Title of Invention	"IMMOBILIZATION OF PROTEINS ONTO SOLID SUPPORT AND ITS PROCESS THEREOF"
Abstract	The present invention relates to an immobilized protein comprising of support linked to the proteins having atleast a free amino functional group through a linking agent having atleast two terminal aldehyde groups and the present invention also relates to a method for obtaining the said immobilized protein by acid treatment of the solid support, followed by contacting with linking agent in aqueous buffer solution to obtain an activated solid support which is linked to the protein having free atleast a amino functional group.

Title of Invention

"IMMOBILIZATION OF PROTEINS ONTO SOLID SUPPORT AND ITS PROCESS THEREOF"

Abstract

The present invention relates to an immobilized protein comprising of support linked to the proteins having atleast a free amino functional group through a linking agent having atleast two terminal aldehyde groups and the present invention also relates to a method for obtaining the said immobilized protein by acid treatment of the solid support, followed by contacting with linking agent in aqueous buffer solution to obtain an activated solid support which is linked to the protein having free atleast a amino functional group.

Full Text	IMMOBILIZATION OF PROTEINS ONTO SOLID SUPPORT Field of invention The present invention relates to an immobilized protein onto a solid support. The invention also relates to an improved method for immobilization of the protein onto a solid support. Background and prior art references Enzymes are proteinaceous catalytic materials that have great industrial potential. The immobilization of enzyme provides reusability and high selectivity with an increase in stability and thus renders it economical. The immobilized enzymes /proteins have been employed in diagnostic kits, medical implants, chemical separation and biosensor. Enzymes are also very expensive materials. They are generally soluble in their respective substrates and except where the conversion product is of great value, recovery of the enzyme for the reuse may be difficult or impossible. To avoid such problems, fixed or immobilized systems have been developed. For the purpose of the immobilization of the enzymes /proteins the procedures such as adsorption, encapsulation and covalent bonding are known in prior art. In the process of immobilizing an enzyme, there are many practical considerations. There should be as little loss of enzyme activity as possible. The cost of immobilization should be low. The carrier material should be one that does not have a deleterious effect on the action of the enzyme during reaction. There are several reports in the literature for immobilization of proteins that are enumerated herein below. Immobilization of Oxalate oxidase Oxalate oxidase from different plant sources has been already immobilized on various supports by different techniques. Some are onto pig intestine membrane (Guilbault et al 1986); onto nylon tubing (Bais et a/,1980); onto polyionic electrolyte ( Raghavan and Tarachand,1986) onto antiserum (Inamdar et a/,1986); onto Polyamide membrane (Assolant et a/,1987); onto concanavalin A (Varalakshmi and Richardson, 1992); onto gelatin (Dinckayan and Telefoncu,1993); onto polyvinyl chloride (PVC) and CA membrane laminate (Reddy et a/,1994); onto AF-Tresyl Toyopearl 650 gel (Yamato et al., 1994); onto collagen membrane (Saka Amini and Vallon,1994); onto polyethylene glycol (PEG) (Varalakshmi et al.,1995); onto acrylamide membrane (Lathika et al, 1995);.onto sodium alginate (Lathika et al., 1995); onto glass beads (Pundir et al, 1993); onto glass beads (Pundir et al 1998). A method of immobilization of oxalate oxidase onto pig intestine membrane was described by Rahni & Guilbault (1986) .They constructed enzyme electrode for oxalate determination in urine. However, this electrode membrane was vulnerable to microbial attack. This method showed less stability of immobilized enzyme (2 month) and less reliability (r = 0.95) in comparison to the present invention (r =0.99). The Sorghum leaf oxalate oxidase (Pundir & Satyapal, 1998) enzyme was immobilized onto alkylamnie glass beads to reduce the cost of oxalate determination in large number of urine samples (Thakur & Pundir, 1999). The drawback associated with glass beads is that glass beads are costly and due to porosity of glass beads the enzyme are entrapped inside the pores and the activity of immobilized enzyme is reduced. The present invention using PVC tube/Plastic strip for immobilization of the enzymes overcomes the problem associated with the prior art. Reddy et al (1994) reported a method for the immobilization of barley oxalate oxidase on polyvinyl chloride (PVC) and cellulose acetate (CA) membrane laminate. The laminate was integrated within an amperometric electrode and a biosensor was developed for oxalate determination via electrochemical oxidation of H2O2 and subsequent current generation. However, this method had some drawbacks like instability, low retention of specific activity and high cost. Varalakshmi et al (1995) described a method of immobilization of beet stem oxalate oxidase onto polyethylene glycol (PEG). Although enzyme retained about 90% of its initial activity, but the stability of immobilized enzyme was up to 3 months only. The present invention overcomes the above described drawbacks associated with the prior art. Immobilization of Upases The Upases from different sources have been immobilized on various supports by different methods for different purposes. Some are onto Sepharose 6-B through covalent coupling (Otero et al, 1988); on polystyrene butadiene rubber using two phase emulsion technique for the hydrolysis of triacetin (Iso et al, 1989); onto polyvinyl chloride (Rucka and Tturkiewicz, 1990) onto microporous polypropylene by adsorption (Montero et al, 1993) onto anion exchange resin and diatomaceous-earth (Mustranat et al, 1993) onto porous polyurethane particles (Wang and Ruchenstein 1993) onto organic polymer beads (Basri et al, 1994) onto polyethylene powder (Watanable et al, 1995) on EP-100 polypropylene powder by adsorption (Thomas Gitlesen et al., 1997); within a phyllosilicate sol-gel matrix (An-fei Hsu, Thomus A. Fogila & Siyan Shen, 2000); on phyllosilicate sol-gel matrix through entrapment technique (Hsau et al, 2000); in a hollow-fibre reactor by covalent coupling (Sehanputri and Hill, 2000); within solid nano composite matrix of tetramethoxy-silane (TMOS) (Schuleit and Luisi, 2001); onto different supports like silica, Sepabeads, CNBR activated Sepharose 4B, HP-20 beads and phenyl-sepharose (Dosaryh & Kaur, 2002) onto 7-Fe2Oa magnetic nanoparticles (Dyal et al, 2003) onto free alkylamine glass beads (Sarita et al, 2000); onto alkylamine glass beads affixed inside a plastic beaker (Arya S et a/,2003). In the present experiment porcine pancreas lipase was immobilized onto egg shell membrane affixed to plastic strip employing this technique Most of these immobilization processes includes physical adsorption which decreased the life of the immobilized enzyme beside this other problems associated are low retention of specific activity and high cost. A method of immobilization by entrapment in chemically inert hydrophobic silica gels, prepared by hydrolysis of alkyl-substituted silanes in the presence of lipase was described by Reetz et al (1996). However, this method involved the entrapment, which does not allow proper interaction of substrate with enzyme. So, it is not an effective methodology for immobilization. Immobilization of alpha-amylase Amylase has been already immobilized on various organic and inorganic supports. Some are polyurethane.polymer. (Storey.et.al,1990) on agar gel (Tonkova et al 1994), on micro spheres (Arica et al 1995), on temperature sensitive membrane (Chen et al, 1998), on polyacrylamide gel by Raviyan et al (2003), on alkylamine glass beads through covalent coupling by Ramesh & Singh (1981), on plastic support (Roig et al, 1993), on coconut fiber by Dey et al (2002). Chen et al (1998) described a method of immobilization of alpha-amylase on to a composite temperature-sensitive membrane by cross-linking. This membrane cannot account for stability, as it is temperature sensitive. A method for immobilization of alpha-amylase on to thermal-responsive composite hydro gel membranes by cross-linking was described by Sun et al (1999). It carried the drawback of thermal dependence and low retention of activity, so this method is not reliable. Raviyan et al (2003) described a method for immobilization of alpha-amylase from Aspergillus oryzae by entrapment in polyacrylamide gel. However the acrylamide is neurotoxin and does not allow proper enzyme substrate interaction. Besides this, handling is tedious. Immobilization of glucose oxidase Wang et al (1995) described a method of immobilization of enzymes covalently on to a cellulose acetate membrane. A glucose oxidase membrane was prepared using this method, which was combined to an oxygen electrode and hydrogen peroxide electrode separately to construct a glucose biosensor. However this was synthetic membrane and could not compete on the ground of cost. This membrane faces the substrate analogy in the case of oxalate oxidase. Zythes et al (1976) described a method of immobilization of glucose oxidase in a polyacrylamized coating obtained by electronically initiated polymerization. It included the expensive chemical treatment and sensitive instrumentation, which increase the cost of immobilization. Degitor & Guly (1981) described a method for immobilization of glucose oxidase on to cellulose by covalent combination with amino ethyl cellulose (AE-Cellulose). However, this method involved expensive chemicals, which increased the cost of immobilization. A method of immobilization of glucose oxidase in a polyacrylamized coating obtained by electronically initiated polymerization was described by Zythes et al (1976). It included the expensive chemical treatment and sensitive instrumentation, which increase the cost of immobilization. Immobilization of macromolecules A report by Amyl Springer et al. (2003) described a method of immobilization of proteins and other macromolecules via interaction of two small synthetic molecules, phenyldiboronic acid (PDBA) and salicylhydroxamic acid (SHA). As this method showed lower activity and stability of enzyme after the immobilization on the solid support, this method is not suited for immobilization of enzymes. Patent documents describing immobilization of proteins: US patent 4251631 discloses immobilization of enzyme onto silica modified vinyl chloride polymer membrane by adsorbing the enzyme. The immobilized enzyme was used to carry out enzymatic reaction by passing a solution of its substrate through a membrane under differential pressure. However, this method has limited application because pressure difference can lead to change in conformation as well as substrate inhibition and increase the cost of procedure. US patent. 