Alkaline Phosphatase
Alkaline Phosphatase
Alkaline Phosphatase
PAPER
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.
Received 28th October 2018
Accepted 16th December 2018
The enzyme activity could be activated by Mg2+, Ca2+, and inhibited by Cu2+, Zn2+, and EDTA. The
Michaelis–Menten constant was 6.05 mg kg1 with dimethoate as the substrate according to the
DOI: 10.1039/c8ra08921c
Lineweaver–Burk plots. These results highlight an important potential use of ALP from LAB for the
rsc.li/rsc-advances cleanup of pesticide pollution in raw materials for the food industry.
354 | RSC Adv., 2019, 9, 354–360 This journal is © The Royal Society of Chemistry 2019
Paper RSC Advances
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 One unit (U) of phosphatase activity is dened as the amount
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
However, the purication of alkaline phosphatase from LAB has
not been reported. 2.3 Preparation of crude enzyme
Alkaline phosphatase (EC 3.1.3.1, ALP) is hydrolase that has The medium was inoculated with Lactobacillus casei. 355 at
been implemented to remove phosphate groups from many 37 C for 24 h in Erlenmeyer asks, and harvested by centrifu-
types of molecules, such as nucleotides, proteins, and alkaloids. gation at 10 000 g for 10 min at 4 C. The cell pellet was
The process of removing the phosphate group is dephosphor- washed three times with carbonate–bicarbonate buffer
ylation. Many bacteria produce ALP, such as Escherichia coli, (10 mmol L1, pH 10.5), resuspended in carbonate–bicarbonate
Bacillus species, Mycobacterium smegmatis, Thermotogamaritime, buffer and agitated for 1 min. The mixture was sonicated 10
Haloarcula marismortui, especially Gram-negative bacteria times (10 s at 15 s interval), as described in a previous study, and
starved of inorganic phosphate.19 LAB is permitted for pro- centrifuged at 10 000 g and 4 C for 15 min.20 The supernatant
cessing in food raw materials. was collected for subsequent enzyme purication.
In order to investigate the fundamental basis of OPPs
biodegradation by the phosphatase from LAB, the purication
of ALP from LAB was developed in this study. More knowledge 2.4 Ammonium sulfate precipitation
about the physicochemical and kinetic characteristics of the
The crude enzyme solution was added ammonium sulfate to the
enzyme is necessary before it can be employed as OPPs
degree of 80% saturation, and was kept at 4 C overnight.
biodegradation tool. The purity of the products was analyzed by
Subsequently, the precipitate was then collected by centrifuga-
electrophoresis, and some characteristics of the puried
tion at 12 000 g for 10 min at 4 C. The protein was dissolved
enzyme were determined. The research targeted critical issues
in minimal amount of Tris–HCl buffer (50 mmol L1, pH 7.4),
of industrial applications including enzymatic catalysis,
and dialyzed against the same buffer for 24 h at 4 C.
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
2.5 ALP purication
successful implementation of OPPs degradation systems under
a variety of eld conditions. Characterizing the enzyme involved The concentrated crude enzyme was loaded onto DEAE
in the application represents new areas for food industry. Sepharose column (16 200 mm) pre-equilibrated with
50 mmol L1 Tris–HCl buffer (pH 7.4). The column was washed
with buffer until no protein was detected in the elutriate. Then
2 Experimental it was eluted using a gradient of 0–1 mol L1 NaCl in the same
buffer at ow rate as 2 mL min1. Fractions of 5 mL were
2.1 Materials collected and analyzed for protein content and enzyme activity.
The lyophilized Lactobacillus casei. 355 was obtained from the Fractions containing ALP were pooled, dialyzed overnight, and
Centre of Key Laboratory of Dairy Science (Harbin, China). p- lyophilized.
Nitrophenyl phosphate was from Sigma Chemical Co. (Saint The lyophilized enzyme was dissolved in Tris–HCl buffer
Louis, MO, USA). DEAE Sepharose Fast Flow and Superdex 75 (50 mmol L1, pH 7.4). The enzyme solution from the previous
were purchased from GE Healthcare Bio-Sciences (Connecticut, step was processed on a column with Superdex 75 (10 300
Faireld, USA). Dimethoate standard was purchased from mm) equilibrated previously with Tris–HCl buffer. Elution was
Sigma Chemical Co. (Saint Louis, MO, USA). All chemicals used performed with the same buffer at a ow rate 0.8 mL min1.
were of analytical agents. Fractions containing ALP were pooled, dialyzed against Tris–
HCl buffer and lyophilized.
The protein concentration was measured following the
2.2 Determination of ALP activity method of Bradford, by using bovine serum albumin as
The activity of ALP was assayed using a colourless substrate p- standard.21
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 L1 p-NPP (prepared in the carbonate 2.6 Sodium dedecylsulfate polyacrylamide gel
buffer containing 1 mmol L1 MgCl2), 1.0 mL carbonate buffer electrophoresis
(10 mmol L1, 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. Aer (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 L1 NaOH and markers with molecular weights of 14.4–97.4 kDa were used in
5 mmol L1 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.
2.7 Enzyme characteristics where [S] is the concentration of substrate, V and Vmax repre-
sented the initial and maximum reactions rate, respectively. Km
2.7.1 Optimum pH and temperature. The enzyme activities
is the Michaelis–Menten constant, which equals to the
of all samples were determined by using p-NPP as ALP substrate
substrate concentration when the rate is half of Vmax.
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 L1), 2.8 Degradation of dimethoate by ALP
Gly–NaOH (pH 8.5–9.5, 50 mmol L1), NaHCO3–Na2CO3 (pH The degradation of dimethoate was performed in buffer solu-
10–11, 10 mmol L1). The optimal temperature of the ALP was tion. A volume of 50 mL buffer was spiked with dimethoate of
determined by performing the standard enzyme assay at the different concentrate and ALP. The reaction mixture was stirred
temperature in the range of 22 to 67 C. at 37 C for 1, 2, 3, 4 h, respectively. Pesticides were extracted by
2.7.2 pH and thermal stability of ALP. For the pH stability a liquid extraction method. The conditions and procedures
determination, the samples were incubated in different buffers were the same as those previously reported.23 Finally, 1 mL of
from pH 6–11 at 4 C for 1 h, the relative residual activity was the extract (7.5 U mg1) was transferred into sample bottle for
assayed under standard conditions as described above. In order GC analysis by Gas Chromatography (Agilent 7890, Agilent
to determine the thermal stability, ALP was incubated for 3 h at Technologies, Inc, Santa Clara, USA) with 30 m 0.250 mm
temperatures in the range of 20 to 67 C, and the relative 0.25 mm capillary column DB-1701.
residual ALP activity was measured.
2.7.3 Effect of metal ions and EDTA on the activity of ALP. 2.9 Statistical analyses
The enzyme was measured in the presence of various metal ions
All the experiments were performed with at least three times. All
(Ca2+, Mg2+, Zn2+, Cu2+), and EDTA with nal concentrations of
the results were expressed as means of all trials. Standard
1–5 mmol L1. The ALP activity was determined by the standard
deviations were determined and reported.
assay as described above, with no metal ions and EDTA addi-
tions as 100%.
2.7.4 Determination of kinetic parameters. Catalytic 3 Results and discussion
activity of ALP was investigated at different p-NPP concentra- 3.1 Purication of ALP from lactic acid bacteria
tions under assay conditions. Km and Vmax were obtained using
The supernatant obtained from cell free extract was concen-
Lineweaver–Burk plots. The reciprocal of substrate concentra-
trated aer precipitation by ammonium sulfate. The protein
tion (1/S) was plotted against the reciprocal of reaction rate (1/V)
solution was then loaded into ion exchange chromatography
according to the following equation:
columns. A peak of the active ALP fraction was collected with
NaCl solution as eluate (Fig. 1A). The active ALP fraction was
1 Km 1 1 further puried by gel ltration chromatography Superdex 75
¼ þ
V Vmax ½S Vmax (Fig. 1B). The results of the purication were summarized in
Table 1. The enzyme was puried 36.07-fold to a specic
Purication step Total activity (units) Total protein (mg) Specic activity (U mg1) Purication fold Recovery (%)
356 | RSC Adv., 2019, 9, 354–360 This journal is © The Royal Society of Chemistry 2019
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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
Ca2+ 91.57 0.001Ae 96.88 0.011Bd 108.43 0.008Bc 119.75 0.011Bb 136.14 0.019Ba
Mg2+ 80.14 0.013Be 113.74 0.009Ad 143.76 0.028Ac 170.21 0.055Ab 188.00 0.002Aa
Zn2+ 47.23 0.007Ca 43.76 0.004Cb 38.11 0.009Cc 29.91 0.002Cd 25.29 0.004Ce
Cu2+ 26.10 0.005Ea 9.00 0.004Db 0Dc 0Dc 0Dc
EDTA 43.88 0.006Da 2.08 0.002Eb 0Dc 0Dc 0Dc
a
Values are given as the means SD. Different letters (A–E, a–e) in the same column and rank indicate signicant 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 signicantly enhanced by Ca2+ and Mg2+.
3.2 Optimal pH and temperature of ALP from lactic acid This enzyme was strongly inhibited by Cu2+, Zn2+ and EDTA. It
bacteria is clear that ALP from LAB is a metalloenzyme according to
The enzyme activities measured at various temperatures and pH these results. Experimental data shows that Ca2+ and Mg2+ has
were showed in Fig. 3. The optimum pH and temperature were clear activation effect to ALP, which may be a metal ion and
determined by the samples with the highest enzyme activity. enzyme active center union to promote the combination
The enzyme activities of the untreated samples were 100% at between the joint of the enzyme and substrate, so as to accel-
the pH stability and temperature tolerance tests. The enzyme erate the enzyme catalytic reaction. Its activity was reduced
had optimal activity at pH 8.5. The activity decreased above or nearly to zero in the presence of EDTA, which chelate the ions to
below pH 8.5, which conrmed that the enzyme was alkaline. destroy the active site of ALP resulting in the loss of enzyme
The activity maintained stable at the range of pH 8.5 to 10 aer activity. The highly sensitive and selective effects of Zn2+, Cu2+,
1 h of incubation in the buffer (Fig. 3a and b). Mg2+, and Ca2+ on the enzyme are based on their inhibiting,
The optimal environmental temperature for application of activating or reactivating effects on the catalytic activity of ALP.
the enzyme was conrmed as 37 C (Fig. 3c). The enzyme is The essential role of Ca2+ and Mg2+ in catalyzing and stabilizing
sensitive to heating aer incubation at various temperatures for the structure of this enzyme was recognized. As a prosthetic
3 h. The enzyme activity was stable below 27 C, with relative group of ALP, Ca2+ is necessary both for the preservation of the
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 puried 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
Fig. 4 Lineweaver–Burk plot for the Michaelis–Menten constant (Km)
cyanobacterium Spirulina platensis as 40 C,17 clam Scrobicularia 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 puried
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.
358 | RSC Adv., 2019, 9, 354–360 This journal is © The Royal Society of Chemistry 2019
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Dimethoate samples 0 1 2 3 4
1 1.089 0.032a 0.992 0.022a 0.877 0.049b 0.786 0.038b 0.662 0.042c
2 2.001 0.031a 1.944 0.016a 1.713 0.009b 1.554 0.035c 1.268 0.023d
3 3.127 0.028a 2.973 0.037a 2.663 0.021b 2.446 0.014c 2.219 0.011d
4 4.012 0.017a 3.838 0.046b 3.456 0.018c 3.147 0.019d 2.974 0.014e
5 5.137 0.006a 4.873 0.036b 4.464 0.017c 4.039 0.042d 3.621 0.034e
a
Values are given as the means SD. Different letters (a–e) in the same rank indicate signicant differences (P < 0.05).
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