Purification and characterization of carbon-phosphorus bond-cleavage enzyme from glyphosate degrading pseudomonas putida T5

Article abstract of DOI:10.1080/10826068.2014.923448

An inducible, carbon-phosphorus bond-cleavage enzyme was purified from cells of Pseudomonas putida T5 grown on N-phosphonomethyl glycine. The native enzyme had a molecular mass of approximately 70 kD and upon sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), yielded a homogeneous protein band with an apparent molecular mass of about 70 kD. Activity of purified enzyme was increased by 627-fold compared to the crude extract and showed pH and temperature optima of approximately 7 and 30°C, respectively. The purified enzyme had an apparent Km and Vmax of 3.7 mM and 6.8 mM/min, respectively, for its sole substrate N-phosphonomethyl glycine. The enzyme was inhibited by phenylmethylsulfonyl fluoride (PMSF), indicating the presence of serine at the active site. The enzyme was not inhibited by SDS, suggesting the absence of disulfide linkage in the enzyme. The enzyme was found to be inhibited by most of the metals studied except Mg²⁺. Detergents studied also inhibited glyphosate acting as a carbon-phosphorus bond-cleavage enzyme. Thus initial characterization of the purified enzyme suggested that it could be used as a potential candidate for glyphosate bioremediation.

Full text of DOI:10.1080/10826068.2014.923448

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Purification and Characterization of
Carbon–Phosphorus Bond-Cleavage
Enzyme From Glyphosate Degrading
Pseudomonas putida T5
a
a
A. Arul Selvi & H. K. Manonmani
a
Fermentation Technology and Bioengineering Department , Central
Food Technological Research Institute (CSIR) , Mysore , Karnataka ,
India
Accepted author version posted online: 19 May 2014.Published
online: 25 Sep 2014.
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To cite this article: A. Arul Selvi & H. K. Manonmani (2015) Purification and Characterization of
Carbon–Phosphorus Bond-Cleavage Enzyme From Glyphosate Degrading Pseudomonas putida T5,
Preparative Biochemistry and Biotechnology, 45:4, 380-397, DOI: 10.1080/10826068.2014.923448
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Preparative Biochemistry & Biotechnology, 45:380–397, 2015
Copyright # Taylor & Francis Group, LLC
ISSN: 1082-6068 print/1532-2297 online
DOI: 10.1080/10826068.2014.923448
Purification and Characterization of Carbon–Phosphorus
Bond-Cleavage Enzyme From Glyphosate Degrading
Pseudomonas putida T5
A. Arul Selvi and H. K. Manonmani
Fermentation Technology and Bioengineering Department, Central Food Technological
Research Institute (CSIR), Mysore, Karnataka, India
An inducible, carbon-phosphorus bond-cleavage enzyme was purified from cells of Pseudomonas
putida T5 grown on N-phosphonomethyl glycine. The native enzyme had a molecular mass
of approximately 70 kD and upon sodium dodecyl sulfate polyacrylamide gel electrophoresis
(
about 70 kD. Activity of purified enzyme was increased by 627-fold compared to the crude
SDS-PAGE), yielded a homogeneous protein band with an apparent molecular mass of
ꢀ
extract and showed pH and temperature optima of approximately 7 and 30 C, respectively.
The purified enzyme had an apparent Km and Vmax of 3.7 mM and 6.8 mM=min, respectively,
for its sole substrate N-phosphonomethyl glycine. The enzyme was inhibited by phenylmethyl-
sulfonyl fluoride (PMSF), indicating the presence of serine at the active site. The enzyme was
not inhibited by SDS, suggesting the absence of disulfide linkage in the enzyme. The enzyme
2
þ
was found to be inhibited by most of the metals studied except Mg . Detergents studied also
inhibited glyphosate acting as a carbon–phosphorus bond-cleavage enzyme. Thus initial
characterization of the purified enzyme suggested that it could be used as a potential candidate
for glyphosate bioremediation.
Keywords bioremediation, carbon-phosphorus bond cleavage enzyme, glyphosate, N-phosphonomethyl
glycine, Pseudomonas putida
INTRODUCTION
Organophosphonates, characterized by the presence of a stable, covalent carbon-to-phosphorus
(C-P) bond, are of widespread occurrence in the environment. Natural and synthetic organo-
phosphonates are of importance, with the latter being utilized extensively in the chemical
[
1]
industries as pesticides, plasticizers, air fuel ingredients, and chemical warfare agents.
Organophosphorus compounds are the most widely used herbicides and insecticides, accounting
[1]
for an estimated 34% of worldwide insecticide sales.
Address correspondence to Haravey K. Manonmani, PhD, Principal Scientist, Fermentation Technology and
Bioengineering Department, Central Food Technological Research Institute (CFTRI), Lab. No. 1, Cheluvamba Mansion,
Mysore, 5700 20 India. E-mail: manonmani_99@yahoo.com
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lpbb.

GLYPHOSATE DEGRADING C-P LYASE
381
Contamination of soil from these pesticides as a result of their bulk handling at the farmyard
or following application in the field or accidental release may lead to contamination of surface
water and groundwater. Contamination of air, water, and terrestrial ecosystems has been reported
to lead to harmful effects on different biota and disruption of biogeochemical cycling by these
[2]
organophosphorus compounds. Organophosphorus compound poisoning is a worldwide
[3,4]
health problem contributing to around 3 million poisonings and 200,000 deaths annually.
These compounds have been implicated in several nerve and muscular diseases in humans,
and their acute adverse effects have been extensively reported by Colborn et al. and
[
5,6]
Ragnarsdottir.
has been reviewed by Galloway and Handy.
Continuous and excessive use of organophosphorus compounds has led to the contamination
Immunotoxicity of organophosphorus compounds in humans and wildlife
[7]
[8–10]
of several ecosystems in different parts of the world.
These compounds possess high
mammalian toxicity and it is therefore essential to remove them from the environment. By
far the most important use of synthetic organophosphonates, however, is as herbicides, with
[
11]
[1]
the world’s leading agrochemical, worth in excess of $1 billion per year It is
glyphosate,
a representative of the phosphonic acid group of compounds, which is characterized by a direct
carbon to phosphorus (C-P) bond. The C-P linkage is chemically and thermally very stable and
renders the molecule much more resistant to nonbiological degradation in the environment than
[12]
its analogues with O-P linkage.
Bioremediation can offer an efficient and cheap option for decontamination of polluted
[
13]
ecosystems and destruction of chemical warfare agents.
been tested for their ability to degrade different organophosphorus pollutants.
Acclimated bacterial cultures have
[14]
It was shown that an obligately thermophilic microorganism could cleave C-P bonds and uti-
lize a number of organophosphonates for growth. Microbial degradation has been considered to
[15]
be the most important of the transformation processes controlling its persistence in soil. It was
observed that mineralization of glyphosate is related to both the activity and biomass of soil
[16]
microorganisms. Several species of Pseudomonas capable of degrading glyphosate have been
[
17,18]
isolated.
The biodegradation of the herbicide glyphosate via the sarcosine pathway by microorganisms
and also the conversion of glyphosate to amino methyl phosphonic acid (AMPA) have been
accepted as mechanisms for detoxification of this herbicide in soil; no microorganism that con-
[19]
clusively exhibited this capability has been isolated from soil.
To date, our understanding of
this phenomenon is based almost exclusively upon work carried out by Monsanto on microbes
within a glyphosate waste treatment plant, which also metabolized the herbicide via this
[
20,21]
pathway.
The present study shows for the first time the conclusive production of sarcosine, which is a
[
22,23]
nontoxic
metabolite of glyphosate degradation by a microorganism. Isolations of micro-
organisms that utilized glyphosate as carbon or nitrogen sources were attempted. Microbes
that degrade phosphonates as phosphorus sources have been isolated and characterized. This
Pseudomonas sp. was observed to grow with glyphosate as a sole phosphorus source with no
accumulating intermediates. To our knowledge, a microbe that accumulates sarcosine as an
intermediate has not been reported. The purposes of this study were (i) to isolate a medium
microorganism capable of degrading glyphosate and (ii) to purify and characterize the first
enzyme capable of breaking C-P bonds.

382
SELVI AND MANONMANI
MATERIALS AND METHODS
Materials
N-Phosphonomethyl glycine (glyphosate) was procured from Monsanto Agricultural Co.,
St. Louis MO. Sarcosine and other pesticides were procured from Serva, Germany. Sepharose
was purchased from Pharmacia. All other chemicals used in these studies were obtained from
standard chemical companies such as Sigma-Aldrich chemical company, E. Merck Laboratories,
and others and were of analytical grade.
Microorganism and Culture Conditions
Pseudomonas putida T5 used in this study was a member of the glyphosate-degrading bacterial
[
24,25]
consortium.
containing 5 ppm glyphosate.
This isolate was maintained on nutrient agar and also on minimal agar
Screening of the Bacterial Cultures
All the individual strains were grown on nutrient broth for 48 hR at room temperature separately.
ꢀ
These were then harvested by centrifugation at 10,000 rpm for 10 min at 4 C; the supernatant
was discarded and the remainder was washed in minimal medium (pH 7.5) of the following
composition (per liter): KH PO , 0.675 g; K HPO , 5.455 g; NH NO , 0.25 g. The cells were
2
4
2
4
4
3
then sonicated in ice-cold phosphate buffer in an ice bath. The harvested cells were resuspended
in a known quantity of minimal medium. The cells were preexposed to 10 ppm glyphosate for
7
After the preexposure, all the consortial cells were harvested by centrifugation at 10,000 rpm for
2 hr. Preexposure of the cells was done by periodic addition of 10 ppm substrate at every 24 hr.
ꢀ
1
0 min at 4 C.
Enzyme Production
ꢀ
Pseudomonas putida T5 was grown in batch cultures at 30 C on an orbital shaker at 150 rpm in
nutrient broth. After 48 hr of growth, the cells were harvested and resuspended in minimal
medium (pH 7.5) of the following composition (per liter): KH PO , 0.675 g; K HPO ,
2
4
2
4
5
up to 72 hr. The flasks were incubated at 30 C on an orbital shaker at 150 rpm.
.455 g; NH NO , 0.25 g. The cells were induced with daily addition of 10 ppm glyphosate
4 3
ꢀ
Enzyme Isolation
ꢀ
The cells were separated from the broth by centrifugation at 4 C, 10,000 rpm, for 15 min. The
supernatant was discarded. Cells were washed well in minimal medium. The cells were then
sonicated in ice-cold phosphate buffer in an ice bath. The homogenate was centrifuged for

GLYPHOSATE DEGRADING C-P LYASE
383
ꢀ
2
0 min at 10,000ꢁ g at 4 C, and the clear supernatant was concentrated using centricon tubes
with 10,000 cutoff, dialyzed against phosphate buffer and designated as crude extract.
Assay of Glyphosate C-P Lyase Activity
Quantitative determination of glyphosate C-P lyase was carried out as follows: The assay
reaction was carried out in a final volume of 1 mL containing 50 mM phosphate buffer
ꢀ
(
pH 7.0), enzyme, and 60 mM glyphosate. The reaction mixture was incubated at 30 C for
ꢀ
6
0 min. After the reaction, residual glyphosate was hydrolysed at 36 C with sodium hypochlorite
to form glycine. The glycine was then reacted with o-phthaldehyde (OPA) in the presence of
ꢀ
b-mercaptoethanol at 55 C to produce a highly fluorescent isoindole for fluorimetric detection
in high-performance liquid chromatography (HPLC).
HPLC was carried out using a cation-exchange analytical column with excitation at 330 nm
and emission at 465 nm. The calibration curve was prepared using glyphosate standard
(Monsonto) hydrolyzed with the already-mentioned protocol. Potassium dihydrogen phosphate
at 0.005 M was used as mobile phase at a flow rate of 1 mL=min.
One unit (U) of glyphosate C-P lyase was defined as the amount of glyphosate C-P lyase that
converts 1 mM of glyphosate per minute under the conditions of the assay.
[26]
Protein content was measured according to Bradford,
as a standard.
using bovine serum albumin (BSA)
Purification of Glyphosate C-P Lyase
Precipitation
Crude enzyme filtrate was precipitated by adding different ice-cold solvents like isopropanol,
ꢀ
ethanol, acetone, and methanol at a 1:2 ratio with constant stirring for 2 hr at 4 C and the
ꢀ
precipitate was separated by centrifugation at 10,000ꢁ g for 20 min at 4 C. The precipitate
was dissolved in a minimal volume of phosphate buffer (50 mM, pH 7.0) and dialyzed against
ꢀ
water for 24 hr at 4 C and assayed for enzyme activity.
Ion-Exchange Chromatography
Methanol-precipitated enzyme, which showed highest activity, was passed through a DEAE
cellulose cation-exchange column using phosphate buffer (50 mM, pH 7.2) for elution. Glypho-
sate C-P lyase activity-containing fractions obtained after the ion-exchange chromatography
were pooled, dialyzed with distilled water, and concentrated with a bench-top lyophilizer.
Size Exclusion Chromatography
The dialyzed and lyophilized sample from the previous step was loaded on 50 mM phosphate
buffer, pH 7.0, preequilibrated Sepharose 6B column (1.1 cm ꢁ 50 cm). The protein elution was
done with the same buffer at a flow rate of 0.4 mL=min. Fractions of 5 mL each were collected.

384
SELVI AND MANONMANI
The absorbance at 280 nm was recorded and active fractions were pooled, concentrated, and
ꢀ
dialyzed against distilled water. This concentrated fraction was stored at ꢂ20 C.
Criteria of Purity
HPLC
Purified enzyme was chromatographed in a Shimadzu high-performance liquid chromatography
(
HPLC) system with reverse-phase C18 column (Thermohypersil, 300ꢁ 4.6 mm, 5 m ODS) using
the following protocol: 1–10min A (0.1% trifluoroacetic acid [TFA]) 100%, 10–70 min, B
acetonitrile 80% containing 0.05% TFA) 100%, 70–75 min, B 100%, 75–80 min A 100%,
(
8
0–90 min A 100%. Detection was carried out at 273 nm.
Polyacrylamide Gel Electrophoresis (PAGE)
The lyophilized C-P lyase enzyme obtained from the Sepharose 6B column was subjected to
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for ascertaining the
homogeneity and to estimate the molecular mass of the protein. SDS-PAGE was performed
on a slab gel containing 10% polyacrylamide, which was employed in order to achieve
[27]
separation of proteins.
Alcohol dehydrogenase (150kD), bovine serum albumin (66 kD),
ovalbumin (43 kD), carbonic anhydrase (29 kD), lactoglobulin (18 kD), cytochrome c (12.4 kD),
and aprotinin (6 kD) were used as reference standards.
Proteins were stained with Coomassie brilliant blue R-250 and also silver nitrate. Native
PAGE of the purified C-P lyase was performed on 7.5% polyacrylamide gel in glycine buffer
ꢀ
[28]
at 5 ꢃ 1 C as described by Gallagher.
Effect of pH and Temperature
Glyphosate C-P lyase activity as a function of pH was determined by using 0.1 M buffers with
pH values of 4.0 to 8.5 for 1 hr. The buffers used were sodium acetate (pH 4.0 to 6.0) and
sodium phosphate (pH 6.5–8.5). Effect of temperature was determined by incubating reaction
ꢀ
ꢀ
mixture from 20 C to 50 C for 1 hr.
Effect of Inhibitors, Detergents, and Metal Salts
Phenylmethanesulfonyl fluoride or phenylmethylsulfonyl fluoride (PMSF), N-bromosuccini-
mide (NBS), urea, ethylenediamine tetraacetic acid (EDTA), and sodium azide were evaluated
at 2 to 5 mM levels for their effectiveness as inhibitors on glyphosate C-P lyase activity using
glyphosate (2 mM) as substrate at optimum pH and temperature for 1 hr. Triton X-100, Tween
2
0, Tween 80, and SDS were tested for their effect on glyphosate C-P lyase activity at 2% level.
Metals including NaCl, KCl, CoCl , CuSO , CuCl , MgSO , Pb(CH COO) ꢄ 3H O, MnSO ꢄ H O,
2
4
2
4
3
2
2
4
2
HgCl , ZnSO ꢄ 7H O, FeCl , NaNO , KNO , FeSO ꢄ H O, and MgCl at 1 mM were evaluated
2
4
2
3
3
3
4
2
2

GLYPHOSATE DEGRADING C-P LYASE
385
for their effect on glyphosate C-P lyase activity by incubating the enzyme at optimum pH and
temperature for 1 hr.
Substrate Specificity
Glyphosate C-P lyase activity was determined with glyphosate, atrazine desethyl, monocroto-
phos, 2,4-D, parathion, methyl parathion, acephate, glyphosine, dichlorvos, atrazine, simazine,
and terbutryn as substrates at a final concentration of 10 mM. The relative activity was expressed
as the percentage ratio of the enzyme activity determined against different structure analogs
of glyphosate to enzyme activity with glyphosate. The rate of the reaction was expressed
as specific activity of the enzyme measured in terms residual or unreacted substrate because
of enzyme action.
Determination of Kinetic Constants
The kinetic constants, Km and Vmax, of the purified glyphosate C-P lyase were determined by
[
29]
the method of Lineweaver and Burk
sate as a substrate. Reactions were performed in 50 mM sodium phosphate buffer (pH 7) at 30 C.
with different concentrations (0.1–1000 mM) of glypho-
ꢀ
RESULTS AND DISCUSSION
Glyphosate C-P Lyase Activity
The culture broth of individual isolates obtained from growth medium (NB) did not show any
glyphosate C-P lyase activity. However, culture broth of the isolates obtained after induction
with glyphosate showed glyphosate C-P lyase activity (Figure 1). Culture broth of Pseudomonas
putida T5 showed maximum activity. Glyphosate C-P lyase activity of the clarified cell
FIGURE 1 Glyphosate degradation in (a) culture broth of bacterial isolates. BC: bacterial culture; BC
flourescens biovar I; BC : Pseudomonas diminuta; BC : Pseudomonas flourescens biovar II; BC
pseudomallei; BC : Pseudomonas putida; BC : Flavobacterium sp.; BC : Vibrio alginolyticus; BC
aeruginosa; BC : Pseudomonas stutzeri; BC10: Pseudomonas flourescens biovar V.
1
: Pseudomonas
: Burkholderia
8
: Pseudomonas
2
3
4
5
6
7
9

386
SELVI AND MANONMANI
1
FIGURE 2 Glyphosate degradation in cell-free extracts of bacterial isolates. BC -BC10 as given in Figure 1.
homogenate (CCH) also designated as cell-free extract (CFE) of individual cells was higher than
that of the culture broth (Figure 2). Culture broth of Pseudomonas putida T5 showed maximum
activity, followed by Ps. aerugenosa T8, Ps. stutzeri T9, and Ps. fluorescens T10 biovar 5.
Burkholderia pseudomallei T4 showed the minimum activity (Figure 1).
Cell-free extract of Ps. putida T5 showed maximum activity, followed by Ps. fluorescens
biovar 2 T3 and Flavobacterium sp. Vibrio algimolyticus T7 5 and Ps. stutzeri T9 gave good
activities. Pseudomonas fluorescens T10 biovar 5 showed the lowest enzyme activity
(Figure 2). In all the cases enzyme activity in induced cells was higher (9.5-fold) than for
uninduced cells (data not shown).
Purification of Glyphosate C-P Lyase Enzyme
The enzyme was purified in three steps. The CFE of Pseudomonas putida T5 was precipitated
with three solvents, of which methanol was observed to be the best (data not shown). The
enzyme precipitate was then passed through a DEAE cellulose column; fractions showing
activity were dialyzed, lyophilized, and loaded onto a Sepharose 6B column equilibrated
with 50 mM phosphate buffer, pH 7.0. The purification steps for glyphosate C-P lyase of
Pseudomonas putida T5 are given in Table 1. The specific activity of the enzyme increased
with every step of purification with a minimum loss in quantity. The purified enzyme after
gel filtration step had a specific activity of 176.6 U (Table 1). After every purification procedure,
the peak fractions with the enzyme activity were analyzed using SDS-PAGE. The enzyme
precipitated out with methanol was observed to have better activity than other precipitants.
TABLE 1
Summary of Steps Employed in Purification of Glyphosate C-P Lyase From Pseudomonas putida T5
Purification steps
Total activity (IU)
Total protein (mg)
Specific activity (U=mg)
Purification (fold)
Crude extract
907.27
1812.04
5218.42
6321.50
3240
480
124
36
0.2800
3.7750
42.084
175.60
1
Methanol precipitation
DEAE-cellulose column
Sepharose 6B
13.482
150.3
627.13

GLYPHOSATE DEGRADING C-P LYASE
387
Removal of the few contaminating proteins using a DEAE cellulose cation-exchange column
was found to be a suitable step. Peak fractions of the DEAE cellulose cation-exchange column
were pooled together and loaded onto an equilibrated Sepharose 6B gel filtration column for
purification, and in the eluent two protein peaks and one enzyme activity peak were obtained
(Figure 3). Peak fractions showing enzyme activity were pooled, dialyzed extensively against
distilled water, and lyophilized.
Criteria of Homogeneity
The homogeneity of the purified protein was assessed by PAGE and HPLC (Figures 4 and 5).
Activity determinations from native cut gel proteins confirmed glyphosate C-P lyase activity;
ꢀ
The dialyzed enzyme was stored in 50 mM phosphate buffer (pH 7.5) for 4 months at 4 C with
less than 10% loss in activity. Purified enzyme was chromatographed in a Shimadzu HPLC
10AT series with an ultraviolet–visible (UV-vis) variable-wavelength detector (SPD-M10AVP,
Shimadzu) using a C-18 reverse-phase column (300 ꢁ 6 mm, 5 mm Hypersil) according to Latha
[
30]
et al. (2012).
Figure 5).
A single peak was observed under the reverse-phase (RP) HPLC conditions
(
Molecular Mass Determination by Size Exclusion Chromatography
The glyphosate C-P lyase from Ps. putida T5 is an active monomer with an approximate total
molecular mass of ꢅ70 kD (Figure 4B). SDS-PAGE revealed only one distinctive band that was
indicated by the pure preparation of glyphosate C-P lyase (Figure 4A). SDS-PAGE showed
a single band with a molecular mass of ꢅ70 kD. Gel filtration indicated the enzyme with
a molecular mass of ꢅ70 kD (Figure 4B). Purified glyphosate C-P lyase was electrophoresed
in native gel. A 1.5% agar layer containing 10 mM glyphosate showed a clear zone after
2
4 hr of incubation, indicating the presence of glyphosate C-P lyase (data not shown).
FIGURE 3 Elution profile of gel filtration sepharose-6B column. Blue line indicates protein estimated at 280 nm; red
line indicates C-P lyase activity.

388
SELVI AND MANONMANI
FIGURE 4 Determination of the molecular weight of the purified glyphosate C-P lyase from Ps. putida T5. (A)
SDS-PAGE was carried out on 10% gel. Lane 1: crude preparation; lane 2: purified glyphosate C-P lyase after DEAE
cellulose; lane 3: purified glyphosate C-P lyase after size-exclusion chromatography; Lane 4: standard protein markers of
different molecular weights. (B) Gel filtration chromatography using Sepharose 6B column. Standard molecular mass
proteins used included chymotrypsinogen (25 kD), carbonic anhydrase (29 kD), ovalbumin (43 kD), BSA (66 kD),
catalase (240 kD), and ferritin (398 kD). (C) Native-PAGE of the purified glyphosate C-P lyase from Ps. putida T5.
Effect of pH, Temperature, and Stability
Glyphosate C-P lyase from Ps putida T5 was active between pH values of 4.0 and 6.0 with an
optimum at pH 7.0 (Figure 6). At lower pH the activity was low. It increased slightly with
increase in pH and reached a maximum at pH 7.0.
The reaction rate of glyphosate C-P lyase was measured at various temperatures ranging from
ꢀ
4
to 45 C; when the enzyme was incubated at different temperatures for 1 hr and then assayed
FIGURE 5 HPLC of purified glyphosate C-P lyase from Ps. putida T5 on C18 reverse-phase column (300 ꢁ 6 mm,
mm Hypersil) at 273 nm.
5

GLYPHOSATE DEGRADING C-P LYASE
389
FIGURE 6 Effect of pH on the activity of glyphosate C-P lyase from Ps. putida T5. Effect of pH was assayed in the pH
range of 5 to 10.
immediately for enzyme activity, there was progressive loss in enzyme activity with increase in
ꢀ
temperature (Figure 5). Maximum activity was obtained at ambient temperature of 28 to 30 C.
At higher temperatures, the reaction rate declined sharply (Figure 7).
In our studies glyphosate C-P lyase enzyme was stable for 2 hr at optimum pH and tempera-
ture (Figures 8 and 9).
Effect of Various Effectors on Glyphosate C-P Lyase Activity
Glyphosate C-P lyase activity was assayed in the presence of different inhibitors=activators
(Tables 2 and 3). Cysteine, PMSF, and iodoacetamide showed 63%, 69%, and 73% inhibition,
respectively. SDS showed only 2% inhibition, indicating the absences of S-S bridges. However,
the inhibition by thiol group blocking reagents, namely, cysteine and iodoacetamide, indicated
the role of sulfhydryl groups in the catalytic activity of the enzyme. Inhibition by PMSF
FIGURE 7 Effect of temperature on the enzyme activity. Enzyme activity was assayed at various temperature ranges
ꢀ
from 4 to 45 C in 50 mM sodium phosphate buffer, pH 7.

390
SELVI AND MANONMANI
FIGURE 8 pH stability of glyphosate C-P lyase enzyme.
indicated involvement of serine in the active site. Detergents such as Tween 80, Tween 20, and
Triton X-100 inhibited the enzyme activity, and there was respectively ꢅ76%, 75%, and 91%
inhibition of enzyme activity. The metal chelating agent EDTA did not affect the glyphosate
C-P lyase activity, which revealed that the enzyme was not a metalloprotein. Decreased enzyme
activity in the presence of NBS (53%) indicated the involvement of tryptophan residues. Urea
also showed inhibitory effects (84%) on enzyme activity.
The influence of various metal ions on the purified glyphosate C-P lyase is presented in
þ
2þ
2þ
þ
þ
2þ
Table 3. Na was detrimental to enzymatic activity. Ca , Cu , K , and Zn inhibited the
2
enzyme activity by 52, 25, 40, and 58%, respectively, whereas Mg did not cause inhibition
of enzyme activity. The activity was almost retained in the presence of Mg . No other metal
studied showed enhancement in enzyme activity. Inhibition of enzyme activity in the presence
2
þ
2
þ
2þ
of Cu and Zn might be indicative of essential vicinal sulfhydryl groups of the enzyme for
productive catalysis.
FIGURE 9 Temperature stability of glyphosate C-P lyase enzyme.

GLYPHOSATE DEGRADING C-P LYASE
391
TABLE 2
Effect of Inhibitors and=or Activators on Glyphosate C-P Lyase Activity
Inhibitors
Relative activity (%)
Control
100.0
98.0
98.0
47.1
37.4
35.2
31.4
27.3
25.7
24.5
16.5
8.4
SDS
EDTA
NBS
L-Cysteine
NAC
PMSF
Iodoacetamide
Tween 20
Tween 80
Urea
Triton X-100
Glutathione
0.0
Substrate Specificity
Substrate specificity of purified enzyme on various substrates is summarized in Table 4. The
purified enzyme had activity against glyphosate and had very low activity against glyphosine.
The enzyme was not reactive to any of the other substrates tested, even though some of the sub-
strates had a C-P bond linkage. This property makes glyphosate C-P lyase from Pseudomonas
putida T5 potentially very useful in remediating glyphosate.
TABLE 3
Effect of Metal Ions on Glyphosate C-P Lyase Activity
Metals
Relative activity (%)
Control
100.0
100.3
99.7
75.5
75.2
63.9
61.4
60.8
59.5
48.7
46.6
42.0
34.1
22.0
0.0
MgSO
MgCl
FeCl
4
2
3
CuSO
KCl
4
BaCl
KNO
2
3
Al
CaCl
NH
ZnSO
NH
2
O
3
2
4
Cl
4
(
4
)
2
SO
OO)
4
Pb(CH
NaCl
3
2

392
SELVI AND MANONMANI
TABLE 4
Effect of Different Substrates on Glyphosate C-P Lyase Activity
Substrate
Specific activity
Glyphosate
65.29
ND
ND
ND
ND
ND
ND
12.70
ND
ND
ND
ND
Atrazine desethyl
Monocrotophos
2
,4-D
Parathion
Methyl parathion
Acephate
Glyphosine
Dichlorvos
Atrazine
Simazine
Terbutryn
Note. ND, none detected.
Kinetics Parameters of Purified Enzyme
The enzyme was assayed at varying concentrations of glyphosate (0.1–1000 mM). The
maximum activity was at 2 mM concentration of glyphosate (Figure 10). This concentration
was used throughout the study. Based on an Eadie–Hofstee plot, the Km and Vmax and were
calculated as 50 mM and 3.42 mM=mL=min, respectively.
Confirmation of the Hydrolytic Mechanism of Glyphosate C-P Lyase
Confirmation of the hydrolytic nature of the carbon–phosphorus bond cleavage activity by purified
enzyme was done by evaluating the products formed by LS-MS=MS. Mass spectral analysis
showed that a peak corresponding to molecular weight of 89 corresponding to sarcosine was formed
as a product of enzyme action on glyphosate. The peak with the molecular weight of ꢅ91 is the
result of adduct formed in the LCMS for the compound sarcosine (Figure 11). No other metabolites
were observed, indicating the catalysis of glyphosate to sarcosine by glyphosate C-P lyase enzyme.
Compounds with a stable carbon–phosphorus bond are extensively used as antibiotics,
[
31,32]
herbicides, adhesives, flame retardants, chelating agents, and so on,
herbicide with a stable carbon–phosphorus bond. C-P lyases are of inherent interest because
and glyphosate is a
[
33]
chemistry of the C-P bond cleavage presently is poorly understood.
To our knowledge this
is the first report on the isolation, purification, and characterization of glyphosate C-P lyase from
bacterial sources. Of the 10 isolates screened for glyphosate C-P lyase production, Pseudomonas
putida T5 showed high enzyme activity. Many bacteria are known to degrade a large number of
structurally different C-P compounds. But only a limited range of bacterial isolates has been
shown to be capable of the degradation of the widely used broad-spectrum phosphonate herbi-
cide glyphosate [N-(phosphonomethyl) glycine]. Pseudomonas sp. has been found to be capable
of utilizing glyphosate as its sole source of phosphorus. Pseudomonas sp. strain PG2982 was
[34]
shown to carry out the cleavage of the C-P bond, giving rise to the formation of sarcosine.

GLYPHOSATE DEGRADING C-P LYASE
393
FIGURE 10 Km and Vmax of the purified enzyme. (A) Plot of the reaction velocities (V) versus substrate concentration
(
S: 0.1–1000 mM) fitted to the Michaelis–Menten equation (maximum activity was at 2 mM concentration of glyphosate).
B) The corresponding Eadie–Hofstee plot (Km ¼ 50 mM and Vmax ¼ 3.42 mmol=mL=min).
(
FIGURE 11 LCMS=MS pattern of sarcosine formed as a product of enzyme action on glyphosate.

394
SELVI AND MANONMANI
Essentially the same pathway of glyphosate metabolism was found in Arthrobacter sp. strain
[
35]
GLP-1.
environmental sources,
characterized. Preliminary attempts to demonstrate in vitro activity of either the glyphosate-
Although many microbes capable of glyphosate utilization have been isolated from
[30]
the enzymes of the degradation of the herbicide have not been
[36]
degrading enzyme or the AMPA-degrading enzyme (C-P lyase) have not met with success.
Cell-free degradation of glyphosate in extracts from any of the glyphosate-degrading
[36]
microorganisms studied so far remains to be demonstrated.
Pseudomonas sp. PG2982 degrade glyphosate by initial cleavage of the C–P bond, resulting
in the production of sarcosine (N-methylglycine) by C–P lyase activity.
has also been reported to degrade glyphosate by this pathway, but it had only one C-P lyase, and
Arthrobacter sp. GLP-1 and
[19,37]
Rhizobium meliloti
[38]
it was able to degrade a wide range of phosphonates.
The enzyme glyphosate C-P lyase was an intracellular enzyme. However, the culture broth
showed very low enzyme activity compared to CCH. The activity in the culture broth could be
due to lysis of the cells and release of the enzymes into the culture broth. The CCH of all induced
cells showed glyphosate C-P lyase activity. Enzyme activity in uninduced cells was extremely
low. That is, induction by glyphosate appeared to be essential for the production of these degra-
dative enzymes. Induction (preexposure) helped in faster degradation of glyphosate without any
lag. The enzyme activity of induced cells was higher than for uninduced cells. Since CCH of
Pseudomonas putida T5 showed maximum activity compared to other isolates, it was selected
for further studies. Phosphonate monoester hydrolase from Pseudomonas sp. PG2982 was cloned
[39]
into E. coli strain CJ236 and the expressed enzyme was purified and characterized.
Glyphosate C-P lyase enzyme of Pseudomonas putida T5 could be purified to homogeneity
in three steps with phosphonate monoester hydrolase (PEH) in 5% ammonium sulphate and
loaded on to a phenyl-Sepharose column equilibrated in 5% ammonium sulfate, 20 mM Bis-Tris
[39]
propane for purification.
The SDS and native gel patterns of the CCH of induced cells
showed a few extra bands and there were eliminations of a few protein bands present in unin-
duced cells (data not shown). This would have facilitated easier purification of the protein, as
other proteins could have been eliminated during induction. In this study, the purity of enzyme
was confirmed by native and SDS-PAGE and HPLC methods. The SDS-PAGE showed the
enzyme to be a monomer of ꢅ70 kD mass. The activity of Pseudomonas putida was ascribed
to the purified enzyme by determination of activity in the region after polyacrylamide gel
[
39]
electrophoresis. The PEH enzyme was composed of 59- and 66-kD polypeptides.
[
Dotson
observed PEH activity to be novel because other known phosphonate ester hydrolases
39]
et al.
were unable to hydrolyze glyceryl glyphosate. Native molecular mass of 240 kD for PEH
enzyme (cloned and expressed enzyme) was determined by chromatography on a Sephacryl
S400 column. A single polypeptide of 66 kD was observed on SDS-PAGE, confirming the
enzyme was a homotetramer. In our studies the molecular mass as determined by SDS-PAGE
and size exclusion chromatography was ꢅ70 kD, indicating that the enzyme is a monomer.
PEH activity was stable over a pH range of 5–10 at 2- or 12-min preincubation at the various
pH conditions. A bell-shaped curve was observed for PEH activity with a maximum activity
[
39]
at pH 9.0.
However, glyphosate C-P lyase enzyme (this study) was stable at pH 7.0 for
þ
2 hr, after which the activity decreased. Na was detrimental to glyphosate C-P lyase enzymatic
2
activity. Ca , Cu , K , and Zn inhibited the enzyme activity, while Mg did not cause
þ
2þ
þ
2þ
2þ
2þ
2þ
inhibition of enzyme activity. However, heavy metal ions such as Cu and Zn were potent
þ
2
inhibitors. Co appeared to stimulate activity of cloned PEH activity. The PEH enzyme has

GLYPHOSATE DEGRADING C-P LYASE
395
[39]
been shown to hydrolyze both glyceryl glyphosate and b-naphthyl phenylphosphonate.
Similarly, glyphosate C-P lyase was found to have high activity against glyphosate and low
activity against glyphosine. Thus, the enzyme had narrow substrate specificity and was not
acting on other C-P bonds.
The occurrence of this unique enzyme that will hydrolyze a C-P bond of glyphosate may find
interesting use in bioremediation of industry effluent or runoff water, as glyphosate is a potent
inhibitor of aromatic amino acid biosynthesis. As the enzyme was active at ambient temperature
ꢀ
of 28–30 C and pH of 7.0, it could be a good candidate for application in glyphosate bioreme-
diation, reducing glyphosate to a nontoxic, easily degradable metabolite.
Our studies on bacterial degradation of glyphosate showed that glyphosate was metabolised
via sarcosine to form glycine.
[24]
Several mechanisms have been envisaged for this conversion.
But in all, the intermediate product of reaction has been shown to be sarcosine as confirmed by
both thin-layer chromatography and amino acid analysis. Since the phosphonomethyl carbon
is lost as a one-carbon unit at the formaldehyde level of oxidation during the conversion
[40]
of sarcosine to glycine,
no radioactivity from this carbon of glyphosate was incorporated
into glycine. Thus, two pathways for the bacterial metabolism of this herbicide with sarcosine
as the intermediate during conversion of glyphosate to glycine by Pseudomonas PG2982
have been confirmed. Glyphosate was found to be converted to aminomethyl phosphonate
[
41]
[42,43]
in a Flauobacterium sp.,
metabolism of glyphosate in Pseudomonas PG2982 was not found to involve the intermediate
as well as in mixed soil cultures of bacteria.
However,
[41]
formation of aminomethyl phosphonate. In Flavobacterium sp.,
conversion of glyphosate
to aminomethyl phosphonate occurred in the presence of inorganic phosphate, whereas
in Pseudomonas PG2982 m glyphosate metabolism to sarcosine was inhibited by inorganic
phosphate. Arthrobacter sp. GLP-1 also appeared to metabolize glyphosate via the sarcosine
[
35]
pathway
and the metabolism was inhibited by inorganic phosphate.
CONCLUSIONS
The occurrence of this unique enzyme that will hydrolyze a C-P bond of glyphosate may find
interesting use in bioremediation of industry effluent or runoff water, as glyphosate is a potent
inhibitor of aromatic amino acid biosynthesis. As the enzyme was active at ambient temperature
ꢀ
of 28–30 C and pH of 7.0, it could be a good candidate for application in glyphosate bioreme-
diation, reducing glyphosate to a nontoxic, easily degradable metabolite.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the director, CFTRI, for providing necessary laboratory
facilities.
FUNDING
Sincere thanks are offered by the first author to the Council of Scientific and Industrial Research
(CSIR), New Delhi, India, for providing the fellowship.

396
SELVI AND MANONMANI
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