5219926 disclose a covalent binding of biopolymer of amino acids on organic polymer surface coated with hydrophilic nonionic polymer having reactive groups. As this method includes attachment of active group (various oxidative groups) which can lead to loss of enzyme activity due to presence of highly reactive oxide group as site of attachment of enzyme. US patent 5705045 describes a method of fabricating a multi biosensor using a carbon paste layer around a insulating substrate (PVC) and by immobilizing the GPT and GOT on amino acid polymer paste layer in carbon paste. This method is limited only to the GPT and GOT only. US patent 5516703 discloses the formation of reactive surface by absorbing a modified block polymeric surfactant containing poly (ethylene oxide) block attached to an end of poly (propylene) centre blocks. This method also has similar limitations which the patent no 521996 contains. US patent 5720969 discloses preparation of perm selective graft that was formed by converting into intermediate reactive sites a portion of Cyano group of a backbone polymer and grafting polyalkyene oxide polymer chain to backbone polymer through a reactive site. The resulting perm selective membrane was formed into hollow fiber or flat sheet for encapsulation of cell. It also shows less stability of enzyme activity on immobilized surface. US patent 5808012 describes the interspreading of a variety of functional thermos table proteins inside thermoplastic after molding /extrusion /casting process. This method has limited application of immobilization of thermo stable proteins and enzyme, while most of protein/enzyme got denatured at that temperature. US patent 5928918 describes a biosensor formation based on enzyme containing polymer matrix on the surface of a supporting layer which contain cross linking groups. This method show less stability of enzyme on surface as well as very costly procedure. US patent 6134461 discloses the formation of a biosensor for the detection of glucose, lactate and oxygen in patients by forming an electrode, in which the conducting material was deposited on the surface of support. Concentration of analyte was measured by forming a potential difference between working electrode and reference electrode. US patent 6130033 describes the manufacturing of small sensor by depositing conductive material in the channels formed on substrate by impact and non-impact methods. As this method involves deposition of conductive material and embedding of enzyme in it, it inhibits direct contact of enzyme to the substrate so this method decreases activity of enzyme. US patent 6670199 discloses the adhesions of the cell and biomolecules to hydrophobic surface and hydrophobic coated surfaces. This method require some specific condition for adhesion of cell and biomolecules which can be possible during cell culture but not during enzyme catalytic reactions. US patent 6180007 discloses the formation of perm selective graft polymer by converting -CH - group of polymer to cyano group and grafting polyalkene oxide polymer chains to backbone of polymer through reactive sites .This method lead to oxidation of active site of enzyme and substrate so it decrease both the activity and stability of enzyme. US patent 4696901 discloses immobilization of organism on a plastic carrier preferably poly tetrafluoroethylene, for this water immiscible hydrocarbon substrate was added in nutrient media of microorganisms which increases the load of microorganisms on carrier. This method is useful for growing of microorganisms and only not for enzyme. US patent 4396716 discloses the immobilization of apyrase (Apyrase is an enzyme which hydrolyze nucleoside triphosphates and diphosphates and are found in all eukaryotes, where they have essential functions in platelet aggregation and neuronal signal termination) on a polyamide surface/polyethylene terphthalate polymer after hydrolytically activating the surface and then using cross linking agent and solution of apyranase. In US patent 5808012, a method is described for the preparation of protein rich thermoplastic. However, this method is applicable for thermo stable polypeptide not for thermo labile protein / enzyme. US patent 4251631 claimed a method for cross linking of enzyme with silica modified poly vinyl membrane. However this method required a pressure differential of 5 to 10 atmospheric pressure, to maintain enzymatic reaction A method of covalently linked biopolymer to solid hydrophilic organic polymer was reported in US specification 5219926, which had limited application for heat stable polypeptide and protein, which remain active at 45 to 55°C. But most of the protein like oxalate oxidase, peroxidase and glucose oxidase, a- amylase are heat labile. So this method had limited applications. However, our method has no such limitations. Another technique for membrane formation by converting unreactive polymer membrane to reactive sites by grafting polyalkene oxide to some cyano group modified reactive sites on backbone of the given polymer had been described in USPTO specification 5720969, but this method was suited for encapsulation of biological organisms and cells but not for enzymatic reaction due to oxidation by reactive sites Compared to above and other relevant searches, the present invention is more fruitful as it covers up all the limitations in these immobilization methods. In most of the prior art designs of various immobilization systems, plastic was used as insulating supports for electrode formation. In other cases, immobilization on PVC was carried out either through surface modification or by addition of other polymer mixture or by affixing some reactive blocks like ethylene oxide etc. Most of prior art processes of immobilization of proteins have major one or more limitations such as: 1) The enzymatic reactions required pressure difference for diffusion of substrate to the enzyme to carry out the reaction. 2) Diffusional resistance of support surface to substrate due to hydrophobicity 5Loss of activity of immobilized proteins within few weeks or in month 3) Very low retention of specific activity of immobilized enzyme 4) Denaturation of protein at the time of protein entrapment 5) In some cases oxidized product are formed (like f^Ch) during enzyme catalysis which may lead to release of enzyme from solid surface into solution and thereby decreasing the stability of the enzyme. Objects of the invention An object of the present invention is to provide an immobilized protein which is easy to handle, cheaper than other immobilized enzymes available in the market, to obtain kits used efficiently in different assays. Another object of the present invention is to provide an improved process for immobilization of proteins onto solid support Another object of the invention is to provide a cost effective support for example plastic strip and PVC material for immobilization of proteins. Yet another object of the invention is to provide an immobilized protein onto the solid support which is reusable. Still another object of the invention is to provide an efficient method for immobilization of alpha- amylase, peroxidase, lipase, glucose oxidase, sorghum oxalate oxidase, human antiserum (IgG) and other proteins onto PVC material and plastic strip with enhanced retention of specific activity of the proteins. Still another object of the present invention is to provide an immobilized oxalate oxidase enzyme which can be used for the assay of urine & serum oxalate by standard colorimetric method. Summary of the invention The present invention relates to an immobilized protein comprising of activated support linked to a protein having atleast a free amino functional group and an improved process for immobilization of said proteins onto a solid support by treating the solid support with inorganic acids followed by treatment with a linking agent having atleast two terminal aldehyde groups in aqueous buffer solution to obtain activated solid support which is further contacted with a proteins having atleast a free amino functional group. Brief description of the drawings Figure 1: Stability results of immobilized amylase onto plastic strip and PVC tube. Figure 2: Stability results of immobilized peroxidase onto plastic strip and PVC tube. Figure 3: Stability results of immobilized lipase onto plastic strip and PVC tube Figure 4: Stability results of immobilized glucose oxidase onto plastic strip and PVC tube Figure 5: Stability results of immobilized oxalate oxidase onto plastic strip & and PVC tube Figure 6: A correlation between urinary oxalate values determined by using Sigma kit method and present immobilized oxalate oxidase enzyme. Figure 7: Standard plot of Oxalate employing grain sorghum leaf oxalate oxidase immobilized on plastic strip or PVC tube Detailed description of the invention In accordance with the object, the present invention provides an immobilized protein comprising of activated support linked to a protein having atleast a free amino functional group through a linking agent having atleast two terminal aldehyde groups. An embodiment of the invention provides a method for obtaining immobilized protein, the said method comprising steps of: a.) treating the support sequentially with inorganic acids in presence of a catalyst; b) treating the support of step (a) with linking agent having atleast two terminal aldehyde groups to obtain activated support, c) contacting the activated support of step (b) with protein having atleast a free amino functional group for 10-12 hrs at a temperature in the range of 30-35°C under dark conditions with continuous gentle shaking to obtain immobilized protein. An embodiment of the invention uses a solid support selected from the group consisting of plastic strip and PVC material. Another embodiment of the invention provides a method wherein the support is first treated with concentrated nitric acid followed by concentrated hydrochloric acid. Yet another embodiment of the invention provides a method wherein the catalyst used may be selected from the group consisting of ferric chloride and other salts of iron. Still another embodiment of the invention provides a method wherein the support is treated with 15% gluteraldehyde in 0.1 M sodium phosphate buffer of pH 6.5 and at temperature in the range of 30-35°C to obtain activated support Still yet another embodiment of the invention provides a method wherein the protein having atleast a free amino functional group is selected from the group consisting of enzyme, antibodies. The enzyme having free amino functional group is selected from the group consisting of oxalate oxidase, alpha amylase, lipase, peroxidase, glucose oxidase and hydrolytic enzymes such as, ligase, lyase and isomerase. The present invention provides an improved method for the immobilization of the proteins onto plastic strip or PVC tube. Immobilization of the protein onto the plastic strip or PVC tube is cost effective and provides the reuse of the enzyme with ease. It is also efficient method; no noticeable change in activity of affixed enzyme is observed even after several months and can be used for immobilization of variety of protein used in diagnostic kits, foods and detergents. For immobilization, Protein is isolated and characterized; the detailed procedure of immobilization of the protein is described in Example 1. Both PVC tube and plastic strip consist of cross linked vinyl polymers, which on treatment with highly concentrated acid lead to breakage of monomer linkage and provide free chloride group for reaction with free aldehyde group of linking agent used. The other aldehyde group of linking agent reacts with free -NH2 group present on surface of protein to form a Schiff "s base and thus lead to immobilization of enzyme. The following chemical reaction are involved stepwise, (a) Breakage of vinyl polymers of PVC material: {-CH2-CH (Cl) -CH2-CH (Cl)-}(n-1) + HNO3 + FeCl3 + HC1 -→ (- CH2CH2 Cl) x*+ (CH3-CH (Cl)y-Where n = x+y - denotes uncertainty in chain length of polymer after breakage, as the site of breakage is not known. (b) Activation of plastic strip and inner wall of PVC tube. - CH2CH2 Cl + OHC-(CH2)3-CHO- → -CH2CH2 (O) C-(CH2)3-CHO (c) Immobilization of enzyme on PVC material: - CH2CH2 (O) C-(CH2)3-CHO +NH2-E - → -CH2 CH2 (O)C(CH2)3 (H)C=N-E Immobilized enzyme By using above method of immobilization many enzymes such as alpha-amylase, lipase, glucose oxidase, peroxidase, oxalate oxidase and other proteins are immobilized. The oxalate oxidase enzyme used is preferably obtained from sorghum sp. The assay of the immobilized enzymes is described Example 2 (I to V). Kinetic properties of the immobilized enzymes are also given in Tables (1 to 5). The present invention also provides an efficient method of immobilization of alpha-amylase, peroxidase, lipase, glucose oxidase , oxalate oxidase, human antiserum (IgG) and other Proteins onto solid support preferably PVC material and plastic strip where there is enhanced retention of specific activity of the enzyme and the immobilized enzyme remains active even after several months. The graph showing stability of five different immobilized enzymes onto plastic strip and PVC tube is shown in Figure 1 to 5. Urinary oxalate is also determined using the immobilized oxalate oxidase enzyme that is described and several analytical parameters are determined to evaluate the efficiency of the method.