Purification and characterization of alkaline phosphatase from lactic acid bacteria

Article abstract of DOI:10.1039/C8RA08921C

Alkaline phosphatase (ALP) excreted from lactic acid bacteria (LAB) showed the ability to degrade organophosphorus pesticides. This study reported the first purification and characterization of ALP from LAB. The molecular weight of ALP was estimated to be

Full text of DOI:10.1039/C8RA08921C

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Purification and characterization of alkaline
phosphatase from lactic acid bacteria
Cite this: RSC Adv., 2019, 9, 354
Yu-Hao Chu,ab Xin-Xin Yu,ab Xing Jin,ab Yu-Tang Wang,ab Duo-Jia Zhao,ab
ab
Po Zhang,ab Guang-Mei Sunab and Ying-Hua Zhang
*
Alkaline phosphatase (ALP) excreted from lactic acid bacteria (LAB) showed the ability to degrade
organophosphorus pesticides. This study reported the first purification and characterization of ALP from
LAB. The molecular weight of ALP was estimated to be 43 kDa measured by SDS-PAGE. The activity of
purified enzyme was determined with the binding of p-nitrophenyl phosphate as the substrate. The
results showed that the optimal temperature for ALP activity was 37 ꢀC, and the optimal pH was 8.5. But
ALP was stable at temperatures below 32 ꢀC. The ALP activity remained at 80% when the pH was 8–9.5.
The enzyme activity could be activated by Mg2+, Ca2+, and inhibited by Cu2+, Zn2+, and EDTA. The
Michaelis–Menten constant was 6.05 mg kgꢁ1 with dimethoate as the substrate according to the
Lineweaver–Burk plots. These results highlight an important potential use of ALP from LAB for the
cleanup of pesticide pollution in raw materials for the food industry.
Received 28th October 2018
Accepted 16th December 2018
DOI: 10.1039/c8ra08921c
rsc.li/rsc-advances
both been characterized from a number of organisms, such as
fungi, bacteria, actinomycetes and viruses.5 An enzyme capable
1 Introduction
of degrading OPPs was produced by Flavobacterium sp. and
identied as Sphingobium fuliginis.6 Spirulina platensis was
capable of excreting alkaline phosphatase showed the ability to
degrade chlorpyrifos.7 With recent advances in molecular
biology, microbial systems and enzymes can be characterized
and utilized more efficiently. Organophosphate hydrolase
encoding gene has been isolated from different species, and has
been sequenced, cloned in different organisms. Engineered
microorganisms have been tested for their ability to degrade
different specic pesticide. But challenge is that they cannot
introduce in the eld and industry because they might cause
some other environmental problems. Limitations of using
microbial enzyme for OPPs degradation in applications con-
sisted of the high cost of preparing pure enzyme due to low
yields and also poor enzyme stability.
Lactic acid bacteria (LAB), as a functional class of micro-
aerophilic Gram-positive bacteria, ferment lactose to primarily
produce lactic acid. LAB has been of major concern to micro-
biologists since human beings began eating fermented foods
via the traditional biotechnology.8,9 LAB plays crucial roles in
manufacturing of fermented milk products, vegetables, meat
and wine.
Organophosphorus compounds are widely used as pesticides in
agriculture. Organophosphorus pesticides (OPPs) are essential
to modern agriculture, and extensively used to control pests and
weeds in order to improve the agricultural harvest. Their
persistent consumption worldwide has led to the accumulation
of these compounds in water and fertile land. This exposure
presents a major hazard to ora, aquatic life, agricultural
products, and eventually human beings.1,2 Over the past decade,
numerous ways have been developed to degrade OPPs,
including physical degradation, chemical oxidation and bio-
logical methods. Recent interest has focused on the use of
microbial organisms and enzymes as an efficient method. In
this scenario, OPPs-degrading enzymes have attracted more
attention as promising candidates due to their high efficiency
compared to other methods.3,4 The identication of microbial
enzymes that have the ability to degrade OPPs has resulted in
encouraging biotechnology platforms with noteworthy
applications.
