Cloning, expression, and characterization of L-asparaginase from a newly isolated Bacillus subtilis B11-06

Article abstract of DOI:10.1021/jf402636w

This study focused on the cloning, overexpression, and characterization of the gene encoding L-asparaginase (ansZ) from a nonpathogenic strain of Bacillus subtilis B11.06. The recombinant enzyme showed high thermostability and low affinity to L-glutamine. The ansZ gene, encoding a putative L-asparaginase II, was amplified by PCR and expressed in B. subtilis 168 using the shuttle vector pMA5. The activity of the recombinant enzyme was 9.98 U/mL, which was significantly higher than that of B. subtilis B11.06. The recombinant enzyme was purified by a two-step procedure including ammonium sulfate fractionation and hydrophobic interaction chromatography. The optimum pH and temperature of the recombinant enzyme were 7.5 and 40 °C, respectively. The enzyme was quite stable at a pH range of 6.0.9.0 and exhibited about 14.7 and 9.0% retention of activity following 2 h incubation at 50 or 60 °C, respectively. The K _m for L-asparagine was 0.43 mM, and the V_max was 77.51 μM/min. Results of this study also revealed the potential industrial application of this enzyme in reducing acrylamide formation during the potato frying process.

Full text of DOI:10.1021/jf402636w

Article
pubs.acs.org/JAFC
Cloning, Expression, and Characterization of L‑Asparaginase from a
Newly Isolated Bacillus subtilis B11−06
Mingmei Jia, Meijuan Xu, Beibei He, and Zhiming Rao*
The Key Laboratory of Industrial Biotechnology, Ministry of Educationand Lab of Applied Microbiology and Metabolic Engineering,
School of Biotechnology, Jiangnan University, Wuxi, Jiangsu Province 214122, China
ABSTRACT: This study focused on the cloning, overexpression, and characterization of the gene encoding L-asparaginase
(
ansZ) from a nonpathogenic strain of Bacillus subtilis B11−06. The recombinant enzyme showed high thermostability and low
affinity to L-glutamine. The ansZ gene, encoding a putative L-asparaginase II, was amplified by PCR and expressed in B. subtilis
68 using the shuttle vector pMA5. The activity of the recombinant enzyme was 9.98 U/mL, which was significantly higher than
1
that of B. subtilis B11−06. The recombinant enzyme was purified by a two-step procedure including ammonium sulfate
fractionation and hydrophobic interaction chromatography. The optimum pH and temperature of the recombinant enzyme were
7
.5 and 40 °C, respectively. The enzyme was quite stable at a pH range of 6.0−9.0 and exhibited about 14.7 and 9.0% retention
of activity following 2 h incubation at 50 or 60 °C, respectively. The K for L-asparagine was 0.43 mM, and the V was 77.51
μM/min. Results of this study also revealed the potential industrial application of this enzyme in reducing acrylamide formation
m
max
during the potato frying process.
KEYWORDS: L-asparaginase II, cloning, expression, Bacillus subtilis, acrylamide
INTRODUCTION
that L-Asn is the key determinant of acrylamide formation in
cereal products. L-Asparaginase can hydrolyze free L-Asn to
aspartic acid, and thus it can decrease acrylamide formation at
■
L-Asparaginase (EC 3.5.1.1; L-asparagine amidohydrolase)
catalyzes the hydrolysis of L-asparagine to L-aspartic acid and
22
1
the source.
ammonia. Aspartate can then be transaminated to oxaloace-
2
,3
Previous studies on L-asparaginase gene expression in Bacillus
tate, an intermediate in the tricarboxylic acid cycle.
Asparaginase exists in various organisms including animals,
plant cells, yeast, fungi, and bacteria. In 1961, Broome
demonstrated that the antilymphoma activity in guinea pig sera
was due to L-asparaginase. In 1963, Mashburn and Wriston
L-
11,23
subtilis focused on the mechanism of gene regulation.
To
our knowledge, there is no research on the properties of the
enzyme derived from a B. subtilis expression system. L-
Asparaginase can be mass produced in B. subtilis in a form
1
,4
5
18
that is safe for use in food production. In the current study, B.
6
reported on the L-asparaginase from an Escherichia coli strain.
subtilis B11−06 was isolated from soil and found to produce a
putative L-asparaginase. The ansZ gene, encoding the putative
L-asparaginase II, was then cloned. Subsequently, ansZ was
Several further microbial strains including Aspergillus terreus,
Erwinia aroideae, Pseudomonas aeruginosa, Serratia marcescens,
Withania somnifera, and Staphylococcus sp. were then shown to
24
inserted into the shuttle expression vector pMA5 and
2
,7,8
produce L-asparaginase.
Campbell confirmed that there
11
expressed in B. subtilis 168. The L-asparaginase II from the
were two isoforms of L-asparaginase, L-asparaginase I and L-
recombinant B. subtilis 168 exhibited higher activity than that of
the wild-type B. subtilis B11−06. We then purified and
characterized the recombinant protein and examined its
industrial application in reducing acrylamide formation during
the potato frying process.
9
asparaginase II, and that these two enzymes were genetically
differentiated from each other. L-Asparaginases derived from
different microorganisms show different properties.
3,10,11
Because the type II enzyme can effectively deplete
cytotoxicity in leukemic cells, it has been widely used for the
chemotherapy of acute lymphoblastic leukemia (ALL) and
shows great commercial value in the pharmaceutical
MATERIALS AND METHODS
■
1
2−14
Strains, Plasmids, and Chemicals. E.coli JM109, B. subtilis 168,
and vector pMA5 were preserved in our laboratory. The restriction
enzymes, T4 DNA ligase, and ExTaq DNA polymerase were obtained
from TaKaRa Bio Co. (Dalian, China). The Mini Chromosome Rapid
Isolation kit, Mini Plasmid Rapid Isolation kit, and Mini DNA Rapid
Purification kit were obtained from Sangon Biotech Co., Ltd.
(Shanghai, China). AKTA prime plus and the Butyl Sepharose HP
column were purchased from GE Healthcare, Inc. (Little Chalfont,
sector.
However, clinical application of the enzyme is
limited by antibody response and by various side effects, which
are attributed to the L-glutaminase activity of this enzyme.
Thus, enzymes that are best suited for ALL treatment should
have high activity, a low K , and a strong affinity for L-
asparagine (L-Asn) over L-glutamine (L-Gln). L-Asparaginase II
from Erwinia chrysanthemi and Escherichia coli has been
acknowledged to best meet these criteria and is widely used
1
5,16
m
1,17,18
in the pharmaceutical industry.
In recent years, L-
Received: June 17, 2013
asparaginase has been shown to reduce acrylamide formation
Revised: September 4, 2013
Accepted: September 4, 2013
Published: September 4, 2013
1
9,20
in starchy foods.
Researchers confirmed that acrylamide
2
1,22
formation in food was related to the Maillard reaction
and
©
2013 American Chemical Society
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U.K.). The Shimadzu LC-20AB high-pressure liquid chromatograph
was purchased from Shimadzu Co. (Kyoto, Japan). The Aminex HPX-
sensitive electrode, which can detect dissolved ammonia accurately in
2
9−31
aqueous solutions.
This apparatus included an ammonia electrode
8
7H Column was purchased from Bio-Rad Laboratories, Inc.
attached to a digital pH meter and a magnetic stirrer. The reaction
mixture (25 mL) consisted of 10 mL of 50 mM Tris-HCl buffer (pH
7.5) and 10 mL of 50 mM L-Asn. Supernatant or cell extract (0.5 mL)
was added, and the reaction proceeded at 37 °C for 15 min. The
reaction was then terminated by addition of 5 mL of a 15% (w/v)
trichloroacetic acid solution. The control tube was terminated before
15 min of incubation. The ammonium electrode was calibrated by
using ammonium chloride with different concentrations. One unit of
the L-asparaginase was defined as the amount of enzyme required to
produce 1 μmol of ammonia per minute under assay conditions. To
evaluate the accuracy of this method, L-asparaginase activity was also
(California, USA). All other chemicals were of analytical grade.
Medium and Culture Conditions. E.coli JM109 and B. subtilis
1
68 were grown at 37 °C in Luria−Bertani (LB) medium during the
1,11
construction of strains.