(Examp!e 3) A comparison of data obtained using sigma kit and the present immobilized oxalate oxidase enzyme provided a good correlation (r=0.99) which evaluated that the present method is more efficient. (Fig 6) No noticeable change in activity of plastic strip and PVC tube bound sorghum oxalate oxidase is observed after 200 uses over a period of 3 months when stored in reaction buffer at 4°C in dark. The invention is illustrated with following examples and should not be construed to limit the scope of the present invention. The present invention has been described in terms of its specific embodiments and certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention EXAMPLES Example 1 Immobilization of the proteins on PVC/ Plastic strip: In present invention protein is immobilized using the following steps. I: Treatment with acid. (a) In this step the solid support is treated with concentrated HNO3 for 5-7 hrs at room temperature followed by many washing with distilled water to remove the excess of acid adhering to the surface, (b) The HNO3 treated solid support is allowed to react with concentrated HC1 in presence of Ferric salt for 2-3 hrs, (c) The treated solid support of step (b) is washed again with distilled water to remove excess acid and Ferric salt. II. Cross linking of membrane: Above treated solid support of step, I (c) is allowed to react with linking agent in buffer solution to obtain activated solid support III. Immobilization of enzyme: Enzyme is immobilized on solid support of step II This experiment involves immobilization of different protein such as alpha-amylase, peroxidase, lipase, glucose oxidase, oxalate oxidase and commercial human antiserum (IgG) onto plastic strip. The antisera are immobilized by incubating the activated strip with the antiserum sample dissolved in buffer for 24 hrs at 4 °C. Example 2 Assay using enzymes I. Assay using a-amylase enzyme: Assay of free α-amylase is carried out by adapting method developed by Mifflin TE, Bruns DE (1987) with modification. (a) Preparation of 0.05 M starch-acetate buffer, (pH 5.6): 0.2 M sodium acetate (91 ml) is added to 9 ml 0.2 M acetic acid. To 2ml of this, 8ml-distilled water is added and its pH is adjusted to 5.6. To this, 0.2 g of starch is added and then boiled, filtered and stored it at 4-6°C until use. (b) Preparation of enzyme solution: α-Amylase powder (5mg) is dissolved in 1ml of 0.05 M starch-acetate buffer, pH 5.6 and stored at 4°C until use. (c) Preparation of DNS (Dinitro Salicylic Acid): Sodium potassium tartarate (20 mg) is dissolved in 50 ml distilled water. A pinch of 2,4- dinitro salicylic acid is dissolved in 20ml of 2N NaOH. These two solutions are mixed and the final volume is made up to 100 ml with distilled water (d) Assay using free a-amylase: Assay of amylase is based on the measurement of glucose and maltose generated from hydrolysis of starch by amylase using DNS reagent. To 1.9 ml of 0.05 M acetate buffer containing 2% starch in a test tube, 0.1 ml enzyme solution is added. For blank, 2.0 ml of reaction buffer containing 2% starch is taken in another test tube. Both the tubes are incubated at 37 °C under continuous stirring conditions for 10 min. The reaction is stopped by adding 0.1 ml 2N NaOH, DNS (0.9 ml) reagent is added to both the tubes, placed in boiling water bath for 5 min & cooled to the room temperature, ASM of red colored reaction mixture is recorded against blank and the amount of glucose generated is extrapolated from standard curve between glucose concentration and A54Q. Enzyme Unit: One unit of enzyme is defined as amount of enzyme required to catalyze the formation of 1 umol of glucose from starch per min. under standard assay conditions. (e) Assay using immobilized a-amylase: It is carried out as described for free enzyme except that immobilized enzyme is used in place of free enzyme and the reaction buffer is increased by 0.1 ml. After the assay the strip is taken off and reaction mixture is transferred to cuvette. In case of PVC tube the reaction mixture is directly transferred to cuvette and As4o of the reaction mixture is read. The enzyme retained 75.3 % and 68.7% of initial activity after immobilization in case of PVC tube and plastic strip respectively. When immobilized a -amylase is incubated in reaction mixture of pH 4.5 at 40 ° C then 0.19 mg, 0.11 mg of glucose is generated by plastic strip, and PVC tube bound enzyme respectively. When immobilized a -amylase is incubated in reaction mixture of pH 5.5 at 40 °C then 0.037mg of glucose is generated by plastic strip bound enzyme and 0.32 of glucose is generated by PVC tube bound enzyme. When immobilized a -amylase is incubated in reaction mixture of pH 5.5 at 35°C then 0.008 mg and 0.012 mg of glucose is generated by plastic strip and PVC bound enzyme respectively. Thus it is observed that pH of 5.5 and 35°C temp is optimum for activity of immobilized enzyme reaction. Various other kinetic properties are described in the table. 1. Table.l. Kinetic properties of α- amylase immobilized on PVC tube/plastic strip (Table Removed) Assay of peroxidase enzyme: The assay of peroxidase based on Trinder's colour reaction is carried out as described by Pundiretal(1999) a) Assay using free enzyme: One mg horseradish peroxidase is dissolved in 10 ml of 0.05 M sodium phosphate buffer, pH 7.0. In a 15 ml conical flask wrapped with black paper, the reaction mixture containing 130 µmol of sodium phosphate buffer (pH 7.0) 25 units of horseradish peroxidase solution, 2.46 µmol of 4-amino phenazone and 10µmol of phenol in a total volume of 3.0 ml is pre incubated at 40°C for 5 min. The reaction is started by adding 10 (amol H2O2 after 20 minutes incubation at 25° C. The absorbance is recorded at 520 nm. Enzyme Unit: One unit of enzyme is defined as amount of enzyme required to catalyze the formation of 1.0 n mol of H2O2 from oxalate per minute under standard assay conditions. (b) Assay using immobilized enzyme: The assay of immobilized horseradish peroxidase is carried out as described for free enzyme except that tube / strip bound enzyme is used in place of free enzyme. To reuse immobilized enzyme the strip / tube is washed with reaction buffer 3-4 times prior its use in next day. The enzyme retained 69.13 % & 64.32% of initial activity after immobilization in case of PVC tube and plastic strip respectively. When immobilized peroxidase is incubated in reaction mixture of pH 4.5 at 40 °C then!6.6 nmol and 20 nmol of H2O2 generated by plastic strip and PVC tube bound enzyme. When immobilized peroxidase is incubated in reaction mixture of pH 6.0 at 40 °C then 243 nmol and 313 nmol of H2O2 generated by plastic strip and PVC tube bound enzyme respectively. When immobilized peroxidase is incubated in reaction mixture of pH 6.0at 35 °C then 146 nmol and 73 nmol of H2O2 generated by plastic strip and PVC bound enzyme. So the PVC and plastic strip bound enzyme show maximum activity at pH of 7.0 and 5.5 when incubated at 40 °C respectively. Other kinetic properties are also studied which are described in the table 2. Table 2: Kinetic properties of horseradish peroxidase immobilized onto PVC /plastic strip (Table Removed) III. Assay using lipase (a) Assay using free lipase: The activity of free lipase is assayed according to Gotthiff Naher with modification. The titration is carried out manually by burette. In a 100 ml conical flask, 5.0 ml olive oil emulsion is added to 5.0 ml of 0.1 M tris buffer (pH 8.0) and incubated at 35 °C for 10 minutes. 1 ml lipase solution (Img/ml) is added and again incubated at 35°C for 20 min. The reaction mixture is then kept at room temperature for 20 min and then added 10 ml of acetone and methanol mixture (1:1) to stop the reaction and titrated against 0.025 N NaOH after adding 1% phenolphthalein as an indicator. The volume of NaOH used in titration is noted. Unit of enzyme: One unit of lipase is defined as the amount of enzyme required to liberate 1 µmole of free fatty acid from olive oil per min under the standard assay conditions (at 35°C and pH 8.0). (b) Assay using immobilized lipase: The assay is carried out as described above except that immobilized enzyme is used in place of free enzyme and the reaction buffer is increased by 1ml. After the final incubation, the strip is taken off and reaction mixture is transferred into titration flask in case of PVC tube the reaction mixture is directly transferred to titration flask. The enzyme retained 70.8 % and 66.2% of initial activity after immobilization in case of PVC tube and plastic strip respectively. Immobilized enzyme is incubated at different pH (5-7) and temperature range (35-45 °C), it is observed from various experiments that pH of 7.0 and 40°C & 45°C is optimum for activity for plastic strip and PVC bound enzyme respectively. Other kinetic properties are also studied which are described in the table 3. Table 3: Kinetic properties of porcine pancreases lipase immobilized on to PVC /plastic strip. (Table Removed) IV Assay using glucose oxidase (GOD) (a) Preparation of colour reagent: The colour reagent is prepared according the method of Bais et al (1980) and consisted of 50 mg 4- aminophenazone, 100 mg solid phenol and 1 mg horseradish peroxidase per 100 ml 0.4M sodium phosphate buffer, pH 7.0. It is stored in amber coloured bottle at 4°C and discarded after one week. . (b) Assay using free glucose oxidase: The assay is carried out in 15 ml test tube wrapped with black paper. The reaction mixture containing 1.7ml 0.05 M sodium phosphate buffer pH 7.0,0.1 ml CuSO4 (10"2M) is preincubated for 5 min at 37° C after addition of 0.1 ml enzyme. The reaction is started by adding 0.1 ml of glucose (Img/ml). After incubating the reaction mixture at 37° C for 10 min under continuous stirring, 1 ml of colour reagent is added and the whole reaction mixture is incubated again at 37° C for 15 min. The reaction mixture is transferred to a cuvette and A520 is recorded. Enzyme Unit: One unit of enzyme is defined as amount of enzyme required to catalyze the formation of 1.0 n mol of H2O1 from oxalate per min. under standard assay conditions. (c) Assay using immobilized enzyme: The assay of immobilized enzyme is performed as described as above except that free enzyme is replaced by immobilized enzyme and reactions mixture is increased by 0.1 ml of reaction buffer and after the assay the plastic strip is taken off and reaction mixture is transferred to cuvette. In case of PVC tube, the reaction mixture is directly transferred to cuvette. The enzyme retained 83.3% and 75% of initial activity after immobilization in case of PVC tube and plastic strip respectively. Immobilized enzyme is incubated at different pH (4.5-5.5) and temperature range (35-45 °C). It is observed from various experiments that pH of 5.5 and 40 °C is optimum for activity of immobilized enzyme. Other kinetic properties are also studied which are described in the table 4. Table4: Kinetic properties of A. niger glucose oxidase immobilized onto Plastic strip/ PVC tube. (Table Removed) V: Assay using oxalate oxidase (a) Preparation of colour reagent: The colour reagent is prepared according the method of Bais et al (1980) and consisted of 50 mg 4- aminophenazone, 100 mg solid phenol and 1 mg horseradish peroxidase per 100 ml 0.4M sodium phosphate buffer, pH 7.0. It is stored in amber coloured bottle at 4°C and discarded after one week. (b) Assay using oxalate oxidase: The assay of oxalate oxidase is carried out as described by Satyapal and Pundir (1993) based on the measurement of H2O2 generated from oxalate by oxalate oxidase with a color reaction containing 4-aminophenazone, phenol and peroxidase as chromogenic system. The assay is carried out in a 15 ml test tube wrapped with black paper. The reaction mixture containing 1.7 ml of 0.05 M sodium succinate buffer (pH 5.0), 0.1 ml CuSO4 solution (10-2 M) and 0.1 ml of crude enzyme is pre incubated at 37°C for 5 min. The reaction is started by adding 0.1 ml oxalate solution (10-2M). After incubation at 38°C for 10 min, under continuous stirring, 1.0 ml color reagent is added and kept at room temperature for 15 min, in dark to develop the colour. The blank contained 1.8 ml 0.05 M sodium succinate buffer pH 5.0, O.lml CuSO4 (10-2M) and 0.1 ml oxalic solution (10-2M). The control consisted of 1.7 ml sodium succinate buffer, pH 5.0, 0.1 ml boiled enzyme solution and O.lml oxalate solution (10 M).The assay for blank/ control is done as described for test sample and A520 is read against blank in Spectronic-20. The content of H2O2 generated in reaction is extrapolated from standard curve between H2O2 cone. Vs A520. Enzyme Unit: One unit of enzyme is defined as the amount of enzyme required to catalyze the formation of 1.0 n mol of H2O2 from oxalate per min. under standard assay conditions. (c) Assay using immobilized enzyme: The assay of immobilized enzyme is performed as described as above except the free enzyme is replaced by immobilized enzyme and reactions mixture is increased by 0.1 ml of reaction buffer. After the assay the plastic strip is taken off, reaction mixture is transferred to cuvette, and As2o is recorded. In case of PVC tube the reaction mixture is directly transferred to cuvette. Immobilized enzyme is incubated at different pH (5.5-6.5) and temperature range (35-45 °C); it is observed from various experiments that pH of 6.5 and 40°C is optimum for activity of immobilized enzyme. Other kinetic properties are also studied which are described in the table 5. Tables: Kinetic properties of sorghum leaf oxalate oxidase immobilized onto PVC /plastic strip (Table Removed) A comparison of various kinetic parameters of grain sorghum leaf oxalate oxidase in free form and that immobilized onto free alkylamine and arylamme glass beads, alkylamine glass beads affixed in a glass beaker and egg membrane affixed on a plastic strip is described in table given below. (Table 6) Table: 6 (Table Removed) A comparison of immobilized sorghum leaf oxalate oxidase onto egg membrane affixed onto plastic strip with that on free alkyl and arylamine glass beads and alkyl amine glass beads affixed in glass beaker is also given in table below (table 7). The table shows better retention of the enzyme onto egg membrane in comparison to other supports and also better specific activity. Table 7 Immobilization of grain sorghum leaf oxalate oxidase