Enzyme based pesticide degradation is an innovative
method for removal of pesticide from polluted environments
and raw material. There are classes of microbial enzymes that
have the ability to degrade harmful OPPs. The most studied and
potential OPPs-degrading enzymes are organophosphorus
hydrolase and organophosphorus acid anhydrolase, which have
Nowadays, LAB was investigated for the effects on OPPs
degradation. For example, Lactobacillus brevis WCP902 and
Lactobacillus plantarum were studied for their abilities to
degrade OPPs in kimchi and wheat dough.10,11 Researchers
measured the degradation kinetics of seven OPPs in yoghurt
fermentation and in skimmed milk cultured with Lactobacillus
sp.12,13 In addition, researchers began to pay more attention to
aKey Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural
University, China. E-mail: yinghuazhang@neau.edu.cn; Fax: +86 451 5519 0340;
Tel: +86 451 55190479
bDepartment of Food Science, Northeast Agricultural University, Harbin 150030, P. R.
China
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phosphatase extracted from bacteria to degrade pesticides.14–16 2401 PC spectrophotometer (Shimadzu Corporation, Kyoto,
Spirulina platensis capable of excreting alkaline phosphatase Japan) at 405 nm.
showed ability to degrade chlorpyrifos.17 The phosphatase
production of the inoculated LAB might be one of the key of enzyme liberating 1 mmol of p-NP per minute at 37 C.
factors responsible for the accelerated OPPs degradation.18
One unit (U) of phosphatase activity is dened as the amount
ꢀ
However, the purication of alkaline phosphatase from LAB has
2.3 Preparation of crude enzyme
not been reported.
Alkaline phosphatase (EC 3.1.3.1, ALP) is hydrolase that has
been implemented to remove phosphate groups from many
types of molecules, such as nucleotides, proteins, and alkaloids.
The process of removing the phosphate group is dephosphor-
ylation. Many bacteria produce ALP, such as Escherichia coli,
Bacillus species, Mycobacterium smegmatis, Thermotogamaritime,
Haloarcula marismortui, especially Gram-negative bacteria
starved of inorganic phosphate.19 LAB is permitted for pro-
cessing in food raw materials.
The medium was inoculated with Lactobacillus casei. 355 at
37 C for 24 h in Erlenmeyer asks, and harvested by centrifu-
ꢀ
gation at 10 000 ꢂ g for 10 min at 4 ꢀC. The cell pellet was
washed three times with carbonate–bicarbonate buffer
(10 mmol Lꢁ1, pH 10.5), resuspended in carbonate–bicarbonate
buffer and agitated for 1 min. The mixture was sonicated 10
times (10 s at 15 s interval), as described in a previous study, and
centrifuged at 10 000 ꢂ g and 4 ꢀC for 15 min.20 The supernatant
was collected for subsequent enzyme purication.
In order to investigate the fundamental basis of OPPs
biodegradation by the phosphatase from LAB, the purication
of ALP from LAB was developed in this study. More knowledge
about the physicochemical and kinetic characteristics of the
enzyme is necessary before it can be employed as OPPs
biodegradation tool. The purity of the products was analyzed by
electrophoresis, and some characteristics of the puried
enzyme were determined. The research targeted critical issues
of industrial applications including enzymatic catalysis,
thermo-stability, the ability to function at high temperature and
extreme pH. The aim of the research provides more cost-
effective and efficient processes, and ultimately allows for
successful implementation of OPPs degradation systems under
a variety of eld conditions. Characterizing the enzyme involved
in the application represents new areas for food industry.
2.4 Ammonium sulfate precipitation
The crude enzyme solution was added ammonium sulfate to the
degree of 80% saturation, and was kept at 4 ꢀC overnight.