Ampicillin (100 μg/mL) or kanamycin (50
μg/mL) was added to the growth medium when necessary. ADS agar
25
(
asparagine dextrose salts agar, g/L): asparagine, 10; dextrose, 2;
K HPO , 1; MgSO , 0.5; agar, 15, and 0.009% phenol red was used as
2
4
4
a pH indicator. The basal medium for producing L-asparaginase
contained (g/L): glucose, 20; soya peptone, 10; corn steep liquor, 15;
K HPO , 2.3; MgSO , 0.75; NaCl, 5, pH 7.0. The optimized medium
2
4
4
3,4
contained (g/L): sucrose, 35; tryptone, 15; urea, 0.8; corn steep
detected by the Nessler assay using Nessler’s reagent. Protein
liquor,12; K HPO , 2.612; KH PO , 2.041; MgSO ·7H O, 1.845;
concentration was determined by the Bradford method with bovine
serum albumin as the standard.
2
4
2
4
4
2
3
2
NaCl, 5; L-Asn, 1, pH 7.0. The temperature and agitation rate were 37
°
C and 160 rpm, respectively, during the cultivation.
Isolation of the Bacteria. Various soil samples were collected
Enzyme Purification. The fermentation broth was centrifuged at
10 000g for 15 min at 4 °C. The supernatant was collected and
precipitated using 80% (w/v) ammonium sulfate. The precipitate was
then dissolved in 20 mM Tris-HCl buffer (pH 7.5) containing 1.5 M
ammonium sulfate. The dissolved enzyme was loaded onto a Butyl
Sepharose HP column equilibrated with the same buffer. Nonabsorbed
proteins were washed off thoroughly with the buffer, and the enzyme
was eluted in a linear gradient of 1.5−0.0 mM ammonium sulfate with
a flow rate of 1.0 mL/min. The active fractions were collected and
dialyzed for an L-asparaginase enzyme assay or were stored at −20 °C
until further analysis.
from different places (Wuxi, China) and put in sterile polythene bags.
The samples were scattered by glass beads and then serially diluted in
sterile distilled water under aseptic conditions. Dilutions were plated
on LB agar plates and incubated at 37 °C for 20−24 h. The individual
colonies were subcultured on ADS agar. Isolates capable of producing
L-asparaginase were selected for further study. The 16S rRNA of the
isolate was amplified by PCR using the universal primers 5′-
AGAGTTTGATCCTGGCTCAG-3′ and 5′-AAGGAGGTGATC-
26
CAGCCGCA-3′.
Cloning and Construction of Recombinant Plasmid pMA5-
ansZ. Total genomic DNA of the isolated B. subtilis B11−06 was
extracted using a Mini Chromosome Rapid Isolation kit. The ansZ
gene was amplified from chromosomal DNA of the B. subtilis B11−06
strain by PCR using ansZ forward (5′-GACGGATCCATGAAAAAA-
CAACGAATGCT-3′) and reverse (5′-GGCACGCGTTTAATACT-
CATTGAAATAAG-3′) primers (underlined parts were the restriction
sites), which were designed according to the L-asparaginase gene of B.
subtilis subsp. subtilis str. 168 (accession number NC-000964) available
from the National Center for Biotechnology Information (NCBI)
Web site (http://www.ncbi.nlm.nih.gov/). The primers annealed to
the 5′- (including Start codon) and 3′-ends (including stop codon) of
the ansZ gene, respectively. The forward primer contained a BamHI
restriction site, while the reverse primer contained a MluI restriction
site. The resulting PCR amplicon and plasmid pMA5 were digested
with BamHI and MluI. The digested fragments were checked by
agarose electrophoresis and purified from the gel using a Mini DNA
Rapid Purification kit. The purified fragment was then ligated into
linearized pMA5. The nucleotide sequence of the ansZ insert was
analyzed by Sangon Biotech. Protein and nucleotide sequence
comparisons were performed using the BLAST server available from
the NCBI Web site (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The
recombinant plasmid was transformed into chemically competent B.
subtilis 168 cells for L-asparaginase production.
Enzyme Characterization. The optimum pH was confirmed in
several buffers (pH 3.0−6.5, 0.05 M citrate−sodium citrate buffer; pH
6.5−9.0, 0.05 M Tris-HCl buffer; pH 9.0−10.0, 0.05 M borax−sodium
hydroxide buffer), while the optimum temperature was examined in 50
mM Tris-HCl buffer (pH 7.5) using the standard reaction mixture.
The pH and thermal stability of L-asparaginase was determined by
incubation at different pH levels at 4 °C for 24 h and at various
temperatures for 10 h. Effects of various compounds on enzyme
activity were examined in the standard reaction mixture supplemented
with various cations and EDTA at concentrations of 1 mM. The
enzyme activity of the recombinant L-asparaginase without the
addition of metal ions or EDTA was used as the control. Kinetic
parameters were performed in 50 mM Tris-HCl buffer (pH 7.5) at 40
°C by changing the concentration of the substrate L-Asn and L-Gln.
27
Eadie−Hofstee plots were used to calculate kinetic parameters K and
m
3
3
V
according to the enzyme reactions.
Raw Potato Preparation and Frying Condition. Potatoes
max
(Fovorita, moisture content of 74 g/100 g) were washed, peeled, and
then sliced (thickness of 2.0 mm) using a slicing machine. All the
potato slices were immersed in distilled water for 2 min to remove the
starch on the surface. Three samples were pretreated prior to frying, as
follows: (i) without any processing; (ii) immersion in distilled water
for 30 min; (iii) immersion in an 8000 U/L L-asparaginase solution for
30 min. All samples were fried at 160 °C for 10 min in an electric fryer.
After frying, the potato slices were dried on paper, cooled to ambient
Expression of ansZ in B. subtilis. The recombinant plasmid
pMA5-ansZ was transformed into E. coli JM109. The plasmid was then
extracted from E. coli JM109 and used to transform B. subtilis 168
34
temperature, and then the acrylamide was extracted for analysis. The
acrylamide content of the fried potato slices purchased from the local
supermarket were also analyzed.
28
using the procedure described by Spizizen. Transformants were
obtained following growth overnight on selective LB medium
supplemented with 50 μg/mL kanamycin. Recombinant cells were
grown in the basal medium (50 mL) for 24 h. The cells were harvested
by centrifugation at 10 000g for 10 min at 4 °C, and the supernatant
was used for the L-asparaginase enzyme activity assay. The pellets were
washed twice with 20 mL of 10 mM PBS buffer (pH 7.4), suspended
in 50 mM Tris-HCl buffer (pH 7.5), and sonicated 10 times for 2 s
with 5 s cooling intervals. Cell extracts were centrifuged for 30 min at
Acrylamide Determination. Acrylamide was analyzed by high-
performance liquid chromatography (HPLC) with ultraviolet
3
4,35
detection at 200 nm, as described previously.
Briefly, 1 g of a
sliced potato sample was crushed by a lab mixer homogenizer and was
homogenized with 1 mL of distilled water. Then, 2 mL of distilled
water was added, and the homogenates were incubated in a water bath
at 70 °C with agitation for 30 min. The homogenates were then
centrifuged at 10 000g for 30 min and were filtered through a 0.45 μm
microporous membrane. The filtrates were transferred to a 10.0 mL
volumetric flask and diluted to mark with distilled water. Hexane (3
mL) was added to 1 mL of the sample solution to extract long-chain
fatty acids. The mixture was shaken thoroughly, and the upper layer
was removed with a pipet. The lower aqueous layer was transferred
into plastic vials (Zhejiang Aijiyen Technology Co., Ltd., Quzhou,
China) for autosampler analysis. Sulfuric acid solution (0.01 M) was
10 000g in a SIGAMA 3K-15 centrifuge (Sigma-Aldrich Co., Ltd.,
Shanghai, China) to remove cell debris. The supernatant was used for
an enzyme activity assay, sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) analysis (12% acrylamide), or were
stored at −20 °C until further analysis.
Enzyme Activity Assay. Enzyme activities of the intracellular and
extracellular parts of the protein were performed using an ammonia-
9
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Journal of Agricultural and Food Chemistry
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used as mobile phase. Samples were then run on an Aminex HPX-87H
(
0
300 mm × 7.8 mm) column with a temperature of 30 °C, flow rate of
.5 mL/min, and a sample size of 20 μL, while ultraviolet detection
was maintained at 200 nm. The experiments were carried out in
triplicate.
RESULTS AND DISCUSSION
■
Isolation of the Bacteria. Four L-asparaginase-producing
strains were isolated from the soil samples. Their enzyme
activities were assayed by an ammonia-sensitive electrode.
Dissolved ammonia, which is produced by the catalysis of L-
asparaginase, was measured using the ammonia-sensitive
electrode, and the maximum enzyme activity of these strains
was 0.039 U/mL. The strain with the greatest activity was
Gram-positive. Sequencing of the 16S rRNA gene revealed that
the isolated strain showed 99% similarity to the same region in
B. subtilis. The strain was named B. subtilis B11−06, and the
sequence was submitted to GenBank with accession number
KF028775.