onto various supports by covalent coupling. (Table Removed) Example 3: Measurement of urinary oxalate by immobilized oxalate oxidase: (a) Collection and storage of urine: The first morning urine samples from apparently healthy and stone patients (both males and females of various age groups) are collected in plastic bottles at local Pt. B.D.S.PGIMS, hospital. The concentrated HCI is added to urine samples to adjust its pH 2.5 and stored at 4°C until use. (b) Pretreatment of urine samples: Various urine samples are pretreated according to method of Buttery and Pannell (1987) with modifications. One ml of acidified urine is added to 1.0 ml of aqueous CaCl2 (5g/l) and its pH is adjusted to 6.0 with IN NaOH. Immediately 10 ml ethanol is added to the tubes, covered with aluminum foil and kept at room temperature for overnight. On next day, the urine sample are centrifuged at 3,000 rpm for 10 min and calcium oxalate precipitates are collected and redissolved in measured quantity of 0.1 N HCI. (c) Preparation of standard curve for urinary oxalate with immobilized oxalate oxidase: It is prepared between different oxalate concentration and activity of sorghum leaf oxalate oxidase immobilized onto various organic supports under optimal assay conditions. Reaction mixture containing 1.8 ml of 0.05 M sodium phosphate buffer pH 6.0, 0.1 ml of CuSO4 (10-2M) in reaction medium wrapped in a black paper is preincubated at 37°C for 7 min. One ml oxalate solution of different concentrations (Final concentration from 0.2 mM to 6 mM) is added to reaction mixture and incubated at 37°C for 7 min under continuous stirring. One ml colour reagent is added to it and kept at room temperature (25 ± 5) °C for 15 min in dark to develop the colour. The reaction mixture is transferred to cuvette slowly and A520 is read in Spectronic-20. A standard curve is plotted between various oxalate concentrations and A520 (Fig.7). (d) Assay using urinary oxalate: The assay of urinary oxalate is carried out in reaction beaker containing immobilized enzyme and wrapped with black paper. Optimal assay conditions are used except that 0.1 ml pretreated urine samples are taken in place of oxalate. The amount of oxalate concentration is calculated from standard curve between oxalate concentration and corresponding A520. The following analytical parameters are determined to evaluate the method. (a) Linearity: A linear relationship is found between oxalate concentrations ranging from 0.02mM to 6mM. The lower detection limit of this method is 1 to 2 µmol/l, which is lower than that of other methods such as isotachophoresis (40 µmol/l) ( Schmidt et al , 1979), enzyme colorimetric method employing sorghum leaf enzyme bound to free alkylamine glass beads (O.46µg/O.lml) (Pundir et al , 1999) and affixed alkylamine glass beads( 0.05 mM/1) (Kumari.M, Pundir,C.S,2004) (b) Recovery: Analytical recovery of added oxalate in urine sample 20mg/l and 30mg/l is 90±2 %( mean ± SD, n=5) which is comparable to earlier reports (Pundir et al, 1998). (c) Precision: To study the reproducibility and reliability of oxalate value in 24 hr urine samples in one run (within batch) and in same urine samples after one week storage at -20 °C (between batches) are determined. The mean oxalate value of these determinations agreed with each other. Within batch and between batch coefficient of variation (cv) for urinary oxalate determinations are are comparable to earlier reports (Pundir et al, 1998, Thakur et al 1999). The oxalate value of 24 hr urine from healthy adults, as measured by the present method is in range, 13.7 to 24.1 mg/24 hr with a mean of 18.9 mg/24 hr, which is in normal established range. (d) Accuracy: To test the accuracy of method ,urinary oxalate value obtained by present method (y) is compared with the those obtained by enzyme colorimetric kit method (Sigma) The correlation coefficient is 0.99, the regression equation being y=1.017x- 0.548. (Figure 6) References : U.S Patent Documents 4251631 Feb, 1981 Simonetal. 435/106 5705045 Jan.,1998, Je-Kyunetal. 204/403 5219926 June, 1993 Lindmanetal. 525/54 5808012 Sept,1998 Donofrio et al. 525/54 Other References 1. Suna Timur and Azmi Telefoncu, "Oxidized carbon powder for enzyme immobilization on screen printed biosensor" World Scientific (2002),21.14,l-5. 2. S.Marino, G.Minervini, M.R.Montereli, "Screen printing of enzyme or selective polymer ins for chemical sensor and biosensor mass production" F.Mazzei & R.Pilloton Eds(2002). 3. Kaetsu I,Kumakura M,Asano M,Yamadia A &Sakurai Y (1980) "immobilization of enzyme for medical use on aplastic surface by radiation induced polymerization at low temperature. J.Biomed.Mater.Res, 14(3): 199-210. 4. Reddy, S.M., S.P.J. Higson., J.M. Christie and P.K. Vadgama (1994) "Selective membrane for the construction and optimization of an amperometric oxalate enzyme electrode." Analyst 119: 949-952. 5. Varalakshmi, P and K.M. Lathika., K.G. Raghavan and B.B. Singh (1995). Altered physicochemical characteristic of polyethlene glycol linked beet stem oxalate oxidase. Biotech. Bioengg 46: 254-257. 6. Ficher (1978) Immobilization of Amylase on Polystyrene Biotechnol. Bioengg. 41: 204-210. 7. Goyal L.Thakur M & Pundir C.S, (1999) Plant Science ,Vol-142 pp 220-228 8. Storey K.B, Duncan J.A, Chakrabarti AC (1990). "Immobilization of amyloglucosidase using two forms of polyurethane polymer." Appl. Biochem. Biotechnol. 23 (3):221-36. 9. Arya S., Kalia V., Chandran P. and Pundir C. S. (2003). Lipase immobilized on walls of plastic beaker: Kinetic properties and applications in cloth washing of oil stained cloth. Ind. J. Chem. Technol. Accepted for publication. 10. Basri, M., K. Ampon., W.M. Yunus., C.N. Razak., A.B. Salleh (1994). Immobilization of hydrophobic lipase derivatives onto organic polymer beads. J. Chem. Technol. Biotechnol. 59 (1): 34-44. 11. Rucka, M. and B. Turkiewicz (1990). Ultrafilterative membranes as carries frees lipase immobilization. Enzyme Microb. Technol. 12 (1): 52:55. 12. Montero, S., A. Blanco., M.D. Virto., L.C. Landeta, I. Agud., R. Colorable., J.M. Lascaray., M.de Renobales. MJ. Lama and J.L. Serra (1993). Immobilization of Candida rugosa lipase and some properties of the immobilized enzyme Enzyme Microb. Technol. 15(3): 239-247. 13. Watanable, T., Y. Suzuki., Y. Sangesaka., M.Kohashi(1995). Immobilization of lipases on polyethylene and application of perilla oil hydrolysis for production of alpha- linolenic acid. J. Nutr, Sci. Vitaminol, Tokyo 41(3) : 307-312. 14. Mauda (1978). Immobilization of enzyme on cellulose. Enzyme Microb. Technol. 20 (4): 117-118 14 Wang et al (1995) Enzyme Microb. Tech. 15(3): 239-247. IS.Raviyan P, Tang J, Rasco BA (2003). Thermal stability of alpha-amylase from Aspergillus oryzae entrapped in polyacrylamide gel. J Agric Food Chem.27;51(18):5462-6. 16.Rahni, M.A.A. and G.G. Guilbault (1986). Immobilized enzyme electrode for the determination of oxalate in urine. Anal. Chem. 58: 523-526 17.Raghavan, K.G. and T.P.A. Devasagayam (1985). Oxalate oxidase from banana peel for determination of urinary oxalate. Clin. Chem. 31(4): 649. IS.Rucka, M. and B. Turkiewicz (1990). Ultrafilterative membranes as carries fes lipase immobilization. Enzyme Microb. Technol. 12 (1): 52:55. 19.Reetz et at (1996), Immobilization by entrapment in chemically inert hydrophobic silica gels prepared by hydrolysis of alkyl-substituted silanes in the presence of lipase 20.Roig et al (1993), covalently immobilized alpha-amylase on new isocyanate, acid chloride and carboxylic acid—activated plastic supports. 21. Saka aminii & Vellon (1994), Immobilization of oxalate oxidase onto collagen membrane by activation of carbonyl group with aid of acyl-azide reaction. 22. Satoh et al (1977) immobilization of Cholesterol oxidase onto collages fibers, which was used. 23. Scandella Land Koruberg, A., (1971). Membrane-bound phospholipase AI purified from Escherichia-coli. Biochemistry 10 (24), 4447-4456 24. Schafhauser (1992) immobilized amyloglucosidase onto granular chicken bone (BIOBONE) by non-covalent interactions. 25. Varalakshmi, P and K.E Richardson (1992). Studies on oxalate oxidase from beet stems upon immobilization on concanavalin A. Biochem. Intern. 26(1): 153-162. 26. Wang et al (1995) immobilization of enzymes covalently on to a cellulose acetate membrane. 27. Walkar JA, Harmon DL(1996). Simple rapid assay for a-amylase in bovine pancreatic juice, J A nim. Sci 47 (3): 658-662. 28. Yotova LK and Ivanov IP, (1996). Simultaneous immobilized glucose oxidase and peroxidase to urea derivative of regenerated acetyl cellulose granules. Appl. Biochem. Biotechnol, 6J.(3):271-2.13(1): 103-12 29. Zythes et al (1976) immobilized glucose oxidase in a polyacrylamized coating obtained by electronically initiated polymerization. 30. Bais et al, (1980) Anal. Chem. 52: 508-511 31. Inamdaref a/(1986). Anal. Lett. 19 and 20: 1987-1989 32. Assolant et al, (1987) Anal. Lett. 20(4): 513-527 33. Reddy et al (1994) Analyst. 119: 949-952 34. Yamato et al., (1994) J. Chromatogr. 656: 29-35 35. Saka Amini and Vallon (1994). Anal. Chim. Acta. 299: 75-79 36. Lathika et al (1995) Biotech. Lett. 17(4): 407-410 37. Pundir et al. (1993). Ind. J. Biochem. Biophys. 30: 54-57. 38 Pundir et al (1998). Biotechnol. Appl. Biochem. 27: 103-107 39. Otero et al, 1988. Appl. Biochem Biotechnol 19(2): 163-175 40. Iso et al, (1989) J. Microencapsul. 6(2): 165-176; 42. Montero et al (1993). Enzyme Microb. Technol. 15 (3): 239-247. 43. Mustranat et al, (1993) Enzyme Microb, Technol. 15(2): 133-139. 44. Wang and Ruchenstein (1993) Enzyme Microb. Tech. 15_(3): 239-247. 45. Basri et al, (1994) J. Chem. Technol. Biotechnol. 59 (1) 34-44 46. Watanable et al (1995) J. Nutr, Sci. Vitaminol, Tokyo 41(3): 307-312., 47. An-fei Hsu, Thomus A. Fogila & Siyan Shen, (2000). Biotechnol. Appl. Biochem. 3J_: 179-183. 48. Hsau et al (2000), Biotechnology and Applied Biochemistry, 31, pp!79 49. Sehanputri and Hill (2000), Biotechnology and Bioengineering, vol .69 pp 450 50. Schuleit and Luisi, (2001) Biotecnol.Bioeng.vol 72(2) pp249-53 51. Dosaryh & Kaur, (2002) Biotechnol.Applied Biochem, vo!36 pp7-12 52. Dyal et al, (2003),J.American Chemical Society, vol-125pp7 53. Sarita et al (2000). Ind. J. Chem technol, 7: 64-67 55. Arica et al (1995) Biomaterials.16 (10):pp761-8., 56. Chen et al (1998), BiotechnolProg. 14(3):473-8. 58. Ramesh & Singh (1981) Enzyme Microb.Technol; 3: 246-247 59. Roig et al (1993)Biomater.Artif.Cell Immobilization Biotechnol. vol 21(4) pp487- 525), 60. Dey et al (2002) Appl Biochem Biotechnol.; 102-103 (1-6):303-13. 62. Goyal L,Thakur M &Pundir C.S,(1999) Anal .Lett., Vol-32 pp 633-647 Claims: I / We claim 1. An immobilized protein comprising of an activated support linked through a linking agent to a protein having atleast a free amino functional group, wherein the said linking agent have at least two terminal aldehyde groups. 2. A method of immobilization of protein of claim 1, the said method comprising steps of: a. treating a support sequentially with inorganic acids in presence of a catalyst, washing with water to remove the excess acid and catalyst, b. treating the washed support of step (a) with linking agent in a buffer to obtain an activated support, and c. contacting the activated support of step (b) with protein having atleast a free amino functional group for 10-12 hrs at a temperature in the range of 30-3 5°C under dark condition with continuous gentle shaking to obtain immobilized protein. 3. A method of claim 1, wherein the support is a solid support selected from the group consisting of plastic strip and PVC material. 4. A method of claim 2 wherein in step (a) the support is first treated with concentrated nitric acid followed by concentrated hydrochloric acid. 5. A method of claim 2, wherein in step (a) the catalyst used may be selected from the group consisting of ferric chloride and other salts of iron. 6. A method of claim 2, wherein in step (b) the linking agent used is selected from the group consisting of glutaraldehyde and Tri (4-formyl phenoxy) cyanurate. 7. A method of claim 2, wherein in step (b) the support is treated with 15% gluteraldehyde in 0.1 M sodium phosphate buffer of pH 6.5 and at a temperature in the range of 30-35 °C. 8. A method of claim 2, wherein in step (c) the protein having free amino functional group is selected from the group consisting of enzyme and antibodies. 9. A method of claim 8 wherein the enzyme having free amino functional group is selected from the group consisting of alpha-amylase, oxalate oxidase, lipase, peroxidase, glucose oxidase and hydrolytic enzymes such as, ligases, lyases and isomerases. 10. A method of claim 8 wherein the antibody immobilised is preferably human antiserum (IgG). 11. An immobilized protein of claim 1, as herein substantially described with reference to examples 12. A method for obtaining immobilized protein of claim 1, as herein substantially described with reference to examples.

