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.

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