Cloning and Construction of Recombinant Plasmid
pMA5-ansZ. The ansZ gene was successfully amplified from
the genomic DNA of the isolated B. subtilis B11−06 strain by
PCR. Electrophoresis revealed that the amplicon consisted of
1
128 bp (Figure 1A), and BLAST analysis showed that the
gene had a high level of similarity to that of several ansZ-
containing strains published in the NCBI database, including B.
subtilis BEST7003 (AP012496.1), B. subtilis subsp. subtilis str.
1
68 (AL009126.3), B. subtilis QB928 (CP003783.1), and B.
subtilis BSn5 (CP002468.1), with similarities of 100%, 100%,
00%, and 99%, respectively. The predicted amino acid
1
sequence of the cloned ansZ gene shared 100% similarity
with B. subtilis 168, and the gene sequence was submitted to
GenBank with accession number KF444946. The digested PCR
amplicon was cloned into the expression vector pMA5 under
the control of the HpaII promoter, resulting in recombinant
vector pMA5-ansZ (Figure 1C). The recombinant plasmid was
digested with BamHI and MluI to produce two major fragments
with sizes of 1128 and 7500 bp (Figure 1B). The result revealed
successful construction of the recombinant plasmid.
Figure 1. PCR and construction of recombinant vector pMA5-ansZ.
(A) Lane 1, DL2000 DNA marker; lane 2, ansZ PCR product. (B)
Lane 1, recombinant vector digested with BamHI; lane 2, recombinant
vector digested with BamHI and MluI; lane 3, DL2000 DNA marker;
lane 4, λ Hind III DNA marker. (C) Ligation of the ansZ gene with
shuttle vector pMA5. The ansZ gene predigested with BamHI and
MluI was ligated into the BamHI and MluI sites of the pMA5 vector.
Expression of the Recombinant Vector in B. subtilis.
The recombinant plasmid pMA5-ansZ was subsequently
transformed into the expression host B. subtilis 168 to construct
the recombinant strain B. subtilis 168/pMA5-ansZ, which
expressed ansZ under the control of the HpaII promoter.
Recombinant B. subtilis was selected with kanamycin and
verified by DNA sequencing. The positive clones showed
intracellular and extracellular enzyme activities of 4.28 and 5.70
U/mL, respectively. The total enzyme activity was substantially
higher than that of the native enzyme producer B. subtilis B11−
production was found to be corn steep liquor > urea > tryptone
> sucrose. Thus, the optimal medium composition (g/L) after
one-factor-at-a-time and orthogonal array design methods were
applied was determined to be sucrose, 35; tryptone, 15; urea,
0.8; corn steep liquor, 12; K HPO , 2.612; KH PO , 2.041;
2
4
2
4
06.
MgSO .7H O, 1.845; NaCl, 5; L-Asn, 1. As a result, the highest
4 2
The one-factor-at-a-time and orthogonal array design
enzyme activity reached 89.48 U/mL in a 5 L fermenter, which
was approximately 8.9-fold higher than the activity in the basal
medium. There are many reports on L-asparaginase production
by recombinant strains. Hatanaka et al. and Eisele et al. cloned
the Streptomyces and Flammulina velutipes L-asparaginase genes
and expressed them in E. coli, resulting in enzyme activities of
36,37
methods were used for medium optimization.
The one-
factor-at-a-time method was used to investigate the effects of
carbon sources, nitrogen sources, inorganic salt, and corn steep
liquor on L-asparaginase production. Of the carbon sources,
sucrose at a concentration of 35 g/L was most suitable for L-
asparaginase production. Of the nitrogen sources, tryptone and
urea were conducive to enzyme production at concentrations of
3
8,39
23.02 and 16 U/mL, respectively.
Gilbert cloned the E.
chrysanthemi asparaginase gene and transformed it into E. coli
and Erwinia. carotovora, with enzyme activities of 49 and 20.2
1
5 and 1 g/L, respectively. Corn steep liquor was used as a
40
growth factor to stimulate cell growth and improve enzyme
activity. Subsequently, the concentrations of sucrose, tryptone,
urea, and corn steep liquor were optimized by the orthogonal
array method. The order of factor effects on L-asparaginase
U/mL, respectively. To the best of our knowledge, the L-
asparaginase-producing system described in the current study
represents the highest level activity of a recombinant strain.
Furthermore, the system is safe, economically viable, and easy
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to scale up. Therefore, the recombinant strain is suitable for
large-scale production of L-asparaginase.
Enzyme Assay. The enzyme activity was tested using an
ammonia electrode. The precision of this method was
investigated by analysis of repeated measurements of L-
asparaginase activity, and the mean enzyme activity was 5.70
U/mL in fermentation broth, with a standard deviation of 0.14.
The accuracy of the ammonia electrode method was checked
by simultaneously determining the L-asparaginase activity with
an ammonia electrode and the Nessler method (Table 1). The
Table 1. Enzyme Assay by Ammonia Electrode and Nessler
Assay
Figure 2. SDS-PAGE analysis of recombinant L-asparaginase. Lane 1,
protein marker; lane 2, B. subtilis 168 cell extract; lane 3, B. subtilis
Nessler assay
ammonia electrode
1
68/pMA5-ansZ cell extract; lane 4, the fermentation supernatant
sample
average activity (U/mL)
average activity (U/mL)
from B. subtilis 168/pMA5-ansZ; lane 5, ammonium sulfate
precipitation; lane 6, butyl Sepharose HP.
one
10.01 ± 0.83
13.33 ± 1.04
11.21 ± 0.87
10.30 ± 0.30
13.49 ± 0.46
11.89 ± 0.27
two
three
mean value determined by the two methods was similar, but the
Nessler assay was less reproducible than the ammonia
electrode. In addition, the Nessler reagent is toxic and has
inferior storage stability. Therefore, the ammonia electrode
method was safe, accurate, and simple to perform compared to
the toxity, complexity, and poor reproducibility of the
traditional Nessler assay.
Purification of the Enzyme. The recombinant L-
asparaginase was purified to homogeneity by a two-step
procedure, with a yield of 26.05% (Table 2). As a result, the
Table 2. Purification of Recombinant L-Asparaginase
total
protein
(mg)
total
activity
(U)
specific
activity
(U/mg)
purification
stage
recovery
(%)
fold
purification
fermentation
supernatant
34.20
816
23.85
100
1
ammonium
sulfate
precipitation
10.75
505.90
47.06
61.99
1.97
butyl Sepharose
HP
2.30
212.60
92.45
26.05
3.88
purified enzyme showed a specific activity of 92.45 U/mg,
which was 3.88 times higher than the crude enzyme from
supernatant from the fermentation broth. A 12% SDS-PAGE
analysis revealed that the subunit of the enzyme had a
molecular mass of about 38 kDa when the gel was stained with
CBB R-250 (Figure 2).
Figure 3. Effects of pH (A) and temperature (B) on recombinant L-
asparaginase activity. (A) Optimal pH was determined by assaying the
enzyme at various pHs (pH 3.0−6.5, 0.05 M citrate−sodium citrate
buffer; pH 6.5−9.0, 0.05 M Tris-HCl buffer; pH 9.0−10.0, 0.05 M
borax−sodium hydroxide buffer), at a temperature of 37 °C. (B)
Influence of temperature on enzyme activity was investigated by
assaying the activity from 20−70 °C in 0.05 M Tris-HCl buffer (pH
Characterization of the Enzyme. The optimum pH of L-
asparaginase for hydrolyzing L-Asn was pH 7.5 (Figure 3A). It
was different from that of the ErA II, which had an optimum
4
2,43
pH of 9.5, but was more similar to that of EcA II (pH 7).
The enzyme showed maximum activity at 40 °C (Figure 3B),
4
,12,39
which was similar to other reported bacterial sources.
The
recombinant enzyme was stable at pH 6.0−9.0 (Figure 4A).
More than 86% of the maximum enzyme activity was retained
after incubation at pH 8.0 for 24 h. The enzyme exhibited high
stability at −20 and 0 °C with only a small decrease in activity
after incubation for 10 h (Figure 4B). It retained about 14.7%
of its residual activity after 2 h incubation in 50 °C and showed
about 9.0% retention of activity after 2 h incubation in 60 °C.
7
.5).
bonds of the enzyme spatial structures. The effects of ions and
EDTA on L-asparaginase were examined (Figure 5), with the
results showing that most metal ions slightly inhibited enzyme
activity; however, strong inhibition was observed in the
41
42
The thermostability was higher than that of YpA and RrA.
Differences in thermostability may relate to the hydrogen
3+
presence of Fe . The addition of EDTA had no effect on
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recombinant enzyme activity. This was similar to findings for
1
7,18,39
BsA I, BsA II, and FvNase.
The K and V values were determined by nonlinear fit
m
max
analysis based on Eadie−Hofstee plots. The recombinant
enzyme hydrolyzed L-Asn with a Michaelis constant K value of
m
0
7
.43 mM (Figure 6A) and a maximum reaction rate Vmax of
7.51 μM/min. The K value showed that the affinity of this
m
recombinant enzyme for L-Asn was slightly lower than that of
EcA II and ErA II,
BsA II.
4
2,43
but it was higher than that of BsA I and
1
7,18
Moreover, the enzyme exhibited very low affinity
for L-Gln. The L-glutaminase activity assayed by the ammonia
electrode was only 1.3% of that of the L-asparaginase activity. It
hydrolyzed L-Gln with a Michaelis constant K value of 43.06
m
mM (Figure 6B). The undesirable side effects of the protein in
clinical use have been attributed to the glutaminase activity of
the enzyme. Gln is an amino group donor for various
biochemical reactions and also the major transport form of
amino nitrogen in the blood. L-Asparaginase with high
glutaminase activity will decrease the plasma Gln levels,
1
5,16
therefore impairing many biochemical functions.
As a
consequence, the recombinant enzyme may be used in the
pharmaceutical industry.
Application of L-Asparaginase in Potato Samples.
HPLC was used to accurately detect the acrylamide in the
samples (Figure 7). After frying, the sample without any
Figure 4. Effects of pH (A) and temperature (B) on recombinant L-
asparaginase stability. (A) Influence of pH on stability of the enzyme
was determined by incubating at different pH conditions at 4 °C for a
certain time and assaying the residual activities. (B) Thermostability of
the enzyme was studied by incubating at different temperatures for a
certain time in pH 7.5 and assaying the residual activities.
Figure 7. Acrylamide content of potato samples subjected to different
treatments prior to frying. All samples, except those obtained from the
supermarket, were fried at 160 °C for 10 min in an electric fryer. (a)
Raw potato without any processing; (b) Raw potato immersed in
distilled water for 30 min; (c) Raw potato immersed in an 8000 U/L L-
asparaginase solution for 30 min; (d) Potato slices purchased from the
local supermarket.
processing contained 2.53 mg/kg of acrylamide (Figure 7a).
Because of the high percentage of reducing sugars and L-Asn in
the raw potato, a high level of acrylamide was produced during
the frying process. The sample treated with distilled water
Figure 5. Effects of different metal ions and EDTA at the same
concentration (1 mM) on L-asparaginase activity (BC represents blank
control: no metal ion or EDTA).
Figure 6. Lineweaver−Burk plots for L-Asn (A) and L-Gln (B).
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Journal of Agricultural and Food Chemistry
Article
showed almost the same acrylamide content, with a level of
enediaminetetraacetic acid; ErA II, Erwinia chrysanthemi L-
asparaginase II; Fvnase, Flammulina velutipes asparaginase;
HPLC, high performance liquid chromatography; L-Asn, L-
asparagine; LB, Luria−Bertani broth; L-Gln, L-glutamine; M,
mol/L; mM, mmol/L; MluI, Endonuclease from Micrococcus
luteus; NCBI, National Center for Biotechnology Information;
PBS, phosphate-buffered saline; PCR, polymerase chain
reaction; RrA, Rhodospirillum rubrum L-asparaginase; SDS-
PAGE, sodium dodecyl sulfate-polyacrylamid gel electro-
phoresis; YpA, Yersinia pseudotuberculosis L-asparaginase
2
.45 mg/kg. However, the sample immersed in 8000 U/L L-
asparaginase solution contained only 0.46 mg/kg acrylamide,
corresponding to a decrease in the acrylamide level of about
8
2% (Figure 7c). This reduction in acrylamide was due to the
decrease of L-Asn, which is catalyzed by L-asparaginase. The
sample obtained from the supermarket had an acrylamide
content of 2.27 mg/kg (Figure 7d), showing that acrylamide
exists in fried potato slices. Paleologos et al. detected
acrylamide in some food samples by normal-phase HPLC.
Pedreschi reported that commercial L-asparaginase could
35
20
diminish the acrylamide content from 2047 to 158 μg/kg.
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