Alkaline Phosphatase

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Purification and characterization of alkaline


Cite this: RSC Adv., 2019, 9, 354
phosphatase from lactic acid bacteria
Yu-Hao Chu,ab Xin-Xin Yu,ab Xing Jin,ab Yu-Tang Wang,ab Duo-Jia Zhao,ab
Po Zhang,ab Guang-Mei Sunab and Ying-Hua Zhang *ab

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.

both been characterized from a number of organisms, such as


1 Introduction fungi, bacteria, actinomycetes and viruses.5 An enzyme capable
Organophosphorus compounds are widely used as pesticides in of degrading OPPs was produced by Flavobacterium sp. and
agriculture. Organophosphorus pesticides (OPPs) are essential identied as Sphingobium fuliginis.6 Spirulina platensis was
to modern agriculture, and extensively used to control pests and capable of excreting alkaline phosphatase showed the ability to
weeds in order to improve the agricultural harvest. Their degrade chlorpyrifos.7 With recent advances in molecular
persistent consumption worldwide has led to the accumulation biology, microbial systems and enzymes can be characterized
of these compounds in water and fertile land. This exposure and utilized more efficiently. Organophosphate hydrolase
presents a major hazard to ora, aquatic life, agricultural encoding gene has been isolated from different species, and has
products, and eventually human beings.1,2 Over the past decade, been sequenced, cloned in different organisms. Engineered
numerous ways have been developed to degrade OPPs, microorganisms have been tested for their ability to degrade
including physical degradation, chemical oxidation and bio- different specic pesticide. But challenge is that they cannot
logical methods. Recent interest has focused on the use of introduce in the eld and industry because they might cause
microbial organisms and enzymes as an efficient method. In some other environmental problems. Limitations of using
this scenario, OPPs-degrading enzymes have attracted more microbial enzyme for OPPs degradation in applications con-
attention as promising candidates due to their high efficiency sisted of the high cost of preparing pure enzyme due to low
compared to other methods.3,4 The identication of microbial yields and also poor enzyme stability.
enzymes that have the ability to degrade OPPs has resulted in Lactic acid bacteria (LAB), as a functional class of micro-
encouraging biotechnology platforms with noteworthy aerophilic Gram-positive bacteria, ferment lactose to primarily
applications. produce lactic acid. LAB has been of major concern to micro-
Enzyme based pesticide degradation is an innovative biologists since human beings began eating fermented foods
method for removal of pesticide from polluted environments via the traditional biotechnology.8,9 LAB plays crucial roles in
and raw material. There are classes of microbial enzymes that manufacturing of fermented milk products, vegetables, meat
have the ability to degrade harmful OPPs. The most studied and and wine.
potential OPPs-degrading enzymes are organophosphorus Nowadays, LAB was investigated for the effects on OPPs
hydrolase and organophosphorus acid anhydrolase, which have degradation. For example, Lactobacillus brevis WCP902 and
Lactobacillus plantarum were studied for their abilities to
a
Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural degrade OPPs in kimchi and wheat dough.10,11 Researchers
University, China. E-mail: [email protected]; Fax: +86 451 5519 0340; measured the degradation kinetics of seven OPPs in yoghurt
Tel: +86 451 55190479 fermentation and in skimmed milk cultured with Lactobacillus
b
Department of Food Science, Northeast Agricultural University, Harbin 150030, P. R. sp.12,13 In addition, researchers began to pay more attention to
China

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 dened 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 purication 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 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 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 puried
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 purication
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
Faireld, 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. 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 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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RSC Advances Paper

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 Purication 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 aer 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 puried by gel ltration chromatography Superdex 75
¼  þ
V Vmax ½S Vmax (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

Table 1 Purification of ALP from lactic acid bacteria

Purication step Total activity (units) Total protein (mg) Specic activity (U mg1) Purication fold Recovery (%)

Crude extract 1876.00 547.55 3.43 1.00 100.00


(NH4)2SO4 1432.80 239.00 5.99 1.74 76.00
DEAE-Sephadex 1064.52 23.06 39.17 11.42 56.74
Superdex 75 445.36 3.60 123.71 36.07 23.74

356 | RSC Adv., 2019, 9, 354–360 This journal is © The Royal Society of Chemistry 2019
Paper RSC Advances

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
Fig. 2 SDS-PAGE (12%) pattern of purified ALP from lactic acid
bacteria. lane 1: marker; lane 2: crude enzyme; lane 3: the supernatant may also exist. A monomeric ALP from Thermotoga neapoli-
after precipitation by ammonium sulfate; lane 4: the sample after DEAE tana with a molecular mass of 45 kDa has been reported.26 B.
Sepharose Fast Flow; lane 5: the sample after Superdex 75. cereus produced extracellular and membrane-bound ALP,
which molecular weight was estimated to be 44 kDa.27 In the
activity of 123.71 U mg1 protein with a yield of 23.74%. The present study, the single predominant band corresponding
relatively low yield is attributed to the fact that only fractions to active ALP was detected on SDS-PAGE, suggesting that the
from the centers of chromatographic peaks and active bands molecular mass of puried alkaline phosphatase is 43 kDa.
were collected to ensure the purication. The enzyme is comparable with that of microbial enzymes,

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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RSC Advances Paper

Table 2 Effect of various metal ions and EDTA on the activity of ALPa

Relative activity (%)


Metal ions
and EDTA 1 mmol L1 2 mmol L1 3 mmol L1 4 mmol L1 5 mmol L1

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 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+.
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 conrmed 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 aer 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 conrmed as 37  C (Fig. 3c). The enzyme is The essential role of Ca2+ and Mg2+ in catalyzing and stabilizing
sensitive to heating aer 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 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
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 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.

358 | RSC Adv., 2019, 9, 354–360 This journal is © The Royal Society of Chemistry 2019
Paper RSC Advances

Table 3 Concentration of dimethoate in buffer solution treated by ALPa

The concentration (mg kg1) at different times (h)

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 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 kg1
and 0.74 mg kg1 h1, 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
Fig. 6 Lineweaver–Burk plot for the Michaelis–Menten constant (Km)
OPPs. The results shown in this study provided unequivocal
and the maximum velocity (Vmax) for the ALP with dimethoate as evidences for the involvement of an ALP that can accelerate
substrate. degradation of OPPs. These ndings make LAB as a tool for
bioremediation by either employing the whole cells in effluent
enzyme structure and for the catalytic activity of ALP. Mg2+ treatment food or by immobilizing the puried enzyme on
controls the occupancy of the catalytic and structural binding a strong matrix to decontaminate polluted raw materials in food
sites, and modulates the enzymatic activity. industry.
It is generally accepted that ions are essential not only for the
catalytic activity of ALP, but also for the stability of the enzyme. 4 Conclusions
The similar results were also in agreement with previous
studies. The ALPs from caespitosus and S. thermophilum are also In this study we reported the purication of ALP from LAB, and
activated by Mg2+.34,35 The stimulation by Mg2+ is non-selective, determined some of their molecular and enzymatic properties.
and Mn2+ or Ca2+ can replace Mg2+ to stimulate the enzyme.36 Because of the preservation of activity at the relatively broad
The possible reason of Zn2+ inhibition of the activity of ALP range of temperature and pH, the isolated ALP from LAB could
could be attributed to the displacement of Mg2+.37 The key be of signicant biotechnological potential. The results of this
amino acids that facilitate the formation of hydrogen bonds paper were to characterize the ALP and to analyze the effect of
between pesticides and the ALP were as Gln375, Asp55 or metals ions on enzyme activity in vitro to estimate its potential
Thr413.38 Binding of the Zn2+ and Cu2+ in the active site might use as OPPs cleaner for food process. The present study will
impede the hydrogen bonds. help in nding an exact correlation between the pesticide
degradation and the ALP in the LAB fermentation.
3.4 Kinetic properties of ALP
The method of initial rates was used in determining the kinetic Conflicts of interest
parameters. The Lineweaver–Burk plot for the ALP with p-NPP
The authors declare that they have no conict of interest.
as substrate was shown in Fig. 4. Km of the enzyme employing p-
NPP as substrate was 2.14 mmol L1, and the Vmax of the
enzyme was 0.19 mmol L1 min1. Acknowledgements
This work was funded by the National Natural Science Foun-
3.5 Accelerated degradation of dimethoate by ALP dation of China (Grant No. 31571930).
Dimethoate was treated through liquid–liquid extraction.
Typical GC proles for the present study are shown in Fig. 5. References
The detection of the dimethoate was in the range of 0.005–
0.010 mg kg1, which ensured a precise measurement of the 1 P. Berny, J. Vet. Pharmacol. Ther., 2007, 30, 93–100.
dimethoate in buffer. The samples with different dimethoate 2 G. M. Calvert, J. Karnik, L. Mehler, J. Beckman, B. Morrissey,
concentrations cultured with ALP at 37  C for 4 h, and the J. Sievert, R. Barrett, M. Lackovic, L. Mabee, A. Schwartz,

This journal is © The Royal Society of Chemistry 2019 RSC Adv., 2019, 9, 354–360 | 359
RSC Advances Paper

Y. Mitchell and S. Moraga-McHaley, Am. J. Ind. Med., 2008, 21 M. M. Bradford, Anal. Biochem., 1976, 72, 248–254.
51, 883–898. 22 U. K. Laemmli, Nature, 1970, 227, 680–685.
3 G. Gotthard, J. Hiblot, D. Gonzalez, M. Elias and 23 L. Bo and X. Zhao, Afr. J. Microbiol. Res., 2010, 4, 1171–1179.
E. Chabriere, PLoS One, 2013, 8, e7799511. 24 T. Ansai, S. Awano, X. Chen, T. Fuchi, T. Arimoto, S. Akifusa
4 R. Iyer, B. Iken and A. Damania, Environ. Microbiol. Rep., and T. Takehara, FEBS Lett., 1998, 428, 157–160.
2013, 5, 787–798. 25 F. M. Hulett, E. E. Kim, C. Bookstein, N. V. Kapp,
5 J. P. Verma, D. K. Jaiswal and R. Sagar, Rev. Environ. Sci. Bio/ C. W. Edwards and H. W. Wyckoff, J. Biol. Chem., 1991,
Technol., 2014, 13, 429–466. 266, 1077–1184.
6 K. Kawahara, A. Tanaka, J. Yoon and A. Yokota, J. Gen. Appl. 26 G. Dong and J. G. Zeikus, Enzyme Microb. Technol., 1997, 21,
Microbiol., 2010, 56(3), 249–255. 335–340.
7 R. R. M. Thengodkar and S. Sivakami, Biodegradation, 2010, 27 S. Kostadinova and M. Marhova, Biotechnol. Biotechnol.
21(4), 637–644. Equip., 2010, 24, 602–606.
8 G. Giraffa, FEMS Microbiol. Rev., 2004, 28, 251–260. 28 A. Barbosa Junior, L. H. S. Guimaraes, H. F. Terenzi,
9 G. W. Tannock, Appl. Environ. Microbiol., 2004, 70, 3189– J. A. Jorge, F. A. Leone and M. L. T. M. Polizeli, Folia
3194. Microbiol., 2008, 53, 509–516.
10 T. M. Dordevic, S. S. Siler-Marinkovic, R. D. Durovic-Pejcev, 29 M. L. Bonet, F. I. Llorca and E. Cadenas, Int. J. Biochem.,
S. I. Dimitrijevic-Brankovic and J. S. G. Umiljendic, Lett. 1992, 24, 839–845.
Appl. Microbiol., 2013, 57, 412–419. 30 L. Guimaraes, H. F. Terenzi, J. A. Jorge, F. A. Leone and
11 S. M. A. Islam, R. K. Math, K. M. Cho, W. J. Lim, S. Y. Hong, M. Polizeli, Folia Microbiol., 2003, 48, 627–632.
J. M. Kim, M. G. Yun, J. J. Cho and H. D. Yun, J. Agric. Food 31 M. T. Mazorra, J. A. Rubio and J. Blasco, Comp. Biochem.
Chem., 2010, 58, 5380–5386. Physiol., Part B: Biochem. Mol. Biol., 2002, 131, 241–249.
12 L. Bo, Y. Zhang and X. Zhao, J. Serb. Chem. Soc., 2011, 76, 32 H. Wu, D. Li, B. Zhu, J. Cheng, J. Sun, F. Wang, Y. Yang,
353–362. Y. Song and C. Yu, Fish. Sci., 2013, 79, 477–485.
13 X. Zhao and J. Wang, Food Chem., 2012, 131, 300–304. 33 K. R. Bogo, D. C. Masui, F. A. Leone, J. A. Jorge and
14 E. S. Gilbert, A. W. Walker and J. D. Keasling, Appl. Microbiol. R. P. M. Furriel, Folia Microbiol., 2006, 51, 431–437.
Biotechnol., 2003, 61, 77–81. 34 L. H. S. Guimaraes, H. F. Terenzi and J. A. Jorge, Folia
15 Y. Kim, J. Ahn, S. Moon and J. Lee, Chemosphere, 2005, 60, Microbiol., 2003, 48(5), 627–632.
1349–1355. 35 L. H. S. Guimaraes, H. F. Terenzi, J. A. Jorge and
16 R. D. Richins, I. Kaneva, A. Mulchandani and W. Chen, Nat. M. L. T. M. Polizeli, J. Ind. Microbiol. Biotechnol., 2001,
Biotechnol., 1997, 15, 984–987. 27(4), 265–270.
17 R. R. M. Thengodkar and S. Sivakami, Biodegradation, 2010, 36 M. Politino, J. Brown and J. J. Usher, Prep. Biochem.
21, 637–644. Biotechnol., 1996, 26(3–4), 171–181.
18 Y. Zhang, D. Xu, J. Liu and X. Zhao, Food Chem., 2014, 164, 37 A. Barbosa, L. H. S. Guimaraes, H. F. Terenzi, J. A. Jorge,
173–178. F. A. Leone and M. L. T. M. Polizeli, Folia Microbiol., 2008,
19 B. L. Wanner, J. Cell. Biochem., 1993, 51, 47–54. 53(6), 509–516.
20 C. Sasikala, S. Jiwal, P. Rout and M. Ramya, World J. 38 Y. H. Chu, Y. Li, Y. T. Wang, B. Li and Y. H. Zhang, Food
Microbiol. Biotechnol., 2012, 28, 1301–1308. Chem., 2018, 254, 80–86.

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