Subsequently, the precipitate was then collected by centrifuga-
tion at 12 000 ꢂ g for 10 min at 4 ꢀC. The protein was dissolved
in minimal amount of Tris–HCl buffer (50 mmol Lꢁ1, pH 7.4),
ꢀ
and dialyzed against the same buffer for 24 h at 4 C.
2.5 ALP purication
The concentrated crude enzyme was loaded onto DEAE
Sepharose column (16 ꢂ 200 mm) pre-equilibrated with
50 mmol Lꢁ1 Tris–HCl buffer (pH 7.4). The column was washed
with buffer until no protein was detected in the elutriate. Then
it was eluted using a gradient of 0–1 mol Lꢁ1 NaCl in the same
buffer at ow rate as 2 mL minꢁ1. Fractions of 5 mL were
collected and analyzed for protein content and enzyme activity.
Fractions containing ALP were pooled, dialyzed overnight, and
lyophilized.
2 Experimental
2.1 Materials
The lyophilized Lactobacillus casei. 355 was obtained from the
Centre of Key Laboratory of Dairy Science (Harbin, China). p-
Nitrophenyl phosphate was from Sigma Chemical Co. (Saint
Louis, MO, USA). DEAE Sepharose Fast Flow and Superdex 75
were purchased from GE Healthcare Bio-Sciences (Connecticut,
Faireld, USA). Dimethoate standard was purchased from
Sigma Chemical Co. (Saint Louis, MO, USA). All chemicals used
were of analytical agents.
The lyophilized enzyme was dissolved in Tris–HCl buffer
(50 mmol Lꢁ1, pH 7.4). The enzyme solution from the previous
step was processed on a column with Superdex 75 (10 ꢂ 300
mm) equilibrated previously with Tris–HCl buffer. Elution was
performed with the same buffer at a ow rate 0.8 mL minꢁ1
.
Fractions containing ALP were pooled, dialyzed against Tris–
HCl buffer and lyophilized.
The protein concentration was measured following the
method of Bradford, by using bovine serum albumin as
standard.21
2.2 Determination of ALP activity
The activity of ALP was assayed using a colourless substrate p-
nitrophenyl phosphate (p-NPP) as per the method, which yiel-
ded yellow p-nitrophenol (p-NP).17 The reaction mixture con-
sisted of 1.0 mL 5 mmol Lꢁ1 p-NPP (prepared in the carbonate
buffer containing 1 mmol Lꢁ1 MgCl2), 1.0 mL carbonate buffer
2.6 Sodium dedecylsulfate polyacrylamide gel
electrophoresis
(10 mmol Lꢁ1, pH 10.5), 1.0 mL crude enzyme solution and Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
distilled water, with a xed nal volume of 5.0 mL. Aer (SDS-PAGE) analysis was performed as per the method.22
a reaction time of 60 min at 37 ꢀC, the reaction was stopped by Sample solutions of 10 mL were loaded into each well. Protein
adding 3.0 mL termination buffer (0.1 mol Lꢁ1 NaOH and markers with molecular weights of 14.4–97.4 kDa were used in
5 mmol Lꢁ1 EDTA). The generated p-NP was measured by UV- analysis.
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Fig. 1 Purification of the ALP from lactic acid bacteria (A) anion exchange chromatography (DEAE-FF); (B) gel filtration molecular sieve Superdex
75.
where [S] is the concentration of substrate, V and Vmax repre-
sented the initial and maximum reactions rate, respectively. Km
is the Michaelis–Menten constant, which equals to the
2.7 Enzyme characteristics
2.7.1 Optimum pH and temperature. The enzyme activities
of all samples were determined by using p-NPP as ALP substrate
and spectrophotometric assay. In order to obtain the optimal
pH of ALP, the enzyme activities were tested in the appropriate
buffers or solution as phosphate buffer (pH 6–8, 20 mmol Lꢁ1),
Gly–NaOH (pH 8.5–9.5, 50 mmol Lꢁ1), NaHCO3–Na2CO3 (pH
10–11, 10 mmol Lꢁ1). The optimal temperature of the ALP was
determined by performing the standard enzyme assay at the
substrate concentration when the rate is half of Vmax
.
2.8 Degradation of dimethoate by ALP
The degradation of dimethoate was performed in buffer solu-
tion. A volume of 50 mL buffer was spiked with dimethoate of
different concentrate and ALP. The reaction mixture was stirred
at 37 ꢀC for 1, 2, 3, 4 h, respectively. Pesticides were extracted by
a liquid extraction method. The conditions and procedures
were the same as those previously reported.23 Finally, 1 mL of
the extract (7.5 U mgꢁ1) was transferred into sample bottle for
GC analysis by Gas Chromatography (Agilent 7890, Agilent
Technologies, Inc, Santa Clara, USA) with 30 m ꢂ 0.250 mm ꢂ
0.25 mm capillary column DB-1701.
ꢀ
temperature in the range of 22 to 67 C.
2.7.2 pH and thermal stability of ALP. For the pH stability
determination, the ꢀsamples were incubated in different buffers
from pH 6–11 at 4 C for 1 h, the relative residual activity was
assayed under standard conditions as described above. In order
to determine the thermal stability, ALP was incubated for 3 h at
temperatures in the range of ꢁ20 to 67 ꢀC, and the relative
residual ALP activity was measured.
2.7.3 Effect of metal ions and EDTA on the activity of ALP.
The enzyme was measured in the presence of various metal ions
(Ca2+, Mg2+, Zn2+, Cu2+), and EDTA with nal concentrations of
1–5 mmol Lꢁ1. The ALP activity was determined by the standard
assay as described above, with no metal ions and EDTA addi-
tions as 100%.
2.7.4 Determination of kinetic parameters. Catalytic
activity of ALP was investigated at different p-NPP concentra-
tions under assay conditions. Km and Vmax were obtained using
Lineweaver–Burk plots. The reciprocal of substrate concentra-
tion (1/S) was plotted against the reciprocal of reaction rate (1/V)
according to the following equation:
2.9 Statistical analyses
All the experiments were performed with at least three times. All
the results were expressed as means of all trials. Standard
deviations were determined and reported.
3 Results and discussion
3.1 Purication of ALP from lactic acid bacteria
The supernatant obtained from cell free extract was concen-
trated aer precipitation by ammonium sulfate. The protein
solution was then loaded into ion exchange chromatography
columns. A peak of the active ALP fraction was collected with
NaCl solution as eluate (Fig. 1A). The active ALP fraction was
further puried by gel ltration chromatography Superdex 75
(Fig. 1B). The results of the purication were summarized in
Table 1. The enzyme was puried 36.07-fold to a specic
1
Km
1
1
¼
ꢂ
þ
V
Vmax ½Sꢃ Vmax
Table 1 Purification of ALP from lactic acid bacteria
Purication step
Total activity (units)
Total protein (mg)
Specic activity (U mgꢁ1
)
Purication fold
Recovery (%)
Crude extract
(NH4)2SO4
DEAE-Sephadex
Superdex 75
1876.00
1432.80
1064.52
445.36
547.55
239.00
23.06
3.60
3.43
5.99
39.17
123.71
1.00
1.74
11.42
36.07
100.00
76.00
56.74
23.74
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The SDS-PAGE pattern of samples at different purication
steps was showed in Fig. 2. Aer the chromatography
process, ALP displayed a single protein band on the electro-
phoresis gel with a molecular weight of about 43 kDa. The
active ALP from LAB appeared on SDS-PAGE gels as a mono-
mer with molecular weight of 43 kDa. The novel alkaline
phosphatase, Pi-alkaline phosphatase showed as homodimer
with molecular weight of about 54 kDa.24 ALP from B. subtilis
is a dimer made up of two subunits with identical molecular
weight between 35 to 70 kDa.25 Although most ALP from
bacterial and vertebrate are homodimers, monomeric forms
may also exist. A monomeric ALP from Thermotoga neapoli-
tana with a molecular mass of 45 kDa has been reported.26 B.
cereus produced extracellular and membrane-bound ALP,
which molecular weight was estimated to be 44 kDa.27 In the
present study, the single predominant band corresponding
to active ALP was detected on SDS-PAGE, suggesting that the
molecular mass of puried alkaline phosphatase is 43 kDa.