Full Text

IMMOBILIZATION OF PROTEINS ONTO SOLID SUPPORT
Field of invention
The present invention relates to an immobilized protein onto a solid support. The invention also relates to an improved method for immobilization of the protein onto a solid support. Background and prior art references
Enzymes are proteinaceous catalytic materials that have great industrial potential. The immobilization of enzyme provides reusability and high selectivity with an increase in stability and thus renders it economical. The immobilized enzymes /proteins have been employed in diagnostic kits, medical implants, chemical separation and biosensor. Enzymes are also very expensive materials. They are generally soluble in their respective substrates and except where the conversion product is of great value, recovery of the enzyme for the reuse may be difficult or impossible.
To avoid such problems, fixed or immobilized systems have been developed. For the purpose of the immobilization of the enzymes /proteins the procedures such as adsorption, encapsulation and covalent bonding are known in prior art.
In the process of immobilizing an enzyme, there are many practical considerations. There should be as little loss of enzyme activity as possible. The cost of immobilization should be low. The carrier material should be one that does not have a deleterious effect on the action of the enzyme during reaction.
There are several reports in the literature for immobilization of proteins that are enumerated herein below. Immobilization of Oxalate oxidase
Oxalate oxidase from different plant sources has been already immobilized on various supports by different techniques. Some are onto pig intestine membrane (Guilbault et al 1986); onto nylon tubing (Bais et a/,1980); onto polyionic electrolyte ( Raghavan and Tarachand,1986) onto antiserum (Inamdar et a/,1986); onto Polyamide membrane (Assolant et a/,1987); onto concanavalin A (Varalakshmi and Richardson, 1992); onto gelatin (Dinckayan and Telefoncu,1993); onto polyvinyl chloride (PVC) and CA membrane laminate (Reddy et a/,1994); onto AF-Tresyl Toyopearl 650 gel (Yamato et al., 1994); onto collagen membrane (Saka Amini and Vallon,1994); onto polyethylene glycol (PEG) (Varalakshmi et al.,1995); onto acrylamide membrane (Lathika et al, 1995);.onto sodium alginate (Lathika et al., 1995);
onto glass beads (Pundir et al, 1993); onto glass beads (Pundir et al 1998).
A method of immobilization of oxalate oxidase onto pig intestine membrane was described by Rahni & Guilbault (1986) .They constructed enzyme electrode for oxalate determination in urine. However, this electrode membrane was vulnerable to microbial attack. This method showed less stability of immobilized enzyme (2 month) and less reliability (r = 0.95) in comparison to the present invention (r =0.99).
The Sorghum leaf oxalate oxidase (Pundir & Satyapal, 1998) enzyme was immobilized onto alkylamnie glass beads to reduce the cost of oxalate determination in large number of urine samples (Thakur & Pundir, 1999). The drawback associated with glass beads is that glass beads are costly and due to porosity of glass beads the enzyme are entrapped inside the pores and the activity of immobilized enzyme is reduced. The present invention using PVC tube/Plastic strip for immobilization of the enzymes overcomes the problem associated with the prior art.
Reddy et al (1994) reported a method for the immobilization of barley oxalate oxidase on polyvinyl chloride (PVC) and cellulose acetate (CA) membrane laminate. The laminate was integrated within an amperometric electrode and a biosensor was developed for oxalate determination via electrochemical oxidation of H2O2 and subsequent current generation. However, this method had some drawbacks like instability, low retention of specific activity and high cost.
Varalakshmi et al (1995) described a method of immobilization of beet stem oxalate oxidase onto polyethylene glycol (PEG). Although enzyme retained about 90% of its initial activity, but the stability of immobilized enzyme was up to 3 months only.
The present invention overcomes the above described drawbacks associated with the prior art. Immobilization of Upases
The Upases from different sources have been immobilized on various supports by different methods for different purposes. Some are onto Sepharose 6-B through covalent coupling (Otero et al, 1988); on polystyrene butadiene rubber using two phase emulsion technique for the hydrolysis of triacetin (Iso et al, 1989); onto polyvinyl chloride (Rucka and Tturkiewicz, 1990) onto microporous polypropylene by adsorption (Montero et al, 1993) onto anion exchange resin and diatomaceous-earth (Mustranat et al, 1993) onto porous polyurethane particles (Wang and Ruchenstein 1993) onto organic polymer beads (Basri et al, 1994) onto polyethylene powder (Watanable et al, 1995) on EP-100 polypropylene powder by adsorption (Thomas Gitlesen et al., 1997); within a
phyllosilicate sol-gel matrix (An-fei Hsu, Thomus A. Fogila & Siyan Shen, 2000); on phyllosilicate sol-gel matrix through entrapment technique (Hsau et al, 2000); in a hollow-fibre reactor by covalent coupling (Sehanputri and Hill, 2000); within solid nano composite matrix of tetramethoxy-silane (TMOS) (Schuleit and Luisi, 2001); onto different supports like silica, Sepabeads, CNBR activated Sepharose 4B, HP-20 beads and phenyl-sepharose (Dosaryh & Kaur, 2002) onto 7-Fe2Oa magnetic nanoparticles (Dyal et al, 2003) onto free alkylamine glass beads (Sarita et al, 2000); onto alkylamine glass beads affixed inside a plastic beaker (Arya S et a/,2003). In the present experiment porcine pancreas lipase was immobilized onto egg shell membrane affixed to plastic strip employing this technique
Most of these immobilization processes includes physical adsorption which decreased the life of the immobilized enzyme beside this other problems associated are low retention of specific activity and high cost.
A method of immobilization by entrapment in chemically inert hydrophobic silica gels, prepared by hydrolysis of alkyl-substituted silanes in the presence of lipase was described by Reetz et al (1996). However, this method involved the entrapment, which does not allow proper interaction of substrate with enzyme. So, it is not an effective methodology for immobilization. Immobilization of alpha-amylase
Amylase has been already immobilized on various organic and inorganic supports. Some are polyurethane.polymer. (Storey.et.al,1990) on agar gel (Tonkova et al 1994), on micro spheres (Arica et al 1995), on temperature sensitive membrane (Chen et al, 1998), on polyacrylamide gel by Raviyan et al (2003), on alkylamine glass beads through covalent coupling by Ramesh & Singh (1981), on plastic support (Roig et al, 1993), on coconut fiber by Dey et al (2002).
Chen et al (1998) described a method of immobilization of alpha-amylase on to a composite temperature-sensitive membrane by cross-linking. This membrane cannot account for stability, as it is temperature sensitive.
A method for immobilization of alpha-amylase on to thermal-responsive composite hydro gel membranes by cross-linking was described by Sun et al (1999). It carried the drawback of thermal dependence and low retention of activity, so this method is not reliable.
Raviyan et al (2003) described a method for immobilization of alpha-amylase
from Aspergillus oryzae by entrapment in polyacrylamide gel. However the acrylamide is neurotoxin and does not allow proper enzyme substrate interaction. Besides this, handling is tedious.
Immobilization of glucose oxidase
Wang et al (1995) described a method of immobilization of enzymes covalently on to a cellulose acetate membrane. A glucose oxidase membrane was prepared using this method, which was combined to an oxygen electrode and hydrogen peroxide electrode separately to construct a glucose biosensor. However this was synthetic membrane and could not compete on the ground of cost. This membrane faces the substrate analogy in the case of oxalate oxidase.
Zythes et al (1976) described a method of immobilization of glucose oxidase in a polyacrylamized coating obtained by electronically initiated polymerization. It included the expensive chemical treatment and sensitive instrumentation, which increase the cost of immobilization.
Degitor & Guly (1981) described a method for immobilization of glucose oxidase on to cellulose by covalent combination with amino ethyl cellulose (AE-Cellulose). However, this method involved expensive chemicals, which increased the cost of immobilization.
A method of immobilization of glucose oxidase in a polyacrylamized coating obtained by electronically initiated polymerization was described by Zythes et al (1976). It included the expensive chemical treatment and sensitive instrumentation, which increase the cost of immobilization.
Immobilization of macromolecules
A report by Amyl Springer et al. (2003) described a method of immobilization of proteins and other macromolecules via interaction of two small synthetic molecules, phenyldiboronic acid (PDBA) and salicylhydroxamic acid (SHA). As this method showed lower activity and stability of enzyme after the immobilization on the solid support, this method is not suited for immobilization of enzymes.
Patent documents describing immobilization of proteins:
US patent 4251631 discloses immobilization of enzyme onto silica modified vinyl chloride polymer membrane by adsorbing the enzyme. The immobilized enzyme was used to carry out enzymatic reaction by passing a solution of its substrate through a membrane under differential pressure. However, this method has limited application

because pressure difference can lead to change in conformation as well as substrate inhibition and increase the cost of procedure.
US patent. 5219926 disclose a covalent binding of biopolymer of amino acids on organic polymer surface coated with hydrophilic nonionic polymer having reactive groups. As this method includes attachment of active group (various oxidative groups) which can lead to loss of enzyme activity due to presence of highly reactive oxide group as site of attachment of enzyme.
US patent 5705045 describes a method of fabricating a multi biosensor using a carbon paste layer around a insulating substrate (PVC) and by immobilizing the GPT and GOT on amino acid polymer paste layer in carbon paste. This method is limited only to the GPT and GOT only. US patent 5516703 discloses the formation of reactive surface by absorbing a modified block polymeric surfactant containing poly (ethylene oxide) block attached to an end of poly (propylene) centre blocks. This method also has similar limitations which the patent no 521996 contains.
US patent 5720969 discloses preparation of perm selective graft that was formed by converting into intermediate reactive sites a portion of Cyano group of a backbone polymer and grafting polyalkyene oxide polymer chain to backbone polymer through a reactive site. The resulting perm selective membrane was formed into hollow fiber or flat sheet for encapsulation of cell. It also shows less stability of enzyme activity on immobilized surface.
US patent 5808012 describes the interspreading of a variety of functional thermos table proteins inside thermoplastic after molding /extrusion /casting process. This method has limited application of immobilization of thermo stable proteins and enzyme, while most of protein/enzyme got denatured at that temperature.
US patent 5928918 describes a biosensor formation based on enzyme containing polymer matrix on the surface of a supporting layer which contain cross linking groups. This method show less stability of enzyme on surface as well as very costly procedure.
US patent 6134461 discloses the formation of a biosensor for the detection of glucose, lactate and oxygen in patients by forming an electrode, in which the conducting material was deposited on the surface of support. Concentration of analyte was measured by forming a potential difference between working electrode and reference electrode.
US patent 6130033 describes the manufacturing of small sensor by depositing
conductive material in the channels formed on substrate by impact and non-impact methods. As this method involves deposition of conductive material and embedding of enzyme in it, it inhibits direct contact of enzyme to the substrate so this method decreases activity of enzyme.
US patent 6670199 discloses the adhesions of the cell and biomolecules to hydrophobic surface and hydrophobic coated surfaces. This method require some specific condition for adhesion of cell and biomolecules which can be possible during cell culture but not during enzyme catalytic reactions.
US patent 6180007 discloses the formation of perm selective graft polymer by converting -CH - group of polymer to cyano group and grafting polyalkene oxide polymer chains to backbone of polymer through reactive sites .This method lead to oxidation of active site of enzyme and substrate so it decrease both the activity and stability of enzyme.
US patent 4696901 discloses immobilization of organism on a plastic carrier preferably poly tetrafluoroethylene, for this water immiscible hydrocarbon substrate was added in nutrient media of microorganisms which increases the load of microorganisms on carrier. This method is useful for growing of microorganisms and only not for enzyme.
US patent 4396716 discloses the immobilization of apyrase (Apyrase is an enzyme which hydrolyze nucleoside triphosphates and diphosphates and are found in all eukaryotes, where they have essential functions in platelet aggregation and neuronal signal termination) on a polyamide surface/polyethylene terphthalate polymer after hydrolytically activating the surface and then using cross linking agent and solution of apyranase.
In US patent 5808012, a method is described for the preparation of protein rich thermoplastic. However, this method is applicable for thermo stable polypeptide not for thermo labile protein / enzyme.
US patent 4251631 claimed a method for cross linking of enzyme with silica modified poly vinyl membrane. However this method required a pressure differential of 5 to 10 atmospheric pressure, to maintain enzymatic reaction
A method of covalently linked biopolymer to solid hydrophilic organic polymer was reported in US specification 5219926, which had limited application for heat stable
polypeptide and protein, which remain active at 45 to 55°C. But most of the protein like oxalate oxidase, peroxidase and glucose oxidase, a- amylase are heat labile. So this method had limited applications. However, our method has no such limitations.
Another technique for membrane formation by converting unreactive polymer membrane to reactive sites by grafting polyalkene oxide to some cyano group modified reactive sites on backbone of the given polymer had been described in USPTO specification 5720969, but this method was suited for encapsulation of biological organisms and cells but not for enzymatic reaction due to oxidation by reactive sites
Compared to above and other relevant searches, the present invention is more fruitful as it covers up all the limitations in these immobilization methods. In most of the prior art designs of various immobilization systems, plastic was used as insulating supports for electrode formation. In other cases, immobilization on PVC was carried out either through surface modification or by addition of other polymer mixture or by affixing some reactive blocks like ethylene oxide etc. Most of prior art processes of immobilization of proteins have major one or more limitations such as:
1) The enzymatic reactions required pressure difference for diffusion of substrate to
the enzyme to carry out the reaction.
2) Diffusional resistance of support surface to substrate due to hydrophobicity 5Loss
of activity of immobilized proteins within few weeks or in month
3) Very low retention of specific activity of immobilized enzyme
4) Denaturation of protein at the time of protein entrapment
5) In some cases oxidized product are formed (like f^Ch) during enzyme catalysis
which may lead to release of enzyme from solid surface into solution and thereby
decreasing the stability of the enzyme.
Objects of the invention
An object of the present invention is to provide an immobilized protein which is easy to handle, cheaper than other immobilized enzymes available in the market, to obtain kits used efficiently in different assays.
Another object of the present invention is to provide an improved process for immobilization of proteins onto solid support
Another object of the invention is to provide a cost effective support for example plastic strip and PVC material for immobilization of proteins.
Yet another object of the invention is to provide an immobilized protein onto the solid support which is reusable.
Still another object of the invention is to provide an efficient method for immobilization of alpha- amylase, peroxidase, lipase, glucose oxidase, sorghum oxalate oxidase, human antiserum (IgG) and other proteins onto PVC material and plastic strip with enhanced retention of specific activity of the proteins.
Still another object of the present invention is to provide an immobilized oxalate oxidase enzyme which can be used for the assay of urine & serum oxalate by standard colorimetric method.
Summary of the invention
The present invention relates to an immobilized protein comprising of activated support linked to a protein having atleast a free amino functional group and an improved process for immobilization of said proteins onto a solid support by treating the solid support with inorganic acids followed by treatment with a linking agent having atleast two terminal aldehyde groups in aqueous buffer solution to obtain activated solid support which is further contacted with a proteins having atleast a free amino functional group.
Brief description of the drawings
Figure 1: Stability results of immobilized amylase onto plastic strip and PVC tube. Figure 2: Stability results of immobilized peroxidase onto plastic strip and PVC tube. Figure 3: Stability results of immobilized lipase onto plastic strip and PVC tube Figure 4: Stability results of immobilized glucose oxidase onto plastic strip and PVC
tube Figure 5: Stability results of immobilized oxalate oxidase onto plastic strip & and PVC
tube Figure 6: A correlation between urinary oxalate values determined by using Sigma kit
method and present immobilized oxalate oxidase enzyme. Figure 7: Standard plot of Oxalate employing grain sorghum leaf oxalate oxidase
immobilized on plastic strip or PVC tube
Detailed description of the invention
In accordance with the object, the present invention provides an immobilized protein comprising of activated support linked to a protein having atleast a free amino
functional group through a linking agent having atleast two terminal aldehyde groups. An embodiment of the invention provides a method for obtaining immobilized protein, the said method comprising steps of:
a.) treating the support sequentially with inorganic acids in presence of a catalyst;
b) treating the support of step (a) with linking agent having atleast two
terminal aldehyde groups to obtain activated support,
c) contacting the activated support of step (b) with protein having atleast a
free amino functional group for 10-12 hrs at a temperature in the range of
30-35°C under dark conditions with continuous gentle shaking to obtain
immobilized protein.
An embodiment of the invention uses a solid support selected from the group consisting of plastic strip and PVC material.
Another embodiment of the invention provides a method wherein the support is first treated with concentrated nitric acid followed by concentrated hydrochloric acid.
Yet another embodiment of the invention provides a method wherein the catalyst used may be selected from the group consisting of ferric chloride and other salts of iron.
Still another embodiment of the invention provides a method wherein the support is treated with 15% gluteraldehyde in 0.1 M sodium phosphate buffer of pH 6.5 and at temperature in the range of 30-35°C to obtain activated support
Still yet another embodiment of the invention provides a method wherein the protein having atleast a free amino functional group is selected from the group consisting of enzyme, antibodies.
The enzyme having free amino functional group is selected from the group consisting of oxalate oxidase, alpha amylase, lipase, peroxidase, glucose oxidase and hydrolytic enzymes such as, ligase, lyase and isomerase.
The present invention provides an improved method for the immobilization of the proteins onto plastic strip or PVC tube.
Immobilization of the protein onto the plastic strip or PVC tube is cost effective and provides the reuse of the enzyme with ease. It is also efficient method; no noticeable change in activity of affixed enzyme is observed even after several months and can be used for immobilization of variety of protein used in diagnostic kits, foods and
detergents.
For immobilization, Protein is isolated and characterized; the detailed procedure of immobilization of the protein is described in Example 1.
Both PVC tube and plastic strip consist of cross linked vinyl polymers, which on treatment with highly concentrated acid lead to breakage of monomer linkage and provide free chloride group for reaction with free aldehyde group of linking agent used. The other aldehyde group of linking agent reacts with free -NH2 group present on surface of protein to form a Schiff "s base and thus lead to immobilization of enzyme.
The following chemical reaction are involved stepwise,
(a) Breakage of vinyl polymers of PVC material:
{-CH2-CH (Cl) -CH2-CH (Cl)-}(n-1) + HNO3 + FeCl3 + HC1 -→
(- CH2CH2 Cl) x*+ (CH3-CH (Cl)y-Where n = x+y
- denotes uncertainty in chain length of polymer after breakage, as the site of breakage is
not known.
(b) Activation of plastic strip and inner wall of PVC tube.
- CH2CH2 Cl + OHC-(CH2)3-CHO- → -CH2CH2 (O) C-(CH2)3-CHO
(c) Immobilization of enzyme on PVC material:
- CH2CH2 (O) C-(CH2)3-CHO +NH2-E - → -CH2 CH2 (O)C(CH2)3 (H)C=N-E
Immobilized enzyme
By using above method of immobilization many enzymes such as alpha-amylase, lipase, glucose oxidase, peroxidase, oxalate oxidase and other proteins are immobilized. The oxalate oxidase enzyme used is preferably obtained from sorghum sp. The assay of the immobilized enzymes is described Example 2 (I to V). Kinetic properties of the immobilized enzymes are also given in Tables (1 to 5).
The present invention also provides an efficient method of immobilization of alpha-amylase, peroxidase, lipase, glucose oxidase , oxalate oxidase, human antiserum (IgG) and other Proteins onto solid support preferably PVC material and plastic strip where there is enhanced retention of specific activity of the enzyme and the immobilized enzyme remains active even after several months.
The graph showing stability of five different immobilized enzymes onto plastic strip and PVC tube is shown in Figure 1 to 5.
Urinary oxalate is also determined using the immobilized oxalate oxidase enzyme
that is described and several analytical parameters are determined to evaluate the efficiency of the method.(Examp!e 3) A comparison of data obtained using sigma kit and the present immobilized oxalate oxidase enzyme provided a good correlation (r=0.99) which evaluated that the present method is more efficient. (Fig 6)
No noticeable change in activity of plastic strip and PVC tube bound sorghum oxalate oxidase is observed after 200 uses over a period of 3 months when stored in reaction buffer at 4°C in dark.
The invention is illustrated with following examples and should not be construed to limit the scope of the present invention. The present invention has been described in terms of its specific embodiments and certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention
EXAMPLES
Example 1
Immobilization of the proteins on PVC/ Plastic strip:
In present invention protein is immobilized using the following steps. I: Treatment with acid.
(a) In this step the solid support is treated with concentrated HNO3 for 5-7 hrs at
room temperature followed by many washing with distilled water to remove
the excess of acid adhering to the surface,
(b) The HNO3 treated solid support is allowed to react with concentrated HC1 in
presence of Ferric salt for 2-3 hrs,
(c) The treated solid support of step (b) is washed again with distilled water to
remove excess acid and Ferric salt.

