI: 10.1021/acs.bioconjchem.7b00217
Bioconjugate Chem. 2017, 28, 1598−1610
Bioconjugate Chemistry Review

(72) Teodor, E., Litescu, S. C., Lazar, V., and Somoghi, R. (2009) (90) Ulu, A., Koytepe, S., and Ates, B. (2016) Design of starch
Hydrogel-magnetic nanoparticles with immobilized L-asparaginase for functionalized biodegradable P(MAA-co-MMA) as carrier matrix for
biomedical applications. J. Mater. Sci.: Mater. Med. 20, 1307−1314. L-asparaginase immobilization. Carbohydr. Polym. 153, 559−572.
(73) Teodor, E., Truica, G., and Lupescu, I. (2008) The release of L- (91) Dzionek, A., Wojcieszyńska, D., and Guzşk, U. (2016) Natural
asparaginase from hydrogel magnetic nanoparticles. Rom. Biotechnol. carriers in bioremediation: A review. Electron. J. Biotechnol. 23, 28−36.
Lett. 13, 3907−3913. (92) Vroman, I., and Tighzert, L. (2009) Biodegradable polymers.
(74) Zhang, Y. Q., Wang, Y. J., Wang, H. Y., Zhu, L., and Zhou, Z. Z. Materials 2, 307−344.
(2011) Highly efficient processing of silk fibroin nanoparticle-L- (93) Bahraman, F., and Iran Alemzadeh, I. (2017) Optimization of L-
asparaginase bioconjugates and their characterization as a drug delivery Asparaginase immobilization onto calcium alginate beads. Chem. Eng.
system. Soft Matter 7, 9728−9736. Commun. 204, 216−220.
(75) Ghosh, S., Chaganti, S. R., and Prakasham, R. S. (2012)
Polyaniline nanofiber as a novel immobilization matrix for the anti-
leukemia enzyme L-asparaginase. J. Mol. Catal. B: Enzym. 74, 132−
(76) Mu, X., Qiao, J., Qi, L., Dong, P., and Ma, H. (2014) Poly(2-
Vinyl-4,4-dimethylazlactone)-functionalized magnetic nanoparticles as
carriers for enzyme immobilization and its application. ACS Appl.
Mater. Interfaces 6, 21346−21354.
(77) Sundaramoorthi, C., Rajakumari, R., Dharamsi, A., and
Vengadeshprabhu, K. (2012) Production and immobilization of L-
Asparaginase from marine source. Int. J. Pharm. Pharm. Sci. 4, 229−
(78) El-Sayed, S. T., Fyiad, A. A., and Gamal-Eldeen, A. M. (2012)
Immobilization, properties and anti-tumor activity of L-Asparaginase
of Vicia faba and Phaseoulus vulgaris Seeds. Aust. J. Basic Appl. 6, 785−
(79) Moharam, M., Gamal-Eldeen, A. M., and El-Sayed, S. T. (2010)
9. Production, immobilization and anti-tumor activity of L-
Asparaginase of Bacillus sp R36. J. Am. Sci. 6, 157−165.
(80) Ahmad, A., Patta, A. M., and Natsır, H. (2013) Purification and
immobilization of L-Asparaginase enzyme from the thermophilic
bacteria bacillus licheniformis Strain HSA3−1a. Int. J. Pharm. Bio Sci. 4,
(81) Muslim, S. N., Al Kadmy, I. M. S, Hussein, N. H., Mohammed
Ali, A. N., and Dwaish, A. S. (2015) Enhancement of the activity and
stability of L-asparaginase food additive purified from acinetobacter
baumannii as anticarcinogenic in processed foods. Int. J. Adv. Chem.
Eng. Biol. Sci. 2, 35−39.
(82) Francois, J., and Fortier, G. (1996) Immobilization of L-
asparaginase into a biocompatible poly(ethylene glycol)-albumin
hydrogel 0.1. preparation and in vitro characterization. Biotechnol.
Appl. Biochem. 23, 221−226.
(83) Francois, J., Durso, E. M., and Fortier, G. (1997) Immobilization
of L-asparaginase into a biocompatible poly(ethylene glycol)-albumin
hydrogel: evaluation of performance in vivo. Biotechnol. Appl. Biochem.
26, 203−212.
(84) Youssef, M. M., and Al-Omair, M. A. (2008) Cloning,
purification, characterization and immobilization of L-asparaginase II
from E. coli W3110. Asian J. Biochem. 3, 337−350.
(85) Karamitros, C. S., Yashchenok, A. M., Möhwald, H., Skirtach, A.
G., and Konrad, M. (2013) Preserving catalytic activity and enhancing
biochemical stability of the therapeutic enzyme asparaginase by
biocompatible multilayered polyelectrolyte microcapsules. Biomacro-
molecules 14, 4398−4406.
(86) Sukhoverkov, K. V., and Kudryashova, E. V. (2015) PEG-
chitosan and glycol-chitosan for improvement of biopharmaceutical
properties of recombinant L-Asparaginase from Erwinia carotovora.
Biochemistry (Moscow) 80, 113−119.
(87) Bahreini, E., Aghaiypour, K., Abbasalipourkabir, R., Mokarram,
A. R., Goodarzi, M. T., and Saidijam, M. (2014) Preparation and
nanoencapsulation of L-asparaginase II in chitosan-tripolyphosphate
nanoparticles and in vitro release study. Nanoscale Res. Lett. 9, 340−
(88) Ulu, A., Koytepe, S., and Ates, B. (2016) Synthesis and
characterization of PMMA composites activated with starch for
immobilization of L-asparaginase. J. Appl. Polym. Sci. 133, n/a.
(89) Ulu, A., Koytepe, S., and Ates, B. (2016) Synthesis and
characterization of biodegradable pHEMA-starch composites for
immobilization of L-asparaginase. Polym. Bull. 73, 1891−1907.

1610 DOI: 10.1021/acs.bioconjchem.7b00217
Bioconjugate Chem. 2017, 28, 1598−1610