The enzyme is comparable with that of microbial enzymes,
Fig. 2 SDS-PAGE (12%) pattern of purified ALP from lactic acid
bacteria. lane 1: marker; lane 2: crude enzyme; lane 3: the supernatant
after precipitation by ammonium sulfate; lane 4: the sample after DEAE
Sepharose Fast Flow; lane 5: the sample after Superdex 75.
activity of 123.71 U mgꢁ1 protein with a yield of 23.74%. The
relatively low yield is attributed to the fact that only fractions
from the centers of chromatographic peaks and active bands
were collected to ensure the purication.
Fig. 3 Effect of pH and temperature on the activity of the ALP from lactic acid bacteria. (a) The optimal pH determined by the enzyme assay at
varied pH; (b) the pH stability determined by the residual activity of ALP after incubating the enzyme with buffers at different pH (6–11) for 1 h; (c)
the optimal temperature for ALP by performing the enzyme assay at 22–67 ꢀC; (d) the thermal stability determined by the residual activity of ALP
after incubating the enzyme at different temperature (22–67 ꢀC) for 3 h.
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Table 2 Effect of various metal ions and EDTA on the activity of ALPa
Relative activity (%)
Metal ions
and EDTA
1 mmol Lꢁ1
2 mmol Lꢁ1
3 mmol Lꢁ1
4 mmol Lꢁ1
5 mmol Lꢁ1
Ca2+
Mg2+
Zn2+
Cu2+
EDTA
91.57 ꢄ 0.001Ae
80.14 ꢄ 0.013Be
47.23 ꢄ 0.007Ca
26.10 ꢄ 0.005Ea
43.88 ꢄ 0.006Da
96.88 ꢄ 0.011Bd
113.74 ꢄ 0.009Ad
43.76 ꢄ 0.004Cb
9.00 ꢄ 0.004Db
2.08 ꢄ 0.002Eb
108.43 ꢄ 0.008Bc
143.76 ꢄ 0.028Ac
38.11 ꢄ 0.009Cc
0Dc
119.75 ꢄ 0.011Bb
170.21 ꢄ 0.055Ab
29.91 ꢄ 0.002Cd
0Dc
136.14 ꢄ 0.019Ba
188.00 ꢄ 0.002Aa
25.29 ꢄ 0.004Ce
0Dc
0Dc
0Dc
0Dc
a
Values are given as the means ꢄ SD. Different letters (A–E, a–e) in the same column and rank indicate signicant differences (P < 0.05).
lower than most mammalian ALP with the molecular weight and catalytic activity of the enzyme. The effects of metal ions at
in the range of 130 to 170 kDa.
different concentrations on ALP activity were shown (Table 2).
The ALP activity was signicantly enhanced by Ca2+ and Mg2+.
This enzyme was strongly inhibited by Cu2+, Zn2+ and EDTA. It
is clear that ALP from LAB is a metalloenzyme according to
these results. Experimental data shows that Ca2+ and Mg2+ has
clear activation effect to ALP, which may be a metal ion and
enzyme active center union to promote the combination
between the joint of the enzyme and substrate, so as to accel-
erate the enzyme catalytic reaction. Its activity was reduced
nearly to zero in the presence of EDTA, which chelate the ions to
destroy the active site of ALP resulting in the loss of enzyme
activity. The highly sensitive and selective effects of Zn2+, Cu2+,
Mg2+, and Ca2+ on the enzyme are based on their inhibiting,
activating or reactivating effects on the catalytic activity of ALP.
The essential role of Ca2+ and Mg2+ in catalyzing and stabilizing
the structure of this enzyme was recognized. As a prosthetic
group of ALP, Ca2+ is necessary both for the preservation of the
3.2 Optimal pH and temperature of ALP from lactic acid
bacteria
The enzyme activities measured at various temperatures and pH
were showed in Fig. 3. The optimum pH and temperature were
determined by the samples with the highest enzyme activity.
The enzyme activities of the untreated samples were 100% at
the pH stability and temperature tolerance tests. The enzyme
had optimal activity at pH 8.5. The activity decreased above or
below pH 8.5, which conrmed that the enzyme was alkaline.
The activity maintained stable at the range of pH 8.5 to 10 aer
1 h of incubation in the buffer (Fig. 3a and b).
The optimal environmental temperature for application of
the enzyme was conrmed as 37 ꢀC (Fig. 3c). The enzyme is
sensitive to heating aer incubation at various temperatures for
ꢀ
3 h. The enzyme activity was stable below 27 C, with relative
80% activity compared to the sample at ꢁ20 ꢀC. The activity
decreased rapidly when the temperature was above 37 ꢀC. 39.0,
80.1 and 84.4% loss in enzymatic activity was observed upon
ꢀ
incubation at 37, 47, 57 C, respectively (Fig. 3d).
The puried ALP from the LAB displayed maximum activity
at pH 8.5 and 37 ꢀC with p-NPP as substrate. The optimal pH of
ALP from Lactobacillus is similar with those of enzymes from
other microbe species such as Rhizopus microsporus,28 Hal-
obacterium halobium,29 lamentous fungus Aspergillus caes-
pitosus.30 The optimal temperature for ALP from LAB
corresponded to the growth temperature of the bacterium. The
results were similar to those reported by others, such as
cyanobacterium Spirulina platensis as 40 ꢀC,17 clam Scrobicularia
Fig. 4 Lineweaver–Burk plot for the Michaelis–Menten constant (Km)
and the maximum velocity (Vmax) for the ALP with p-NPP as substrate.
plana as 30–40 C,31 Stichopus japonicus as 40 C,32 but lower
than that of the enzyme from Neurospora crassa as 65 ꢀC,33
ꢀ
ꢀ
Thermotoga neapolitana as 85 C.26 The activity of the puried
ꢀ
ALP has shown to be stable during storage, and capable of
keeping activity at low temperature and over a wide range of pH.
These properties would make it ideal for the enzyme to be
immobilized on a matrix. The low temperature stability also
indicates that the biomass should be cultivated in low
temperature fermentation.
3.3 Effects of EDTA and metal ions on ALP activity
It has certain signicance to explore the characteristics of ALP
and to investigate the role of ions on the molecular structure Fig. 5 Typical GC profiles of dimethoate for a standard solution.
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Table 3 Concentration of dimethoate in buffer solution treated by ALPa
The concentration (mg kgꢁ1) at different times (h)
Dimethoate samples
0
1
2
3
4
1
2
3
4
5
1.089 ꢄ 0.032a
2.001 ꢄ 0.031a
3.127 ꢄ 0.028a
4.012 ꢄ 0.017a
5.137 ꢄ 0.006a
0.992 ꢄ 0.022a
1.944 ꢄ 0.016a
2.973 ꢄ 0.037a
3.838 ꢄ 0.046b
4.873 ꢄ 0.036b
0.877 ꢄ 0.049b
1.713 ꢄ 0.009b
2.663 ꢄ 0.021b
3.456 ꢄ 0.018c
4.464 ꢄ 0.017c
0.786 ꢄ 0.038b
1.554 ꢄ 0.035c
2.446 ꢄ 0.014c
3.147 ꢄ 0.019d
4.039 ꢄ 0.042d
0.662 ꢄ 0.042c
1.268 ꢄ 0.023d
2.219 ꢄ 0.011d
2.974 ꢄ 0.014e
3.621 ꢄ 0.034e
a
Values are given as the means ꢄ SD. Different letters (a–e) in the same rank indicate signicant differences (P < 0.05).
dimethoate concentration in the samples had decreasing trend
along with the reaction time (Table 3). Fig. 6 showed the Line-
weaver–Burk plot for the ALP with dimethoate as substrate. The
Km and the Vmax of the ALP for dimethoate were 6.05 mg kgꢁ1
and 0.74 mg kgꢁ1 hꢁ1, respectively.
The evidence of OPPs biodegradation by the puried enzyme
from an edible microbial organism was provided in this study.
Since LAB can be used in the food processing for a variety of raw
materials, using LAB for OPPs biodegradation appears to be an
inexpensive and ideal option for feed and fodder to degrade
OPPs. The results shown in this study provided unequivocal
evidences for the involvement of an ALP that can accelerate
degradation of OPPs. These ndings make LAB as a tool for
bioremediation by either employing the whole cells in effluent
treatment food or by immobilizing the puried enzyme on
a strong matrix to decontaminate polluted raw materials in food
industry.
Fig. 6 Lineweaver–Burk plot for the Michaelis–Menten constant (Km)
and the maximum velocity (Vmax) for the ALP with dimethoate as
substrate.
enzyme structure and for the catalytic activity of ALP. Mg2+
controls the occupancy of the catalytic and structural binding
sites, and modulates the enzymatic activity.
It is generally accepted that ions are essential not only for the
catalytic activity of ALP, but also for the stability of the enzyme.
The similar results were also in agreement with previous
studies. The ALPs from caespitosus and S. thermophilum are also
4 Conclusions
In this study we reported the purication of ALP from LAB, and
determined some of their molecular and enzymatic properties.
Because of the preservation of activity at the relatively broad
range of temperature and pH, the isolated ALP from LAB could
be of signicant biotechnological potential. The results of this
paper were to characterize the ALP and to analyze the effect of
metals ions on enzyme activity in vitro to estimate its potential
use as OPPs cleaner for food process. The present study will
help in nding an exact correlation between the pesticide
degradation and the ALP in the LAB fermentation.
activated by Mg2+ 34,35 The stimulation by Mg2+ is non-selective,
.
and Mn2+ or Ca2+ can replace Mg2+ to stimulate the enzyme.36
The possible reason of Zn2+ inhibition of the activity of ALP
could be attributed to the displacement of Mg2+
.
The key
37
amino acids that facilitate the formation of hydrogen bonds
between pesticides and the ALP were as Gln375, Asp55 or
Thr413.38 Binding of the Zn2+ and Cu2+ in the active site might
impede the hydrogen bonds.
3.4 Kinetic properties of ALP
Conflicts of interest
The method of initial rates was used in determining the kinetic
parameters. The Lineweaver–Burk plot for the ALP with p-NPP
as substrate was shown in Fig. 4. Km of the enzyme employing p-
NPP as substrate was 2.14 mmol Lꢁ1, and the Vmax of the
The authors declare that they have no conict of interest.
enzyme was 0.19 mmol Lꢁ1 minꢁ1
.
Acknowledgements
This work was funded by the National Natural Science Foun-
dation of China (Grant No. 31571930).
3.5 Accelerated degradation of dimethoate by ALP
Dimethoate was treated through liquid–liquid extraction.
Typical GC proles for the present study are shown in Fig. 5.
The detection of the dimethoate was in the range of 0.005–
0.010 mg kgꢁ1, which ensured a precise measurement of the
dimethoate in buffer. The samples with different dimethoate
concentrations cultured with ALP at 37 ꢀC for 4 h, and the
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