II. Cross linking of membrane: Above treated solid support of step, I (c) is allowed to
react with linking agent in buffer solution to obtain activated solid support
III. Immobilization of enzyme: Enzyme is immobilized on solid support of step II
This experiment involves immobilization of different protein such as alpha-amylase,
peroxidase, lipase, glucose oxidase, oxalate oxidase and commercial human
antiserum (IgG) onto plastic strip. The antisera are immobilized by incubating the
activated strip with the antiserum sample dissolved in buffer for 24 hrs at 4 °C.
Example 2 Assay using enzymes
I. Assay using a-amylase enzyme:
Assay of free α-amylase is carried out by adapting method developed by Mifflin TE, Bruns DE (1987) with modification.
(a) Preparation of 0.05 M starch-acetate buffer, (pH 5.6): 0.2 M sodium acetate
(91 ml) is added to 9 ml 0.2 M acetic acid. To 2ml of this, 8ml-distilled water is
added and its pH is adjusted to 5.6. To this, 0.2 g of starch is added and then
boiled, filtered and stored it at 4-6°C until use.
(b) Preparation of enzyme solution: α-Amylase powder (5mg) is dissolved in
1ml of 0.05 M starch-acetate buffer, pH 5.6 and stored at 4°C until use.
(c) Preparation of DNS (Dinitro Salicylic Acid): Sodium potassium tartarate
(20 mg) is dissolved in 50 ml distilled water. A pinch of 2,4- dinitro salicylic
acid is dissolved in 20ml of 2N NaOH. These two solutions are mixed and the
final volume is made up to 100 ml with distilled water
(d) Assay using free a-amylase: Assay of amylase is based on the measurement
of glucose and maltose generated from hydrolysis of starch by amylase using
DNS reagent. To 1.9 ml of 0.05 M acetate buffer containing 2% starch in a test
tube, 0.1 ml enzyme solution is added. For blank, 2.0 ml of reaction buffer
containing 2% starch is taken in another test tube. Both the tubes are incubated at
37 °C under continuous stirring conditions for 10 min. The reaction is stopped by
adding 0.1 ml 2N NaOH, DNS (0.9 ml) reagent is added to both the tubes, placed
in boiling water bath for 5 min & cooled to the room temperature, ASM of red
colored reaction mixture is recorded against blank and the amount of glucose
generated is extrapolated from standard curve between glucose concentration and
A54Q.
Enzyme Unit: One unit of enzyme is defined as amount of enzyme required to catalyze the formation of 1 umol of glucose from starch per min. under standard assay conditions.
(e) Assay using immobilized a-amylase: It is carried out as described for free
enzyme except that immobilized enzyme is used in place of free enzyme and the
reaction buffer is increased by 0.1 ml. After the assay the strip is taken off and
reaction mixture is transferred to cuvette. In case of PVC tube the reaction
mixture is directly transferred to cuvette and As4o of the reaction mixture is read.
The enzyme retained 75.3 % and 68.7% of initial activity after immobilization in
case of PVC tube and plastic strip respectively.
When immobilized a -amylase is incubated in reaction mixture of pH 4.5 at 40 ° C then 0.19 mg, 0.11 mg of glucose is generated by plastic strip, and PVC tube bound enzyme respectively. When immobilized a -amylase is incubated in reaction mixture of pH 5.5 at 40 °C then 0.037mg of glucose is generated by plastic strip bound enzyme and 0.32 of glucose is generated by PVC tube bound enzyme. When immobilized a -amylase is incubated in reaction mixture of pH 5.5 at 35°C then 0.008 mg and 0.012 mg of glucose is generated by plastic strip and PVC bound enzyme respectively. Thus it is observed that pH of 5.5 and 35°C temp is optimum for activity of immobilized enzyme reaction.
Various other kinetic properties are described in the table. 1. Table.l. Kinetic properties of α- amylase immobilized on PVC tube/plastic strip
(Table Removed)
Assay of peroxidase enzyme:
The assay of peroxidase based on Trinder's colour reaction is carried out as described by
Pundiretal(1999)
a) Assay using free enzyme: One mg horseradish peroxidase is dissolved in 10 ml of 0.05 M sodium phosphate buffer, pH 7.0. In a 15 ml conical flask wrapped with black paper, the reaction mixture containing 130 µmol of sodium phosphate buffer (pH 7.0) 25 units of horseradish peroxidase solution, 2.46 µmol of 4-amino phenazone and 10µmol of phenol in a total volume of 3.0 ml is pre
incubated at 40°C for 5 min. The reaction is started by adding 10 (amol H2O2 after 20 minutes incubation at 25° C. The absorbance is recorded at 520 nm. Enzyme Unit: One unit of enzyme is defined as amount of enzyme required to catalyze the formation of 1.0 n mol of H2O2 from oxalate per minute under standard assay conditions.
(b) Assay using immobilized enzyme: The assay of immobilized horseradish peroxidase is carried out as described for free enzyme except that tube / strip bound enzyme is used in place of free enzyme. To reuse immobilized enzyme the strip / tube is washed with reaction buffer 3-4 times prior its use in next day. The enzyme retained 69.13 % & 64.32% of initial activity after immobilization in case of PVC tube and plastic strip respectively.
When immobilized peroxidase is incubated in reaction mixture of pH 4.5 at 40 °C then!6.6 nmol and 20 nmol of H2O2 generated by plastic strip and PVC tube bound enzyme. When immobilized peroxidase is incubated in reaction mixture of pH 6.0 at 40 °C then 243 nmol and 313 nmol of H2O2 generated by plastic strip and PVC tube bound enzyme respectively. When immobilized peroxidase is incubated in reaction mixture of pH 6.0at 35 °C then 146 nmol and 73 nmol of H2O2 generated by plastic strip and PVC bound enzyme. So the PVC and plastic strip bound enzyme show maximum activity at pH of 7.0 and 5.5 when incubated at 40 °C respectively. Other kinetic properties are also studied which are described in the table 2.
Table 2: Kinetic properties of horseradish peroxidase immobilized onto PVC /plastic strip
(Table Removed)
III. Assay using lipase
(a) Assay using free lipase: The activity of free lipase is assayed according to Gotthiff Naher with modification. The titration is carried out manually by burette. In a
100 ml conical flask, 5.0 ml olive oil emulsion is added to 5.0 ml of 0.1 M tris buffer (pH 8.0) and incubated at 35 °C for 10 minutes. 1 ml lipase solution (Img/ml) is added and again incubated at 35°C for 20 min. The reaction mixture is then kept at room temperature for 20 min and then added 10 ml of acetone and methanol mixture (1:1) to stop the reaction and titrated against 0.025 N NaOH after adding 1% phenolphthalein as an indicator. The volume of NaOH used in titration is noted.
Unit of enzyme: One unit of lipase is defined as the amount of enzyme required to liberate 1 µmole of free fatty acid from olive oil per min under the standard assay conditions (at 35°C and pH 8.0).
(b) Assay using immobilized lipase: The assay is carried out as described above except that immobilized enzyme is used in place of free enzyme and the reaction buffer is increased by 1ml. After the final incubation, the strip is taken off and reaction mixture is transferred into titration flask in case of PVC tube the reaction mixture is directly transferred to titration flask. The enzyme retained 70.8 % and 66.2% of initial activity after immobilization in case of PVC tube and plastic strip respectively.
Immobilized enzyme is incubated at different pH (5-7) and temperature range (35-45 °C), it is observed from various experiments that pH of 7.0 and 40°C & 45°C is optimum for activity for plastic strip and PVC bound enzyme respectively. Other kinetic properties are also studied which are described in the table 3.
Table 3: Kinetic properties of porcine pancreases lipase immobilized on to PVC /plastic strip.
(Table Removed)
IV Assay using glucose oxidase (GOD)
(a) Preparation of colour reagent: The colour reagent is prepared according the method of Bais et al (1980) and consisted of 50 mg 4- aminophenazone, 100 mg solid
phenol and 1 mg horseradish peroxidase per 100 ml 0.4M sodium phosphate buffer, pH 7.0. It is stored in amber coloured bottle at 4°C and discarded after one week. .
(b) Assay using free glucose oxidase: The assay is carried out in 15 ml test tube
wrapped with black paper. The reaction mixture containing 1.7ml 0.05 M sodium
phosphate buffer pH 7.0,0.1 ml CuSO4 (10"2M) is preincubated for 5 min at 37° C after
addition of 0.1 ml enzyme. The reaction is started by adding 0.1 ml of glucose (Img/ml).
After incubating the reaction mixture at 37° C for 10 min under continuous stirring, 1 ml
of colour reagent is added and the whole reaction mixture is incubated again at 37° C for
15 min. The reaction mixture is transferred to a cuvette and A520 is recorded.
Enzyme Unit: One unit of enzyme is defined as amount of enzyme required to catalyze the formation of 1.0 n mol of H2O1 from oxalate per min. under standard assay conditions.
(c) Assay using immobilized enzyme: The assay of immobilized enzyme is
performed as described as above except that free enzyme is replaced by immobilized
enzyme and reactions mixture is increased by 0.1 ml of reaction buffer and after the
assay the plastic strip is taken off and reaction mixture is transferred to cuvette. In case
of PVC tube, the reaction mixture is directly transferred to cuvette. The enzyme retained
83.3% and 75% of initial activity after immobilization in case of PVC tube and plastic
strip respectively.
Immobilized enzyme is incubated at different pH (4.5-5.5) and temperature range (35-45 °C). It is observed from various experiments that pH of 5.5 and 40 °C is optimum for activity of immobilized enzyme.
Other kinetic properties are also studied which are described in the table 4.
Table4: Kinetic properties of A. niger glucose oxidase immobilized onto Plastic strip/ PVC tube.
(Table Removed)
V: Assay using oxalate oxidase
(a) Preparation of colour reagent: The colour reagent is prepared according the
method of Bais et al (1980) and consisted of 50 mg 4- aminophenazone, 100 mg solid
phenol and 1 mg horseradish peroxidase per 100 ml 0.4M sodium phosphate buffer, pH
7.0. It is stored in amber coloured bottle at 4°C and discarded after one week.
(b) Assay using oxalate oxidase: The assay of oxalate oxidase is carried out as
described by Satyapal and Pundir (1993) based on the measurement of H2O2 generated
from oxalate by oxalate oxidase with a color reaction containing 4-aminophenazone,
phenol and peroxidase as chromogenic system. The assay is carried out in a 15 ml test
tube wrapped with black paper. The reaction mixture containing 1.7 ml of 0.05 M
sodium succinate buffer (pH 5.0), 0.1 ml CuSO4 solution (10-2 M) and 0.1 ml of crude
enzyme is pre incubated at 37°C for 5 min. The reaction is started by adding 0.1 ml
oxalate solution (10-2M). After incubation at 38°C for 10 min, under continuous stirring,
1.0 ml color reagent is added and kept at room temperature for 15 min, in dark to
develop the colour. The blank contained 1.8 ml 0.05 M sodium succinate buffer pH 5.0,
O.lml CuSO4 (10-2M) and 0.1 ml oxalic solution (10-2M). The control consisted of 1.7 ml
sodium succinate buffer, pH 5.0, 0.1 ml boiled enzyme solution and O.lml oxalate
solution (10 M).The assay for blank/ control is done as described for test sample and
A520 is read against blank in Spectronic-20. The content of H2O2 generated in reaction is
extrapolated from standard curve between H2O2 cone. Vs A520.
Enzyme Unit: One unit of enzyme is defined as the amount of enzyme required to catalyze the formation of 1.0 n mol of H2O2 from oxalate per min. under standard assay conditions.
(c) Assay using immobilized enzyme: The assay of immobilized enzyme is
performed as described as above except the free enzyme is replaced by immobilized
enzyme and reactions mixture is increased by 0.1 ml of reaction buffer. After the assay
the plastic strip is taken off, reaction mixture is transferred to cuvette, and As2o is
recorded. In case of PVC tube the reaction mixture is directly transferred to cuvette.
Immobilized enzyme is incubated at different pH (5.5-6.5) and temperature range (35-45
°C); it is observed from various experiments that pH of 6.5 and 40°C is optimum for
activity of immobilized enzyme.
Other kinetic properties are also studied which are described in the table 5.
Tables: Kinetic properties of sorghum leaf oxalate oxidase immobilized onto PVC /plastic strip
(Table Removed)
A comparison of various kinetic parameters of grain sorghum leaf oxalate oxidase in free form and that immobilized onto free alkylamine and arylamme glass beads, alkylamine glass beads affixed in a glass beaker and egg membrane affixed on a plastic strip is described in table given below. (Table 6)
Table: 6
(Table Removed)
A comparison of immobilized sorghum leaf oxalate oxidase onto egg membrane affixed onto plastic strip with that on free alkyl and arylamine glass beads and alkyl amine glass beads affixed in glass beaker is also given in table below (table 7). The table shows better retention of the enzyme onto egg membrane in comparison to other supports and also better specific activity.
Table 7 Immobilization of grain sorghum leaf oxalate oxidase onto various supports by covalent coupling.
(Table Removed)
Example 3:
Measurement of urinary oxalate by immobilized oxalate oxidase:
(a) Collection and storage of urine: The first morning urine samples from
apparently healthy and stone patients (both males and females of various age groups) are
collected in plastic bottles at local Pt. B.D.S.PGIMS, hospital. The concentrated HCI is
added to urine samples to adjust its pH 2.5 and stored at 4°C until use.
(b) Pretreatment of urine samples: Various urine samples are pretreated
according to method of Buttery and Pannell (1987) with modifications. One ml of
acidified urine is added to 1.0 ml of aqueous CaCl2 (5g/l) and its pH is adjusted to 6.0
with IN NaOH. Immediately 10 ml ethanol is added to the tubes, covered with
aluminum foil and kept at room temperature for overnight. On next day, the urine sample
are centrifuged at 3,000 rpm for 10 min and calcium oxalate precipitates are collected
and redissolved in measured quantity of 0.1 N HCI.
(c) Preparation of standard curve for urinary oxalate with immobilized
oxalate oxidase: It is prepared between different oxalate concentration and activity of
sorghum leaf oxalate oxidase immobilized onto various organic supports under optimal
assay conditions. Reaction mixture containing 1.8 ml of 0.05 M sodium phosphate buffer
pH 6.0, 0.1 ml of CuSO4 (10-2M) in reaction medium wrapped in a black paper is
preincubated at 37°C for 7 min. One ml oxalate solution of different concentrations
(Final concentration from 0.2 mM to 6 mM) is added to reaction mixture and incubated
at 37°C for 7 min under continuous stirring. One ml colour reagent is added to it and
kept at room temperature (25 ± 5) °C for 15 min in dark to develop the colour. The
reaction mixture is transferred to cuvette slowly and A520 is read in Spectronic-20. A
standard curve is plotted between various oxalate concentrations and A520 (Fig.7).
(d) Assay using urinary oxalate: The assay of urinary oxalate is carried out in
reaction beaker containing immobilized enzyme and wrapped with black paper. Optimal
assay conditions are used except that 0.1 ml pretreated urine samples are taken in place of oxalate. The amount of oxalate concentration is calculated from standard curve between oxalate concentration and corresponding A520.
The following analytical parameters are determined to evaluate the method.
(a) Linearity: A linear relationship is found between oxalate concentrations
ranging from 0.02mM to 6mM. The lower detection limit of this method is 1 to 2 µmol/l,
which is lower than that of other methods such as isotachophoresis (40 µmol/l) ( Schmidt
et al , 1979), enzyme colorimetric method employing sorghum leaf enzyme bound to free
alkylamine glass beads (O.46µg/O.lml) (Pundir et al , 1999) and affixed alkylamine glass
beads( 0.05 mM/1) (Kumari.M, Pundir,C.S,2004)
(b) Recovery: Analytical recovery of added oxalate in urine sample 20mg/l and
30mg/l is 90±2 %( mean ± SD, n=5) which is comparable to earlier reports (Pundir et al,
1998).
(c) Precision: To study the reproducibility and reliability of oxalate value in
24 hr urine samples in one run (within batch) and in same urine samples after one week
storage at -20 °C (between batches) are determined. The mean oxalate value of these
determinations agreed with each other. Within batch and between batch coefficient of
variation (cv) for urinary oxalate determinations are are comparable to earlier reports (Pundir et al, 1998, Thakur et al 1999). The oxalate
value of 24 hr urine from healthy adults, as measured by the present method is in range,
13.7 to 24.1 mg/24 hr with a mean of 18.9 mg/24 hr, which is in normal established
range.
(d) Accuracy: To test the accuracy of method ,urinary oxalate value obtained by
present method (y) is compared with the those obtained by enzyme colorimetric kit
method (Sigma) The correlation coefficient is 0.99, the regression equation being
y=1.017x- 0.548. (Figure 6)
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62. Goyal L,Thakur M &Pundir C.S,(1999) Anal .Lett., Vol-32 pp 633-647 Claims:

I / We claim
1. An immobilized protein comprising of an activated support linked through a
linking agent to a protein having atleast a free amino functional group, wherein
the said linking agent have at least two terminal aldehyde groups.
2. A method of immobilization of protein of claim 1, the said method comprising
steps of:
a. treating a support sequentially with inorganic acids in presence of a
catalyst, washing with water to remove the excess acid and catalyst,
b. treating the washed support of step (a) with linking agent in a buffer to
obtain an activated support, and
c. contacting the activated support of step (b) with protein having atleast a
free amino functional group for 10-12 hrs at a temperature in the range of
30-3 5°C under dark condition with continuous gentle shaking to obtain
immobilized protein.
3. A method of claim 1, wherein the support is a solid support selected from the
group consisting of plastic strip and PVC material.
4. A method of claim 2 wherein in step (a) the support is first treated with
concentrated nitric acid followed by concentrated hydrochloric acid.
5. A method of claim 2, wherein in step (a) the catalyst used may be selected from
the group consisting of ferric chloride and other salts of iron.
6. A method of claim 2, wherein in step (b) the linking agent used is selected from
the group consisting of glutaraldehyde and Tri (4-formyl phenoxy) cyanurate.
7. A method of claim 2, wherein in step (b) the support is treated with 15%
gluteraldehyde in 0.1 M sodium phosphate buffer of pH 6.5 and at a temperature
in the range of 30-35 °C.
8. A method of claim 2, wherein in step (c) the protein having free amino functional
group is selected from the group consisting of enzyme and antibodies.
9. A method of claim 8 wherein the enzyme having free amino functional group is
selected from the group consisting of alpha-amylase, oxalate oxidase, lipase,
peroxidase, glucose oxidase and hydrolytic enzymes such as, ligases, lyases and
isomerases.
10. A method of claim 8 wherein the antibody immobilised is preferably human
antiserum (IgG).
11. An immobilized protein of claim 1, as herein substantially described with
reference to examples
12. A method for obtaining immobilized protein of claim 1, as herein substantially
described with reference to examples.

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http://ipindiaonline.gov.in/patentsearch/GrantedSearch/viewdoc.aspx?id=E50UVDa9OsaNAopcS2mblw==&loc=+mN2fYxnTC4l0fUd8W4CAA==

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Patent Number

269204

Indian Patent Application Number

2393/DEL/2004

PG Journal Number

42/2015

Publication Date

16-Oct-2015

Grant Date

09-Oct-2015

Date of Filing

30-Nov-2004

Name of Patentee

M.D. UNIVERSITY,

Applicant Address

BIOSCIENCES, BIOCHEMISTRY RESEARCH LABORATORY, ROHTAK 124 001 (HARYANA),INDIA

Inventors:

#	Inventor's Name	Inventor's Address
1	PUNDIR, CHANDRA SHEKHAR	BIOSCIENCES, BIOCHEMISTRY RESEARCH LABORATORY, ROHTAK 124 001 (HARYANA),INDIA
2	BHAMBI, MANU	BIOSCIENCES, BIOCHEMISTRY RESEARCH LABORATORY, ROHTAK 124 001 (HARYANA),INDIA
3	CHAUHAN, NAR SINGH	BIOSCIENCES, BIOCHEMISTRY RESEARCH LABORATORY, ROHTAK 124 001 (HARYANA),INDIA

PCT International Classification Number

C12N 11/08

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA