Title of Invention

A PROCESS FOR THE DEVELOPMENT OF IMMOBILIZED ENZYMES

Abstract A process for the preparation of bio-sensor columns containing an inert carrier impregnated with purified PMA-AChE in a proportion of 1:100 comprising adding an aliquot of PMA-AChE to an inert carrier and suspended in phosphate buffer, stirring the mixture and loading the same in a column.
Full Text FIELD OF THE INVENTION
This invention relates to a process for the development of
immobilized enzymes.
This invention further relates to the development of immobilized
enzymes to be used for the detection of products or substrates
such as organophasphate and carbamate in environmental water
samples.

PRIOR ART
Immobilized enzymes are known to have practical advantages (Lilly
et al., 1966; Goldstein and Katchalski, 1968; Goldman et al,
1971; Brummer et al., 1972; Goodson et al., 1973; Alsen et al.,
1975; Ngo and Laidler, 1975; Campbell et al., 1977; Sundaram
et al., 1978,1979; Christensen and Riedel, 1980 ) in detecting
substrates, inhibitors, antibodies etc., in trace amounts.
Immobilized cholinesterases from various tissue/animal sources
can also be utilized in the determination of specific
anticholinesterase compounds . in environmental samples (Alsen
et al., 1981).

Numerous biosensors have been developed to determine
biomolecules which are applicable in areas of food fermentation,
medicine, health care, defence and process control. Among
these,only certain of the biosensors have been commercialized
which are based on immobilized enzymes (Turner et al., 1987).
In the recent years biosensor development has gained much
attention specially in the area of bioanalytical chemistry by
applying micro and nanofabrication techniques (Gopal and
Heiduschka, 1995).
Immobilized ChE was first used by Bauman et al., (1965;1967)
by physical entrapment of horse serum ChE in starch gel on the
surface of an open pore polyurethane foam. This system was not
widely acceptable for environmental analysis because it could
be used only once and enzyme foam pads had to be replaced every
hour.
Goodson and Jacobs (1972) developed a slightly better detection
system using immobilized ChE foam pads but the detection level
remained at only 0.2 ppm of 2,2-dichlorovinyl dimethyl phosphate'
(DDVP). Later, Goodson and Jacobs (1976) used immobilized
electric eel ChE and bovine erythrocyte ChE to develop an
electrochemical detection system. Again these devices were not

able to detect very low levels of Paraoxon (0.1 ppm) , Diazinon
(1.2 ppm), Systox (1.4 ppm), Dursban (4.5 ppm) (all
organophosphates) and Sevin (20 ppm) and Temik (0.5ppm), all
carbamates.
The potentiometric pH-sensitive AChE biosensor (Tran-Minh et
al., 1990) detecting. 10-10 M Malathion, Methylparathion, and
Paraoxon or. the amperoraetric sensor capable of detecting 0.05
uM Dimethoate and 5uM Trichlorofon (Wollenberger et al., 1991)
were cost intensive because of their limited shelf life, other
detecting systems such as the butyrylcholinesterase ion selective
field effect transistors (BChE ISFETs) of Hendji et al., 1991

could detect 10-6 M of Trichlorofon and 10—8 M Methylparaoxon.
Similar systems used by Vlasov et al., 1991 not only had a low
detection level (10-6 M DDVP) but require extremely sophisticated
instruments and trained hands to operate the system. The tandem
packed bed reactors with human RBC AChE and choline oxidase
in a switching flow system (Salamoun and Remien 1994) has not
been used to detect anticholinesterase agents in drinking waters
owing to its low detection level giving rise to false negative
analyses. In a biosensor based on the inhibition of
AChE-catalysed hydrolysis of 4-aminophenylacetate (La Rosa et
al., 1995) the calibration graphs were found to be linear only
— 7 -5
for a short range (10 M to 10 M). Other amperometric detection
systems have also been described but none of those had a
detection level lower than 10-11 M tested for Chlorophos (Budnikov
et al., 1991). Thus none of the earlier biosensors had a
detection level as low as 10-13 M tested for two organophosphates,
Metacid 50 and Vapona.

The above biosensors and techniques suffer from the disadvantages
of being cost intensive and require extremely sophisticated
instruments, posses low sensitivity and cannot be used in the
field.
OBJECT OF THE INVENTION
It is an object of the invention to propose a simple and
economical tool or kit which Mill be sufficiently sensitive to
very low levels of contamination by pesticide residues.
Another object of this invention is to propose an immobilization
technique for immobilized enzyme designed into a workable,
reliable and reproducible tool for the detection of very low
levels of organophosphate and carbamate residues in environmental
samples.
Thus according to this invention is provided a process for the
preparation of immobilised PMA—AChE, comprising coupling
polymaleinie anhydride (PMA) with AChe followed by stirring
mixture and washing to remove the unbound protein from bound PMA—
AChE.

According to this invention is further provided an enzyme-sensor
using the immobilized enzyme, for the detection of
organophosphate and carbamate compound in environmental Mater
samples.
In accordance with this invention, a constant activity of
purified AChE Mas maintained by using human serum albumin during
covalent coupling of the purified AChE to PMA at a ratio for
example 1;100.
In an embodiment of the invention a small aliquot of PMA—AChE is
mixed with celite, an inert carrier and a small glass column
(ENZCOL) loaded with this mixture, is used to detect the
catalytic activity by employing the perfusion system.

The ENZCOL also maintains the substrate, pH and temperature
optima throughout the duration of the assays at the initial
level. Consequently there is no change in the pattern of the
inhibition kinetics of the anticholinesterase compounds.
In another embodiment of the present invention, an aliquot of
PMA-AChE is soaked on silica gel strips and allowed to dry.
These ENZSTRIPS are stored at for example 4°C until further
use and do not require any sophisticated instruments for activity
determination and thus can be conveniently used as dipsticks.
A kit can be prepared including the substrate and a colour code
so that the degree of contamination of water samples can be
assessed very easily from the degree of change in the yellow
colour of the ENZSTRIP reacted water sample.
The immobilization of lyophilized goat brain AChE was done in
two sequential steps:
i. covalent coupling of polymaleinic anhydride (PMA) with AChE;
ii. removal of unbound protein from bound PMA-AChE by repetitive
washing.

A 10-fold purification of the enzyme AChE was obtained over
the crude homogenate which may be used for immobilization and
development of the biosensor. On immobilization of the purified
goat brain AChE to PMA, the protein provided is covalently
bound to the polyanionic carrier.
Covalent coupling of the enzyme with PMA is effected under
a continuous stirred condition at pH 8.0, and 40°C for 5 h
followed by raising the temperature to 3 5°C and pH to 8.0 for
another 1^ h. The operating parameters are described only as
by way of example.An aliquot of the final washed PMA-AChE
suspension was used to prepare small double-jacketed glass
columns (ENZCOL). The quantitation system is based on perfusion
of substrate before and after inhibitor perfusion. Aliquots
of the effluent collected for ten minutes of substrate perfusion
before and after inhibitor perfusion were measured
spectrophotometrically (405 nm) for thiocholine produced by
the catalytic activity of the PMA-AChE column. VThe difference
between the initial and pesticide perfused catalytic activity
of PMA-AChE gives an accurate measure of inhibition following
the typical inhibition kinetics of organophosphates and
carbamates. Calibration graphs were constructed for measurement
of pesticidal contamination in environmental sample. The
immobilizePA- could also be used in the form of a strip (as
commonly .used in clinical kits), ENZSTRIP.

Further objects and advantages of this invention will be more
apparent from the ensuing description when read in conjunction
with the accompanying drawings wherein:
Fig. 1 shows the operation protocol of ENZCOL.
Fig- 2 shows the operation protocol of ENZSTRIP.
Fig. 3 is a standard curve for OP pesticide with moles of
tnetacid-50 along the abscessa and activity percentage along
the ordinate.
Fig. 4 shows a standard curve for CA pesticide with moles of
carbaryl along the abscussa and activity percentage along the
ordinate.
Experiaent with soil and water in the field and the laboratory
The field study was carried out in a field of the Department
of Zoology, Visva Bharati, Santiniketan. Size of "the selected
plots were 6 ft x 4 ft. The texture of the soil was sandy loam.
One plot was kept as untreated and two other plots were treated
with Metacid-50 and Carbaryl at their agricultural doses, 500
ppm and 6.250 ppm respectively. In the field condition, the

run off water was collected in the respective collecting pits.
Bucket experiments were conducted under laboratory condition.
Perforated buckets filled with soil upto a height of 8 cm were
placed on trays. Water was allowed to pass through the soil
which was collected in the tray for both field and laboratory
experiments soil sampling size was 5x5x5 cm. Soil and water
samples were collected at regular intervals of 1,3,7,15 and
21 day of pesticide application. Soil samples were dissolved
in water and both soil and water samples were centrifuged to
have clear supernatants. All experimental plots were pre-checked
and found to be free from any pesticidal contamination,
Physicochemical properties of the soil were also studied during
sampling. In order to reach an ideal active condition of the
column 1 ml of the soil extract and run off water were sitably
diluted with perfusion fluid to carry on the perfusions as
standardised for the commercial formulation.
Preliminary experiments concerning proportionality between enzyme
concentration and activity, temperature and ionic requirements
were conducted to optimize the catalytic property of the column.
This immobilized PMA bound enzyme demonstrated optimum activity
at a substrate concentration of 8x10-3 M, pH of 7.6, temperature
of 40°C and activator concentrations of 4 mM MgCl., and 30 mM
NaCl. Contrastingly, the purified enzyme exhibited optimum
substrate concentration of 2x10-3 M, pH at 8.0, temperature at
40°C and activator concentrations at 2 mM MgCl and 15 mM NaCl.

Methyl parathion (50 EC) exhibited 21% to 50% inhibition at
the concentration of 3x10-12M to 3x10-10 M (Fig.3). On the other
hand carbaryl depicted 9% to 46% inhibition respectively from
3x10-9M to 3x10 M and no inhibition at a concentration of 3x10-
10M (Fig.4). Although both the pesticides are specific AChE
inhibitors, the pesticide belonging to the carbamate group is
found to be less inhibitory than the organophosphate. Thus the
biosensor is capable of detecting inhibition caused by methyl
parathion (50 EC) at fg level and carbamate (50 WDP) at pg level
present in the perfusion solution. On the contrary, the free
enzyme is unable to detect such low levels of the pesticides.
Besides, there is no other known detection system which can
correctly estimate fg/pg doses of contamination.
The physicochemical parameters of soil fluctuated without any
notable difference between untreated and pesticide treated plots
and buckets. Under both field and laboratory conditions (Table
2), Metacid-50 (methyl parathion 50EC) disappeared completely
from both soil and water within 3 days of application whereas
Carbaryl was detected in the soil and run off water upto 7 days
after treatment. Both Metacid-50 and Carbaryl disappeared very
rapidly in soil and run off water and this is probably due to
the surface application of pesticides, high temperature and
high moisture during the initial phase of the experiment. This
biosensor appeared to- be stable for 100 perfusions after which
it could be stored at 277°K for six months.

Finally, the present results show that the PMA bound AChE-Celite
system, is a potential tool for analytical purpose. The stability
of the column (ENZCOL) over long and continuous perfusion period
enables the detection of very low residual level of
methylparathion and carbaryl both in soil and run off water.
Moreover, the dipstick method of detection could also be applied
by using the PMA-AChE soaked in silica gel strips (ENZSTRIP).
The dipstick method is very simple and can be used by
nontechnical personnel if a kit is prepared containing a colour
.code to determine the rate of inhibition. Thus this technology
can be used as a biosensor of pesticide pollution in the
environment.





The invention will now be explained in greater detail with
the help of the following non-limiting examples.
All solutions were prepared with extra pure Milli Q water
(Millipore). Acetylthiocholine iodide, 5-5'-dithiobis
2-nitrobenzoic acid, human serum albumin were purchased from
Sigma, USA. Triton-X 100, ammonium sulfate, sodium chloride,
magnesium chloride, di-sodium hydrogen orthrophosphate, sodium
carbonate, potassium sodium tartarate were procurred from E.Merck
(India) Ltd. Si-sodium tetraborate and boric acid were from
Glaxo Laboratories Ltd. All chemicals were of analytical grade.
The goat brain cerebeller AChE was purified following the method
of Ord and Thompson (1951) with minor modifications. Briefly,
the cerebellum of the brain was dissected immediately from
freshly sacrified goat since this region possessed maximal AChE
activity (Guhathakurta et al., 1984). A 5% homogenate prepared
in Milli Q water was centrifuged at 4000 rpm, The supernatant
was brought to pH 4.5 and Triton X-100 was added followed by
centrifugation. The clear supernatant obtained was fractionated
with 25 and 50% saturation of ammonium sulfate and the pellet
was centrifuged at 10,000 rpm. The final, precipitate was
dissolved in water and dialysed overnight.

AchE activity was determined by measuring the hydrolysis of
acetylthiocholine iodide by the method of Ellman et al. , (1961).
Aliquots of both brain homogenate and purified AChE Mere
separately added to the reaction mixture (final volume 3 ml)
containing phosphate buffer (pH 7.6, 0.05 m) with activators
(NaCl 15 mM, HgC12) mH) thiol indicator (DTNB, 0.2S mil) and the
substrate, acetylthiocholine iodide, (1x10 M) for the homogenate
and 4x10 M for the purified AChE repsectively in the reaction
mixture). The enzyme reaction was allowed to progress of 40 C for
5 min and the rate of enzyme reaction was determined in a Beckman
Spectrophotometer (DU 640) at 405 nm. Protein contents were
determined by the method of Lowry et al., (1951) using BSA as a
standard.
Polymaleinic anhydride (PHA) 200 mg in 10ml phosphate buffer (ph
7.0 50 mH) is vigorously stirred with 10 ml purified goat brain

AChE: Human senw albumin (1:100) in a standard solution
(phosphate buffer, pH 7.6,50 mM with 150 mM sodium chloride and
20 mM Magnesium chloride) in a reaction vessel maintained at 4
degree Celsius for 5th during which the pH was adjusted to 8.0
with 1 M NaOH solution. The temperature was then raised to 35
degree Celsius and stirring continued for another 21 h at pH
8.0. This gives the immobilized PMA—AChE product which is
thoroughly washed alternately with 2 M NaCl, borate buffer; 2 mM
NaCl, borate buffer by alternate stirring and centrifuging at 10
degree Celsius, 3800 xg for 20 min. The final pellet was
collected and filtered by passing 300 ml Standard Solution over a
D3 filter under vacuum. The immobilized enzyme was then collected
from the filter and suspended in 50 ml of Standard solution and
stored at 4 degree celsius for future use.

An aliquot containing 600 ug/ml PMA-AChE was added to 250 mg
of Celite and suspended in 10 ml phosphate buffer (50mM, pH
7.6) at room temperature. After vigorous stirring for 30 min.
the whole suspension was poured carefully int a double jacketed
glass column (1 cm x 6 cm) maintained at 40°C. The outlet of
the column was closed by a D.. glass filter overlayered with
washed sea sand upto a height of 0.5 cm in order to achieve
the desired length of 2.5 cm which was found to be most suitable
for the present experiments. The total bed volume as measured
by cresol red was found to be 0.8 ml.
All solutions were perfused by means of a Gilson peristaltic
pump maintaining the following order of perfusions. The enzyme
column was first equilibrated with phosphate buffer (50 mM,
pH 7.6) and a perfusion fluid consisting of the phosphate buffer
containing 30 mM NaCl and 4 mM MgCl2 for 20 and 30 min.
respectively. The substrate was prepared in the perfusion fluid
and perfused for 10 min at a flow rate of 0.3 ml/min at 40°C.
The activity of the enzyme was measured by assaying
spectrophotometricaLly the reaction of an aliquot of the effluent
with Ellman's reagent. Pesticide inhibitors of varying
concentrations were prepared in the perfusion fluid and perfused
at a rate of 0.3 ml/min for a constant time period of 5 min
followed by perfusion of the substrate and assayed in the same
manner as before (Fig.1).

An aliquot of the PMA-AChE was also soaked on silica yel strips
having a size of 5x25 mm and allowed to dry. The ENZSTFUPs were
stored at 4°C until further use. The procedure of detection
is simplified in the dipstick method (Fig.2). Briefly, the strip
is first dipped in the substrate solution for 10 min t note
the uninhibited enzyme activity. Catalytic action of the enzyme
will produce a yellow colour on addition of the thiol indicator
which is measured at 405 nm as described before. To determine
the inhibition by pesticides the ENZSTRIP was first dipped in
the inhibitor solution for varying time periods and then in
the substrate followed by DTNB solution as described earlier.
The difference in the 0 D at 405 nm in the untreated and treated
samples were calculated and the percentage inhibition determined
at different time of treatments. Calibration graphs were
constructed and used for determining the contamination level
of the pesticides.

I CLAIM:
1. A process for the preparation of bio-sensor columns an inert
carrier impregnated with purified PMA-AChE in a proportion of
1:100 comprising adding an aliquot of PMA-AChE to an inert
carrier and suspended in phosphate buffer, stirring the mixture
and loading the same in a column.
2. The process as claimed in claim 1, wherein said inert carrier is
celite.
3. The process as claimed in claim 1, wherein said glass column is a
double jacketed column.
4. The process as claimed in claim 1, wherein 50 to 500 ml of PMA-
AChE is used in the column.
5. The process as claimed in claim 1, wherein 200 to 300 mg celite is
used in the column.
6. The process as claimed in claim 1, wherein said glass column is
maintained at a temperature 4°C.
7. The process as claimed in claim 1, wherein the outlet of said glass
column is closed by a glass filter overlayered with sea-sand.
8. The process as claimed in claim 1, wherein the column has a
dimension of 2 cm by 6 cm.

9. A process for the preparation of strips impregnated with PMA-AChE
comprising soaking an aliquot of PMA-AChE on silica gel strips and
drying the same.
10. The process as claimed in claim 9, wherein said strips are made of
silica gel dried on aluminium.
11. The process as claimed in claim 9, wherein the strips have a size of 5
to 6 cm by 1 cm.


A process for the preparation of bio-sensor columns containing an inert carrier
impregnated with purified PMA-AChE in a proportion of 1:100 comprising adding
an aliquot of PMA-AChE to an inert carrier and suspended in phosphate buffer,
stirring the mixture and loading the same in a column.

Documents:

505-KOL-2004-ABSTRACT 1.1.pdf

505-kol-2004-abstract.pdf

505-KOL-2004-CLAIMS 1.1.pdf

505-kol-2004-claims.pdf

505-kol-2004-correspondence.pdf

505-kol-2004-correspondence1.1.pdf

505-KOL-2004-DESCRIPTION (COMPLETE) 1.1.pdf

505-kol-2004-description (complete).pdf

505-KOL-2004-DRAWINGS 1.1.pdf

505-kol-2004-drawings.pdf

505-KOL-2004-EXAMINATION REPORT REPLY RECIEVED.pdf

505-kol-2004-examination report1.1.pdf

505-KOL-2004-FORM 1 1.1.pdf

505-kol-2004-form 1.pdf

505-kol-2004-form 18.1.pdf

505-kol-2004-form 18.pdf

505-KOL-2004-FORM 2 1.1.pdf

505-kol-2004-form 2.pdf

505-kol-2004-form 26.pdf

505-kol-2004-form 3.1.pdf

505-kol-2004-form 3.pdf

505-kol-2004-granted-abstract.pdf

505-kol-2004-granted-claims.pdf

505-kol-2004-granted-description (complete).pdf

505-kol-2004-granted-drawings.pdf

505-kol-2004-granted-form 1.pdf

505-kol-2004-granted-form 2.pdf

505-kol-2004-granted-specification.pdf

505-KOL-2004-OTHERS 1.1.pdf

505-KOL-2004-PA.pdf

505-kol-2004-reply to examination report.pdf

505-kol-2004-specification.pdf


Patent Number 248949
Indian Patent Application Number 505/KOL/2004
PG Journal Number 37/2011
Publication Date 16-Sep-2011
Grant Date 14-Sep-2011
Date of Filing 24-Aug-2004
Name of Patentee SHELLEY BHATTACHARYA
Applicant Address PROFESSOR OF ZOOLOGY ENVIRONMENTAL TOXICOLOGY LABORATORY VISVA BHARATI UNIVERSITY, SANTINIKETAN
Inventors:
# Inventor's Name Inventor's Address
1 SHELLEY BHATTACHARYA PROFESSOR OF ZOOLOGY ENVIRONMENTAL TOXICOLOGY LABORATORY VISVA BHARATI UNIVERSITY, SANTINIKETAN 731235
PCT International Classification Number C08F 2/00
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA