Patent Publication Number: US-5834273-A

Title: Heat-stable and water soluble modified enzymes

Description:
This application is a continuation of application Ser. No. 07/857,424 filed Mar. 26, 1992, now abandoned. 
    
    
     BACKGROUND OF THE INVENTION 
     This invention relates to a modified enzyme remarkably improved in stability in an aqueous solution. 
     Recently, noticing excellent catalytic function, effective applications of enzymes outside of living bodies have been tried actively. For example, various methods for measuring micro-components applying catalytic reactions of enzymes are widely practiced in the fields of clinical chemistry, biochemistry, food chemistry, food industry, and the like. But the enzymes used in these measuring methods are low in stability in aqueous solutions. Thus, the usable time of prepared enzyme solutions is short. In order to use effectively, it is necessary to prepare such aqueous enzyme solutions at the time of use in necessary amounts. 
     In order to improve the stability of enzymes in aqueous solutions, there have been proposed various methods, for example, a method of adding as a stabilizing agent for enzymes a saccharide such as sucrose, maltose, etc., a protein such as albumin, skim milk, etc., a salt such as Ca 2+ , Mg 2+ , etc., a reducing agent such as 2-mercaptoethanol, etc., a polyol such as polyethylene glycol, etc., a substrate of enzyme, a coenzyme, a chelating agent, etc.; a method of fixing an enzyme on a suitable carrier; a method of modifying amino acid sequence of an enzyme by means of genetic engineering; etc. Since there is no general rule which method is suitable for a specific enzyme, a stabilizing method is selected for individual enzymes by trial and error. Among these stabilizing methods, the method of fixing on a carrier is frequently used recently and known as a method having a relatively high stabilizing effect (German Patent No. 2,919,622, etc.). But even this method has a problem in that when an active center of enzyme or an amino acid residue relating to expression of enzymatic activity such as a substrate binding site is modified, the enzymatic activity is not expressed. Thus, even if a binding method, a binding agent, a crosslinking agent, and the like are studied for selecting the best conditions, such conditions are not always applicable for every enzyme as in the other methods. 
     On the other hand, in order to solve the above mentioned problems, there is also proposed a method of screening enzymes, which are good in stability in an aqueous solution and have properties suitable for quantitatively determining the desired microcomponents in living body samples, from various microorganisms such as thermophilic bacteria. But this method not only requires much money and time but also gives no guarantee for finding precisely enzymes suitable for the purpose. Thus, this method is not a usable method. 
     SUMMARY OF THE INVENTION 
     It is an object of the present invention to provide a modified enzyme remarkably improved in stability in an aqueous solution overcoming the problems mentioned above. 
     The present invention provides a modified enzyme comprising an enzyme selected from the group consisting of an enzyme generating hydrogen peroxide, ascorbate oxidase, peroxidase, urease, catalase, and glycerokinase bound to a polysaccharide having a plurality of carboxyl groups a polyamino acid having a plurality of carboxyl groups or a synthetic polymer having a plurality of carboxyl groups via a crosslinking agent capable of binding a carboxyl group and an amino group. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a graph showing pH stability curves obtained in Experiment 3. 
     FIG. 2 is a graph showing pH activity curves obtained in Experiment 3. 
     FIG. 3 is a graph showing temperature activity curves obtained in Experiment 3. 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     According to the present invention, an enzyme to be modified by binding a polysaccharide having a plurality of carboxyl groups, a polyamino acid having a plurality of carboxyl groups or a synthetic polymer having a plurality of carboxyl groups via a crosslinking agent capable of binding a carboxyl group and an amino group, is selected from the group consisting of an enzyme generating hydrogen peroxide, ascorbate oxidase (E.C.1.10.3.3, hereinafter referred to as &#34;AOD&#34;), peroxidase (E.C.1.11.1.7, hereinafter referred to as &#34;POD&#34;), urease (E.C.3.5.1.5, hereinafter referred to as &#34;URS&#34;), catalase (E.C.1.11.1.6, hereinafter referred to as &#34;CAT&#34;), and glycerokinase (E.C.2.7.1.30, hereinafter referred to as &#34;GK&#34;). The modified enzyme is remarkably high in stability in an aqueous solution and excellent in resistance to heat, proteases, modifying agents, and has the same fundamental properties such as Km values, optimum pH range, etc., at the time of enzymatic reaction as the non-modified enzymes. 
     As the enzyme generating hydrogen peroxide, there can be used enzymes which generate hydrogen peroxide by reaction with a substrate. Preferable examples of such enzymes are choline oxidase (E.C.1.1.3.17, hereinafter referred to as &#34;COD&#34;), glucose oxidase (E.C.1.1.3.4, hereinafter referred to as &#34;GOD&#34;), xanthine oxidase (E.C.1.2.3.2, hereinafter referred to as &#34;XOD&#34;), oxalate oxidase (E.C.1.2.3.4, hereinafter referred to as &#34;OOD&#34;), sarcosine oxidase (E.C.1.5.3.1, hereinafter referred to as &#34;SAO&#34;), uricase (E.C.1.7.3.3, hereinafter referred to as &#34;US&#34;), etc. 
     Further, the origin of the enzyme generating hydrogen peroxide, AOD, POD, URS, CAT and GK is not particularly limited and any ones derived from animals, plants, microorganisms can be used. Further, the purity of enzymes is not a problem particularly. Needless to say, it is preferable not to contain proteases. 
     As the crosslinking agent capable of binding a carboxyl group and an amino group, there can be used compounds which can bind a carboxyl group and an amino group. Preferable examples of the crosslinking agent are carbodiimide, carbodiimide derivatives such as dicyclo-hexylcarbodiimide, di-p-toluoylcarbodiimide, benzyl-dimethylaminopropylcarbodiimide (BDC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (WSC), etc.; Woodward reagent, etc. 
     As the polysaccharide having a plurality of carboxyl groups, the polyamino acid having a plurality of carboxyl groups, or a synthetic polymer having a plurality of carboxyl groups, there can be used natural polysaccharides having carboxyl groups such as alginic acid, pectic acid, protuberic acid, hyaluronic acid, chondroitin, heparin, etc.; polysaccharides introducing carboxyl groups thereinto artificially such as carboxymethyl cellulose, carboxymethyl dextran, etc.; polyamino acids such as polyglutamic acid, polyaspartic acid, polyamino acids containing a plurality of glutamic acid residues and/or a plurality of aspartic acid residues, etc.; synthetic polymers such as ethylene-maleic acid copolymer, methylvinylether-maleic acid copolymer, styrene-maleic acid copolymer, etc. 
     As the polysaccharides introducing carboxyl groups thereinto artificially, there can also be used those obtained by activating a polysaccharide such as dextran, dextran sulfate, pullulan, Ficoll, starch, dextrin, cellulose, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or the like by a method using cyanogen bromide (Nature, vol. 214, page 1302, 1967), epichlorohydrine (Infect. Immun., vol. 20, page 867, 1978), 1-cyano-4-dimethylaminopyridinium salt, 2,4,6-trichloro-1,3,5-triazine (J. Solid-Phase Biochem., vol. 4, page 233, 1979), or periodic acid (Proc, Natl. Acod. Sci. U.S.A., vol. 73, page 2128, 1976), followed by reaction with an amino acid. As the amino acid, there can be used compounds having one or more amino groups and one or more carboxyl groups. Preferable examples of such amino acids are β-alanine, 4-aminobutyric acid, 5-aminovaleric acid, 6-aminocaproic acid, p-aminophenyl-acetic acid, p-aminobenzoic acid, glutamic acid, aspartic acid, etc. 
     Further, as the polysaccharides introducing carboxyl groups thereinto artificially, there can also be used those obtained by reacting the polysaccharides mentioned above directly with an acid anhydride such as maleic anhydride, succinic anhydride, phthalic anhydride, pyromellitic anhydride, mellitic anhydride, trimellitic anhydride, etc. 
     The direct reaction with an acid anhydride is explained in detail below. 
     To a solution obtained by dissolving a polysuccharide in a solvent such as water, a pyro-phosphate buffer, etc. containing no amine so as to make the concentration usually 0.5 to 10% by weight, preferably 4 to 8% by weight, an acid anhydride such as maleic anhydride, succinic anhydride, phthalic anhydride, pyromellitic anhydride, mellitic anhydride or trimellitic anhydride is added gradually in a suitable amount while maintaining the pH usually at 7 to 10, preferably 8 to 9 using a pH-stat. After the addition, stirring is continued until the pH of reaction solution does not vary. Then, the reaction solution is desalted by dialysis, gel filtration, etc., followed by concentration or freeze drying to obtain the desired polysaccharide introducing carboxyl groups thereinto artificially. According to this process, carboxyl groups can be introduced into all the hydroxyl groups in the starting polysaccharide. By properly selecting and adjusting the kind and amount of acid anhydride, the introduced amount of carboxyl group can be adjusted. Thus, the kinds and molar ratio of the polysaccharide and acid anhydride can be selected properly depending on purposes. 
     The molecular weight of polysaccharide into which carboxyl groups are to be introduced is usually about 4,000 to 10,000,000, preferably 10,000 to 500,000. 
     In the case of polysaccharide, the amount of carboxyl group is usually 0.1 to 5, preferably 0.2 to 4, more preferably 2 to 4 per constituting sugar unit (glucose residue, glucosamine residue, etc.). On the other hand, in the case of polyamino acid, it is preferable to use that having glutamic acid residue and/or aspartic acid residue in an amount of 25% or more as the amino acid residue. In the case of the synthetic polymer, it is preferable to use the polymer having the constituting unit containing carboxyl groups in an amount of 25% or more based on the total weight of the polymer. 
     The modified enzyme can be synthesized as follows. A suitable amount of polysaccharide having a plurality of carboxyl groups, polyamino acid having a plurality of carboxyl groups or synthetic polymer having a plurality of carboxyl groups (hereinafter referred to as &#34;carboxy high polymer&#34;) is dissolved in water or a buffer solution such as a phosphate buffer, quinoline buffer, etc., followed by addition of a crosslinking agent in an amount of usually 1 to 10 equivalent weight, preferably 3 to 7 equivalent weight per equivalent weight of the carboxyl group in the carboxy high polymer. Then, an enzyme to be modified is added to the solution in an amount of preferably 0.1 to 10 parts by weight, more preferably 0.5 to 3 parts by weight per part by weight of the carboxy high polymer. The reaction is carried out at a pH of preferably 4.0 to 8.0, more preferably 5.0 to 7.5 using a pH-stat or the like and preferably 1° to 40° C., more preferably 4° to 30° C. for preferably 1 to 48 hours, more preferably 10 to 24 hours. After the reaction, the reaction solution is purified by dialysis, gel filtration, or the like to obtain the modified enzyme of the present invention. Needless to say, the purification to obtain the modified enzyme having desired molecular weight can be carried out by gel filtration chromatography, etc. 
     The modified enzyme of the present invention has almost the same fundamental properties at the time of enzymatic reaction such as Km values, optimum pH range, etc. as the original enzyme, and is remarkably improved in resistance to heat, proteases, denaturants, etc. and in storage stability in aqueous solutions. Therefore, when a diagnostic reagent is prepared using the modified enzyme of the present invention, the resulting diagnostic reagent is not only usable for a long period of time in the form of solution but also able to be reduced in initial changing amount of enzyme due to almost no change in enzymatic activity. 
     The modified enzyme of the present invention can be stored as a freeze dried product, which has good solubility when dissolved at the time of use. 
     The present invention is illustrated by way of the following Examples and Experiments, in which all percents are by weight unless otherwise specified. 
     In the following Examples and Experiments, the enzymatic activity was measured as follows: 
     Measuring Method of AOD Activity 
     (Substrate solution) 
     A substrate solution was prepared by dissolving 140 mM of ascorbic acid in 50 mM of phosphate buffer (pH 5.6). 
     (Measuring procedure) 
     After well mixing 3.0 ml of a substrate buffer solution previously preheated at 25° C. with 40 μl of a sample, a change of absorbance at 295 nm of the resulting solution was measured using a spectrophotometer controlled at 25° C. AOD activity was obtained by inserting the obtained changing rate of absorbance per minute (ΔE) into the following equation (1): 
     
         AOD activity (U/ml)=3.04÷2.67÷0.04×ΔE  (1) 
    
     Measuring Method of COD Activity 
     The method described in Method of Enzymatic Analysis, vol. 2, pages 172-173, 1983. 
     Measuring Method of GK Activity 
     The method described in Method of Enzymatic Analysis, vol. 2, pages 216-217, 1983. 
     Measuring Method of GOD Activity 
     The method described in Method of Enzymatic Analysis, vol. 2, pages 201-202, 1983. 
     Measuring Method of US Activity 
     The method described in Method of Enzymatic Analysis, vol. 3, pages 500, 1963. 
     Measuring Method of SAO Activity 
     The method described in Method of Enzymatic Analysis, vol. 17A, pages 976, 1970. 
     Measuring Method of XOD Activity 
     The method described in Method of Enzymatic Analysis, vol. 3, pages 212, 1983. 
     EXAMPLE 1 
     Preparation of Modified AOD 
     (1) In 20 ml of 0.2M pyrophosphate buffer solution (pH 9.0), 1 g of dextran (molecular weight 100,000 to 200,000) was dissolved. To the resulting solution, 6 g of pyromellitic anhydride was added gradually while maintaining the pH of the solution at 8 to 9 using a pH-stat. Then, the reaction was continued at room temperature with stirring for 2 hours. The resulting reaction solution was placed in a dialysis tube and dialyzed against water (3 liters×5 times) to obtain pyromellitic acid-modified dextran. The amount of carboxyl group in the pyromellitic acid-modified dextran was 3.0 per glucose residue constituting the pyromellitic acid-modified dextran. 
     (2) A solution obtained by dissolving 10 mg of AOD (mfd. by Eastman Kodak Co., drived from cucumber) in 1 ml of 5 mM of phosphate buffer solution (pH 6.0) and dialyzed against a phosphate buffer solution several times. The resulting solution was added to a solution obtained by adding 180 mg of WSC (1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide) to 6 ml of 20 mM phosphate buffer solution containing 10 mg of pyromellitic acid-modified dextran obtained in (1) mentioned above with stirring, followed by reaction at 5° C. for 24 hours. The resulting reaction solution was placed in a dialysis tube and dialyzed against 20 mM phosphate buffer solution (pH 7.0) to obtain the desired modified AOD (enzymatic activity recovery 65%). When the resulting modified AOD was analyzed by using high-pressure liquid chromatography (HPLC), the molecular weight was about 700,000. Further, unreacted AOD was hardly observed. 
     Experiment 1 
     Study of Thermal Stability 
     Thermal stability of the modified AOD obtained in Example 1 and the intact AOD was compared. 
     (Procedure) 
     In 20 mM phosphate buffer solution (pH 7.0), predetermined AOD was dissolved so as to make the amount 5 U/ml, and stored at predetermined temperatures for 10 minutes, followed by rapid cooling. A change of AOD activity before and after treatment was measured. 
     (Results) 
     The results are shown in Table 1. 
     
                       TABLE 1                                                     
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Treating         Residual activity (%)                                    
temperature      Modified Intact                                          
(°C.)     AOD      AOD                                             
______________________________________                                    
50               100      100                                             
55               100      100                                             
60               100      22.3                                            
65               100      3.2                                             
70               100      0                                               
75               93.4     0                                               
80               41.5     0                                               
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     The figures in Table 1 mean residual activity when AOD activity before treatment is taken as 100. 
     As is clear from the results of Table 1, the modified AOD of the present invention is remarkably improved in thermal stability compared with the intact AOD. 
     Experiment 2 
     Study of Storage Stability in Solution 
     Storage stability in a solution of the modified AOD obtained in Example 1 and the intact AOD was compared. 
     (Procedure) 
     In 20 mM phosphate buffer solution (pH 7.0), predetermined AOD was dissolved so as to make the amount 5 U/ml, and stored at predetermined temperatures for predetermined days. A change of AOD activity was measured. 
     (Results) 
     The results are shown in Table 2. 
     
                       TABLE 2                                                     
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Stored                                                                    
temper-                                                                   
       Residual activity (%)                                              
ature  4° C.     30° C.                                     
                                     45° C.                        
Stored Modified Intact  Modified                                          
                               Intact                                     
                                     Modified                             
                                            Intact                        
days   AOD      AOD     AOD    AOD   AOD    AOD                           
______________________________________                                    
2      --       --      --     --    100    69.8                          
5      --       --      --     --    100    15.4                          
10     --       --      94.0   89.2  89.1   7.9                           
15     99.8     91.8    --     --    50.2   0                             
20     --       --      81.2   69.5  40.5   0                             
30     --       --      77.0   59.7         --                            
50     99.9     81.8    70.0   25.4         --                            
80     --       --      34.3    2.9         --                            
90     92.3     75.1    --     --           --                            
110    90.1     72.7    --     --           --                            
141    90.5     73.3    --     --           --                            
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     The figures in Table 2 mean residual activity after predetermined days when AOD activity immediately after the preparation of AOD solution is taken as 100. 
     As is clear from the results of Table 2, the storage stability in the solution of the modified AOD of the present invention is remarkably improved compared with the intact AOD. 
     Experiment 3 
     Study of Resistance to Denaturant 
     Resistance to a denaturant (urea) of the modififed AOD obtained in Example 1 was compared with the intact AOD. 
     (Procedure) 
     In a urea solution with a predetermined concentration, predetermined AOD was dissolved so as to make the amount 5 U/ml, and stored at 30° C. for 16 hours. A change of AOD activity was measured. 
     (Results) 
     The results are shown in Table 3. In Table 3, the figures mean the residual activity after treatment with the denaturant, when the AOD activity immediately after the preparation of the AOD solution is taken as 100. 
     
                       TABLE 3                                                     
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Concentration  Residual activity (%)                                      
of urea (M)    Modified AOD                                               
                          Intact AOD                                      
______________________________________                                    
0              100        100                                             
2              100        88.4                                            
3              100        44.6                                            
4              100        5.5                                             
6              45.6       0                                               
8              3.0        0                                               
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     As is clear from the results of Table 3, the modified AOD of the present invention is remarkably improved in resistance to the denaturant (urea) compared with the intact AOD. 
     The Michaelis constant, pH stability, optimum pH and optimum temperature of the modified AOD and the intact AOD were obtained by a conventional method. The resulting Michaelis constants are shown in Table 4. The resulting pH stability is shown in FIG. 1. The resulting pH activity is shown in FIG. 2. The resulting temperature activity is shown in FIG. 3. In FIGS. 1, 2 and 3, the curve ---- means the results of the intact AOD and the curve --□-- means the results of the modified AOD. 
     
                       TABLE 4                                                     
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Substrate      Ascorbic acid                                              
                          Oxygen                                          
______________________________________                                    
Modified AOD   0.26 mM    0.098 mM                                        
Intact AOD     0.50 mM    0.068 mM                                        
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     As is clear from the results shown in Table 4, and FIGS. 1, 2 and 3, the modified AOD has almost the same or superior fundamental properties as enzyme compared with the intact AOD. 
     Experiment 4 
     Study of Resistance to Subtilisin 
     Resistance to subtilisin of the modified AOD obtained in Example 1 and the intact AOD was compared. 
     (Procedure) 
     To a solution obtained by dissolving 20 μg of predetermined AOD in 1 ml of 0.1M tris(hydroxymethyl)-aminomethane.hydrochloric acid buffer solution (pH 8.0), 10 μl of a solution containing 14.4 U/ml of subtilisin (mfd. by ICN Biochemicals Co.) was added and incubated at 37° C. for a predetermined time. 
     A change of residual activity at the time of incubation was measured. 
     (Results) 
     The results are shown in Table 5. In Table 5, the figures mean the residual activity after the treatment with subtilisin, when the AOD activity immediately after the preparation of AOD solution is taken as 100. 
     
                       TABLE 5                                                     
______________________________________                                    
Treating                                                                  
time         Residual activity (%)                                        
(min.)       Modified AOD                                                 
                        Intact AOD                                        
______________________________________                                    
0            100        100                                               
5            91.0       42.2                                              
25           87.6       33.5                                              
90           86.0       28.5                                              
150          82.3       23.1                                              
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     As is clear from the results of Table 5, the modified AOD of the present invention is remarkably improved in resistance to subtilisin compared with the intact AOD. 
     Resistance to proteases other than subtilisin such as trypsin, papain, chymotrypsin, and thermolysin was also examined. Both the modified AOD and the intact AOD were stable to these proteases. 
     EXAMPLE 2 
     Preparation of Modified GK 
     (1) In 4 ml of 0.2M pyrophosphate buffer solution (pH 10.0), 200 mg of dextran (molecular weight 69,000) was dissolved. While maintaining the pH of the resulting solution at 8 to 9 using a pH-stat, 440 mg of succinic anhydride was added to the solution gradually. Then, the reaction was continued at room temperature for 2 hours with stirring. The resulting reaction solution was place in a dialysis tube and dialyzed against pure water (1 liter×5 times) to obtain succinic acid-modified dextran. The amount of carboxyl group in the resulting succinic acid-modified dextran was 1.6 per glucose residue constituting the succinic acid-modified dextran. 
     (2) In 1 ml of 10 mM phosphate buffer solution (pH 5.5), 10 mg of GK (mfd. by Toyobo Co., Ltd., derived from Cellulomonas sp.) was dissolved. The resulting solution was dialyzed against the phosphate buffer solution several times. The resulting solution was added to a solution obtained by adding 10 mg of WSC to 6 ml of 10 mM phosphate buffer solution containing 10 mg of the succinic acid-modified dextran obtained in (1) mentioned above with stirring, followed by reaction at room temperature for 2 hours. The resulting reaction solution was packed in a dialysis tube and dialyzed against 50 mM Tris buffer solution (pH 8.0) to obtain the desired modified GK (enzyme activity recovery 27.2%). 
     Experiment 5 
     Study of Thermal Stability 
     Thermal stability of the modified GK obtained in Example 2 and the intact GK was compared. 
     (Procedure) 
     A solution obtained by dissolving a predetermined GK so as to make the amount 1 U/ml in 20 mM phosphate buffer solution (pH 7.0) and incubated at 55° C. or 60° C. to assay the GK activity after a predetermined time. Based on the assayed values, a regression line showing a relationship between a time elapsed and a logarithm of GK activity at each time was obtained, and the gradient of regression line was defined as a first-order rate constant of thermoinactivation. 
     (Results) 
     The results are shown in Table 6. 
     
                       TABLE 6                                                     
______________________________________                                    
Treating       First-order rate constant of                               
temperature    thermoinactivation (min.sup.-1)                            
(°C.)   Modified GK                                                
                          Intact GK                                       
______________________________________                                    
55             0.0084     0.078                                           
60             0.015      0.24                                            
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     As is clear from the results of Table 6, the modified GK of the present invention is remarkably improved in the thermal stability compared with the intact GK. 
     Further, the modified GK obtained in Example 2 and the intact GK were dissolved in 20 mM phosphate buffer solution (pH 7.0) so as to make the amount 1 U/ml and stored at 4° C. for predetermined days to measure the GK activity. 
     The results are shown in Table 7, wherein the figures mean the residual activity after predetermined days when the GK activity immediately after the preparation of GK solution is taken as 100. 
     
                       TABLE 7                                                     
______________________________________                                    
               Residual activity (%)                                      
Storing days   Modified GK                                                
                          Intact GK                                       
______________________________________                                    
20             95.8       78.0                                            
40             88.1       69.4                                            
60             81.3       62.6                                            
80             75.5       58.0                                            
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     As is clear from the results of Table 7, the modified GK of the present invention is remarkably improved in stability in the aqueous solution compared with the intact GK. 
     EXAMPLE 3 
     Preparation of Modified AOD 
     (1) To 11 ml of 0.2M N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonate (BES) buffer solution (pH 7.5) containing 260 mg of dialdehyde form dextran (molecular weight 100,000) obtained by oxidation with meta-periodic acid according to the method described in Proceedings National Academy Science USA, vol. 73, page 2128, 1976, 1 g of 6-aminocaproic acid was added, followed by stirring at room temperature for 24 hours. To the resulting reaction solution, 50 mg of sodium borohydride was added and reacted. Then, the resulting solution was placed in a dialysis tube and dialyzed against pure water (1 liter×5 times) to give 6-aminocaproic acid-modified dextran. The amount of carboxyl group in the resulting 6-aminocaproic acid-modified dextran was 0.3 per glucose residue constituting the 6-aminocaproic acid-modified dextran. 
     (2) To 1 ml of an aqueous solution containing 10 mg of the 6-aminocaproic acid-modified dextran obtained in (1) mentioned above, 40 mg of WSC and 10 mg of AOD (mfd. by Eastman Kodak Co., derived from pumpkin) were added, followed by reaction at 10° C. for 1 hour while maintaining the pH at 5.0 using a pH-stat. The resulting reaction solution was packed in a dialysis tube and dialyzed against a 10 mM phosphate buffer solution (pH 7.0) to give the desired modified AOD (enzyme activity recovery 49.1%). 
     Experiment 6 
     Study of Thermal Stability 
     The modified AOD obtained in Example 3 and the intact AOD were compared in thermal stability. 
     (Procedure) 
     A solution obtained by dissolving a predetermined AOD in an amount of 4 U/ml in 20 mM phosphate buffer solution (pH 7.0) was incubated at 45° C., 56° C. or 60° C. and subjected to measurement of AOD activity after a period of predetermined time. Based on the measured values, a regression line showing a relationship between a time elapsed and logarithms of AOD activity at each time was obtained, and the gradient of regression line was defined as a first-order rate constant of thermoinactivation. 
     (Results) 
     The results are shown in Table 8. 
     
                       TABLE 8                                                     
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Treating      First-order rate constant                                   
temperature   of thermoinactivation                                       
(°C.)  Modified AOD                                                
                         Intact AOD                                       
______________________________________                                    
45            1.9 × 10.sup.-4                                       
                         8.5 × 10.sup.-4                            
56            1.0 × 10.sup.-3                                       
                         1.3 × 10.sup.-2                            
60            2.0 × 10.sup.-3                                       
                         --                                               
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     As is clear from the results of Table 8, the modified AOD of the present invention is remarkably improved in the thermal stability compared with the intact AOD. 
     EXAMPLE 4 
     Preparation of Modified GOD 
     (1) To a solution obtained by dissolving 1 g of pullulan (molecular weight 200,000) in 70 ml of aqueous solution of 2M sodium carbonate, 1 ml of accetonitrile containing 1 g of bromine cyanide was added and reacted for 2 minutes. The reaction solution was dialyzed against an aqueous solution of 0.1M sodium bicarbonate (containing 0.5M sodium chloride). To the resulting activated pullulan solution, 6 g of 5-aminovaleric acid was added and reacted at 4° C. overnight, followed by dialysis against water to give 5-aminovaleric acid-modified pullulan. The amount of carboxyl group in the obtained 5-aminovaleric acid-modified pullulan was 0.2 per glucose residue constituting the 5-aminovaleric acid-modified pullulan. 
     (2) A solution obtained by dissolving 15 mg of GOD (mfd. by Toyobo Co., Ltd., derived from Aspergillus niger) in 10 mM phosphate buffer solution (pH 7.0) was added to a solution obtained by adding 10 mg of WSC to 6 ml of 20 mM phosphate buffer solution containing 10 mg of the 5-aminovaleric acid-modified pullulan obtained in (1) mentioned above, and reacted at 4° C. for 24 hours. The resulting reaction mixture was desalted with column chromatography  filler Sephadex G-25, a trade name, mfd. by Pharmacia; eluent 10 mM acetate buffer solution (pH 5.5)! to give the desired modified GOD (enzyme activity recovery 72.0%). 
     Experiment 7 
     Study of Thermal Stability 
     Thermal stability of the modified GOD obtained in Example 4 was compared with the intact GOD. 
     (Procedure) 
     A solution obtained by dissolving 1.5 U/ml of a predetermined GOD in 20 mM phosphate buffer solution (pH 7.0) was incubated at 60° C. and subjected to measurement of GOD activity after a predetermined time. Based on measured values, a regression line showing a relationship between a time elapsed and logarithms of GOD activity at each time was obtained. The gradient of the regression line was defined as a first-order rate constant of thermoinactivation. 
     (Results) 
     The results are shown in Table 9. 
     
                       TABLE 9                                                     
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Treating      First-order rate constant                                   
temperature   of thermoinactivation                                       
(°C.)  Modified GOD                                                
                         Intact GOD                                       
______________________________________                                    
60            0.012      0.25                                             
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     As is clear from the results of Table 9, the modified GOD of the present invention is remarkably improved in thermal stability compared with the intact GOD. 
     EXAMPLE 5 
     Preparation of Modified COD 
     The process of Example 1(1) was repeated except for using 1 g of Ficoll (molecular weight 400,000) in place of using the dextran to give a pyromellitic acid-modified Ficoll. To 2.7 ml of water containing 5 mg of the pyromellitic acid-modified Ficoll, 65 mg of WSC was added. To the resulting solution maintained at pH 7.0 using a pH-stat, 0.5 ml of 10 mM phosphate buffer solution (pH 7.0) containing 5 mg of COD (mfd. by Toyo Jozo Co., Ltd., derived from Arthrobactor sp.) was added and reacted at 4° C. for 17 hours. The resulting reaction solution was dialyzed against 10 mM Tris(hydroxymethyl)-aminomethane.hydrochlorate buffer solution (pH 8.0) to give the desired modified COD (enzyme activity recovery 51.7%). 
     Experiment 8 
     Study of Thermal Stability 
     Thermal stability of the modified COD obtained in Example 5 and the intact COD was compared. 
     (Procedure) 
     A solution obtained by dissolving 1 U/ml of a predetermined COD in 20 mM phosphate buffer solution (pH 7.0) was incubated at a predetermined temperature and subjected to measurement of COD activity after a predetermined time. Based on the measured values, a regression line showing a relationship between a time elapsed and logarithms of COD activity at each time was obtained. The gradient of the regression line was defined as a first-order rate constant of thermoinactivation. 
     (Results) 
     The results are shown in Table 10. 
     As is clear from the results of Table 10, the modified COD of the present invention is remarkably improved in thermal stability compared with the intact COD. 
     
                       TABLE 10                                                    
______________________________________                                    
Treating      First-order rate constant                                   
temperature   of thermoinactivation                                       
(°C.)  Modifed COD                                                 
                         Intact COD                                       
______________________________________                                    
40            8.1 × 10.sup.-4                                       
                         1.3 × 10.sup.-2                            
45            2.8 × 10.sup.-3                                       
                         1.5 × 10.sup.-1                            
50            4.4 × 10.sup.-3                                       
                         --                                               
54            2.5 × 10.sup.-2                                       
                         --                                               
60            1.4 × 10.sup.-1                                       
                         --                                               
______________________________________                                    
 
    
     Experiment 9 
     Study of Storage Stability in Solution 
     Storage stability in a solution of the modified COD obtained in Example 5 and the intact COD was compared. 
     (Procedure) 
     A solution obtained by dissolving 1.5 U/ml of a predetermined COD in 50 mM 3-(N-morpholino)propane-sulfonate (MOPS) buffer solution (pH 7.7) was stored at a predetermined temperature for a predetermined time to measure the change of COD activity. 
     (Results) 
     The results are shown in Table 11, wherein the figures show COD residual activity after predetermined time, when the COD activity immediately after the preparation of the COD solution is taken as 100. 
     
                       TABLE 11                                                    
______________________________________                                    
              Residual activity (%)                                       
Stored days   Modified COD                                                
                         Intact COD                                       
______________________________________                                    
0             100        100                                              
5             100        43.4                                             
10            86.0       15.0                                             
20            74.1       6.2                                              
35            72.5       3.1                                              
______________________________________                                    
 
    
     As is clear from the results of Table 11, the modified COD of the present invention is remarkably improved in storage stability in the solution compared with the intact COD. 
     Comparative Example 1 
     (Preparation of Crosslinked AOD) 
     To 1 ml of 10 mM phosphate buffer solution (pH 7.0) dissolving 4.1 mg of AOD (mfd. by Eastman Kodak Co., derived from pumpkin), 50 μl of a solution of 2.5% glutaraldehyde was added and reacted at room temperature for 3 hours stirring. After the reaction, the reaction solution was dialyzed against 20 mM phosphate buffer solution (pH 7.0) to give crosslinked AOD (enzyme activity recovery 73.4%). 
     (Measurement of First-order Rate Constant of Thermoinactivation)) 
     Thermal stability of the crosslinked AOD and the intact AOD compared by means of the first-order rate constant of thermoinactivation. The measuring a procedure is as follows. 
     A solution obtained by dissolving 4 U/ml of a predetermined AOD in 20 mM phosphate buffer solution (pH 7.0) was incubated at a predetermined temperature, followed by measurement of AOD activity after predetermined days. Based on the measured values, a regression line showing a relationship between a time elapsed and logarithms of AOD activity at each time was obtained. The gradient of the regression line was defined as a first-order rate constant of thermoinactivation. 
     The results are shown in Table 12. 
     
                       TABLE 12                                                    
______________________________________                                    
               First-order rate constant                                  
Treating       of thermoinactivation                                      
temperature    Crosslinked                                                
(°C.)   AOD       Intact AOD                                       
______________________________________                                    
4              1.4 × 10.sup.-6                                      
                         1.4 × 10.sup.-6                            
30             1.4 × 10.sup.-5                                      
                         1.4 × 10.sup.-5                            
56             1.3 × 10.sup.-3                                      
                         2.1 × 10.sup.-2                            
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     As is clear from Table 12, the crosslinked AOD is remarkably improved in the storage stability in the solution at a high temperature (56° C.) compared with the intact AOD, but it is almost the same as the intact AOD in the storage stability when stored at 4° C. and 30° C. 
     Comparative Example 2 
     Preparation of Dextran-modified Glucoamylase 
     (1) A solution in an amount of 2 ml of 0.1M acetate buffer solution (pH 5.0) containing 26 mg of dialdehyde form dextran (molecular weight 100,000) obtained by oxidation with metaperiodic acid according the method described in Proceedings National Academy Science USA, vol. 73, page 2128, 1976, and 1 ml of 50 mM acetate buffer solution (pH 5.5) containing 26 mg of glucoamylase (derived from Saccharomyces, hereinafter referred to as &#34;GA&#34;) were mixed. To the resulting mixture, 100 μl of borane-pyridine complex was added and reacted at 37° C. for 20 hours. After the reaction, the reaction solution was dialyzed against 10 mM acetate buffer solution (pH 5.5) to give the desired modified dextran-modified GA (enzyme activity recovery 80.6%). 
     (2) The process of Example 1(2) was repeated except for using 10 mg of GA in place of AOD to give the desired dextran-modified GA (enzyme activity recovery 47.4%). 
     The stability in a solution of the dextran-modified GA obtained (1) and (2) mentioned above and the intact GA was compared. 
     (Procedure) 
     A solution obtained by dissolving 2 U/ml of a predetermined GA in 50 mM acetate buffer solution (pH 4.5) was treated at a predetermined temperature for 10 minutes to obtain the residual activity of GA. 
     (Results) 
     The results are shown in Table 13. 
     
                       TABLE 13                                                    
______________________________________                                    
             Treating temperature (°C.)                            
Enzyme       50     55         60   65                                    
______________________________________                                    
Intact GA    100    66.1       20.0 0.1                                   
Comparative  87.3   85.9       56.3 45.9                                  
Example 2(1)                                                              
Comparative  47.4   3.7        0    0                                     
Example 2(2)                                                              
______________________________________                                    
 
    
     As is clear from the results of Table 13, the stability of the dextran-modified GA obtained in the process of (2) mentioned above is worse than that of the dextran-modified GA obtained in the process (1) mentioned above. 
     This means that when the kind of enzyme to be treated is changed from the specific enzymes usable in the present invention, no effect is obtained (rather, a reverse effect is obtained). 
     Comparative Example 3 
     Preparation of Modified AOD by Borane-pyridine Complex Method 
     A 0.1M phosphate buffer solution (pH 7.5) in an amount of 0.8 ml containing 10 mg of dialdehyde form dextran (molecular weight 100,000) obtained by oxidation with metaperiodic acid according to the method described in Proceedings National Academy Science USA, vol. 73, page 2128, 1976, was mixed with 1 ml of 10 mM phosphate buffer solution (pH 7.5) containing 10 mg of AOD (mfd. by Eastman Kodak Co.). To the resulting mixture, 20 μl of borane-pyridine complex was added and reacted at room temperature for 24 hours. After the reaction, the reaction solution was dialyzed against 20 mM phosphate buffer solution (pH 7.0) to give the desired dextran-modified AOD (enzyme activity recovery 5.7%). 
     As is clear from the results mentioned above, the method of binding dextran to AOD using the borane-pyridine complex is so low in the enzyme activity recovery that it is not practical. 
     EXAMPLE 6 
     Preparation of Modified COD 
     To a solution obtained by adding 50 mg of WSC to 2 ml of 20 mM phosphate buffer solution (pH 5.5) containing 10 mg of sodium polyaspartic acid (molecular weight 16,400), 1 ml of 5 mM phosphate buffer solution (pH 7.0) containing 10 mg of COD (mfd. by Toyo Jozo Co., Ltd.) was added, followed by reaction at 4° C. for 24 hours. After the reaction, the reaction solution was dialyzed against 50 mM MOPS buffer solution (pH 7.7) to give the desired modified COD (enzyme activity recovery 63.8%). 
     The modified COD thus obtained and the intact COD were subjected to measurement of first-order rate constant of thermoinactivation at 56° C. in 50 mM MOPS buffer solution (pH 7.7). The results are shown in Table 14. 
     
                       TABLE 14                                                    
______________________________________                                    
Treating     First-order rate constant                                    
temperature  of thermoinactivation                                        
(°C.) Modified COD                                                 
                        Intact COD                                        
______________________________________                                    
56           0.046      Deactivated                                       
                        completely                                        
                        within 1 minute.                                  
______________________________________                                    
 
    
     As is clear from the results of Table 14, the modified COD of the present invention is remarkably improved in thermal stability compared with the intact COD. 
     EXAMPLE 7 
     Preparation of Modified POD 
     To 2 ml of an aqueous solution containing 5 mg of pyromellitic acid-modified dextran obtained in Example 1(1), 65 mg of WSC was added. To the resulting solution maintained at pH 6.0 using a pH-stat, 0.5 ml of 10 mM phosphate buffer solution (pH 7.0) containing 5 mg of POD (mfd. by Sigma Chemical Co.) was added and reacted at 5° C. for 24 hours. The resulting reaction solution was packed in a dialysis tube and dialyzed against 20 mM phosphate buffer solution (pH 7.0) to give the desired modified POD (enzyme activity recovery 80.6%). 
     The thus obtained modified POD and the intact POD were dissolved in 20 mM phosphate buffer solution (pH 7.0) in predetermined concentrations, respectively, and stored at 45° C. to measure the change of residual activity. The results are shown in Table 15. 
     As is clear from the results of Table 15, the modified POD of the present invention is remarkably improved in thermal stability compared with the intact POD. 
     
                       TABLE 15                                                    
______________________________________                                    
              Residual activity (%)                                       
Stored days   Modified POD                                                
                         Intact POD                                       
______________________________________                                    
18            49         31                                               
55            37         24                                               
78            24         10                                               
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     EXAMPLE 8 
     Preparation of Modified US 
     To 2 ml of aqueous solution containing 5 mg of pyromellitic acid-modified dextran obtained in Example 1(1), 65 mg of WSC was added. To the resulting solution maintained at pH 6.0 using a pH-stat, 0.5 ml of 10 mM phosphate buffer solution (pH 7.0) containing 5 mg of US (mfd. by Toyobo Co., Ltd., derived from Candida sp.) was added and reacted at 5° C. for 24 hours. The resulting reaction solution was packed in a dialysis tube and dialyzed against 20 mM phosphate buffer solution (pH 7.0) to give the desired modified US (enzyme activity recovery 27.6%). 
     The thus obtained modified US and the intact US were dissolved in 20 mM phosphate buffer solution (pH 7.0) in an amount of 1 U/ml, respectively, and stored at a predetermined temperature for 30 min. to measure the change of residual activity. The results are shown in Table 16. 
     As is clear from the results of Table 16, the modified US of the present invention is remarkably improved in thermal stability compared with the intact US. 
     
                       TABLE 16                                                    
______________________________________                                    
Predetermined                                                             
temperature     Residual activity (%)                                     
(°C.)    Modified US                                               
                          Intact US                                       
______________________________________                                    
65              98        65                                              
75              65        15                                              
80              30        0                                               
______________________________________                                    
 
    
     EXAMPLE 9 
     Preparation of Modified SAO 
     To 2 ml of aqueous solution containing 5 mg of pyromellitic acid-modified dextran obtained in Example 1(1), 65 mg of WSC was added. To the resulting solution maintained at pH 6.0 using a pH-stat, 0.5 ml of 10 mM phosphate buffer solution (pH 7.0) containing 10 mg of SAO (mfd. by Toyobo Co., Ltd., derived from Arthrobactor sp.) was added and reacted at 5° C. for 24 hours. The resulting reaction solution was packed in a dialysis tube and dialyzed against 20 mM phosphate buffer solution (pH 7.0) to give the desired modified SAO (enzyme activity recovery 19.5%). 
     The thus obtained modified SAO and the intact SAO were dissolved in 20 mM phosphate buffer solution (pH 7.0) in an amount of 20 U/ml, respectively, and stored at a predetermined temperature for 30 min. to measure the change of residual activity. The results are shown in Table 17. 
     As is clear from the results of Table 17, the modified SAO of the present invention is remarkably improved in thermal stability compared with the intact SAO. 
     
                       TABLE 17                                                    
______________________________________                                    
Predetermined                                                             
temperature    Residual activity (%)                                      
(°C.)   Modified SAO                                               
                          Intact SAO                                      
______________________________________                                    
40             100        78                                              
45             100        35                                              
50             99         0                                               
______________________________________                                    
 
    
     EXAMPLE 10 
     Preparation of Modified XOD 
     To 2 ml of aqueous solution containing 5 mg of pyromellitic acid-modified dextran obtained in Example 1(1), 65 mg of WSC was added. To the resulting solution maintained at pH 6.0 using a pH-stat, 0.5 ml of 10 mM phosphate buffer solution (pH 7.0) containing 10 mg of XOD (mfd. by Boehringer Mannheim GmbH., derived from cow milk) was added and reacted at 50° C. for 24 hours. The resulting reaction solution was packed in a dialysis tube and dialyzed against 20 mM phosphate buffer solution (pH 7.0) to give the desired modified XOD (enzyme activity recovery 24.0%). 
     The thus obtained modified XOD and the intact XOD were dissolved in 20 mM phosphate buffer solution (pH 7.0) in an amount of 0.2 U/ml, respectively, and stored at a predetermined temperature for 75 min. to measure the change of residual activity. The results are shown in Table 18. 
     As is clear from the results of Table 18, the modified XOD of the present invention is remarkably improved in thermal stability compared with the intact XOD. 
     
                       TABLE 18                                                    
______________________________________                                    
Predetermined                                                             
temperature    Residual activity (%)                                      
(°C.)   Modified XOD                                               
                          Intact XOD                                      
______________________________________                                    
56             75.6       40.5                                            
58             60.0       31.2                                            
60             53.5       23.4                                            
______________________________________                                    
 
    
     EXAMPLE 11 
     Preparation of Modified AOD 
     (1) In 5 ml of 0.27 pyrophosphate buffer solution (pH 9.0), 0.1 g of α-, β- or γ-cyclodextrin (mfd. by Wako Pure Chemical Industries, Ltd.) was dissolved. To the resulting solution, 0.5 g of pyromellitic anhydride was added gradually while maintaining the pH of the solution at 8 to 9 using a pH-stat. Then, the reaction was continued at room temperature with stirring for 2 hours. The resulting solution was desalted by column chromatography  filler: Sephadex G-25, mfd. by Pharmacia; eluent: water! to give pyromellitic acid-modified α-, β- and γ-cyclodextrin. 
     (2) A solution obtained by dissolving 10 mg of AOD (mfd. by Boehringer Mannheim GmbH, derived from pumpkin) in 1 ml of 5 mM phosphate buffer solution (pH 6.0) was dialyzed against the phosphate buffer solution several times. The resulting solution was added to a solution obtained by adding 100 mg of WSC to 5 ml of 20 mM phosphate buffer solution containing 10 mg of pyromellitic acid-modified α-, β- or γ-cyclodextrin  hereinafter referred to as &#34;PM-α-CD, PM-β-CD and PM-γ-CD, respectively! obtained in (1) mentioned above with stirring, followed by reaction at 5° C. for 24 hours. The resulting reaction solution was placed in a dialysis tube and dialyzed against 20 mM phosphate buffer solution (pH 7.0) to obtained the desired modified AOD. The enzyme activity recovery was as follows: 
     
         ______________________________________                                    
PM-α-CD-modified AOD                                                
                   32.5%                                                  
PM-β-CD-modified AOD                                                 
                   31.6%                                                  
PM-γ-CD-modified AOD                                                
                   31.2%                                                  
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     When the resulting modified AOD was analyzed by using high-pressure liquid chromatography (HPLC), individual molecular weights were about 400,000. Further, unreacted AOD was hardly observed. 
     Experiment 10 
     Study of Thermal Stability 
     Thermal stability of the modified AOD obtained in Example 11 and the intact AOD was compared. 
     The procedure was the same as Experiment 1 except for making the treating time 35 minutes. 
     The results are shown in Table 19. 
     
                       TABLE 19                                                    
______________________________________                                    
           Residual activity (%)                                          
Treating          PM-α-CD-                                          
                              PM-β-CD-                               
                                     PM-γ-CD-                       
temperature                                                               
           Intact modified    modified                                    
                                     modified                             
(°C.)                                                              
           AOD    AOD         AOD    AOD                                  
______________________________________                                    
56         0      72.7        70.4   69.5                                 
61         0      86.3        78.5   76.2                                 
______________________________________                                    
 
    
     As is clear from the results of Table 19, the modified AODs of the present invention are remarkably improved in thermal stability compared with the intact AOD. 
     Experiment 11 
     Study of Storage Stability in Solution 
     Storage stability in a solution of the modified AODs obtained in Example 11 and the intact AOD was compared. 
     (Procedure) 
     In 20 mM phosphate buffer solution (pH 7.0), predetermined AOD was dissolved so as to make the amount 5 U/ml, and stored at 30° C. for predetermined days. A change of AOD activity was measured. 
     (Results) 
     The results are shown in Table 20, wherein the figure mean residual activity after predetermined days when AOD activity immediately after the preparation of AOD solution is taken as 100. 
     
                       TABLE 20                                                    
______________________________________                                    
         Residual activity (%)                                            
                PM-α-CD-                                            
                            PM-β-CD-                                 
                                   PM-γ-CD-                         
Stored   Intact modified    modified                                      
                                   modified                               
days     AOD    AOD         AOD    AOD                                    
______________________________________                                    
7        89.3   98.7        101.0  100                                    
14       74.4   96.0        93.0   93.8                                   
21       66.6   84.2        85.9   83.5                                   
32       55.3   81.3        80.7   79.9                                   
______________________________________                                    
 
    
     As is clear from the results of Table 20, the storage stability in the solution of the modified AODs of the present invention is remarkably improved compared with the intact AOD. 
     EXAMPLE 12 
     In 20 mM phosphate buffer solution (pH 6.0), each 10 mg of sodium heparin (mfd. by Wako Pure Chemical Industries, Ltd.), pectic acid (mfd. by Wako Pure Chemical Industries, Ltd.) or ethylene-maleic acid copolymer (hereinafter referred to as &#34;EMAC&#34;, mfd. by Aldrich Chemical Co.) was dissolved. To this, 100 mg of WSC was added, followed by addition of a solution obtained by dissolving 10 mg of AOD (mfd. by Boehringer Mannheim GmbH, derived from pumpkin) in 1 ml of 5 mM phosphate buffer (pH 6.0) and reaction at room temperature for 6 hours. The resulting reaction solution was packed in a dialysis tube and dialyzed against a 20 mM phosphate buffer solution (pH 7.0) to give the desired modified AOD. 
     The activity recoveries of AODs are shown in Table 21. When the resulting modified AODs were analyzed by using high-pressure liquid chromatography (HPLC), the molecular weights are about 400,000 to 600,000, respectively. Further, unreacted AOD was hardly observed. 
     
                       TABLE 21                                                    
______________________________________                                    
                 Activity recovery (%)                                    
______________________________________                                    
Heparin-modified AOD                                                      
                 35.9                                                     
Pectic acid-modified AOD                                                  
                 42.6                                                     
EMAC-modified AOD                                                         
                 33.8                                                     
______________________________________                                    
 
    
     Experiment 12 
     Study of Thermal Stability 
     Thermal stability of the modified AODs obtained in Example 12 and the intact AOD was compared. 
     The procedure was the same as Experiment 1 except for making the treating time 35 minutes. 
     The results are shown in Table 22. 
     
                       TABLE 22                                                    
______________________________________                                    
           Residual activity (%)                                          
                             Pectic                                       
Treating          Heparin-   acid-  EMAC-                                 
temperature                                                               
           Intact modified   modified                                     
                                    modified                              
(°C.)                                                              
           AOD    AOD        AOD    MOD                                   
______________________________________                                    
56         0      65.6       89.9   75.8                                  
61         0      19.7       13.7   63.6                                  
______________________________________                                    
 
    
     As is clear from the results of Table 22, the modified AODs of the present invention are remarkably improved in thermal stability compared with the intact AOD. 
     Experiment 13 
     Study of Storage Stability in Solution 
     Storage stability in a solution of the modified AODs obtained in Example 12 and the intact AOD was compared. 
     (Procedure) 
     In 20 mM phosphate buffer solution (pH 7.0), predetermined AOD was dissolved so as to make the amount 5 U/ml, and stored at 30° C. for predetermined days. A change of AOD activity was measured. 
     (Results) 
     The results are shown in Table 23, wherein the figures mean residual activity after predetermined days when AOD activity immediately after the preparation of AOD solution is taken as 100. 
     
                       TABLE 23                                                    
______________________________________                                    
         Residual Activity (%)                                            
                            Pectic                                        
                Heparin-    acid-  EMAC                                   
Stored   Intact modified    modified                                      
                                   modified                               
days     AOD    AOD         AOD    AOD                                    
______________________________________                                    
7        89.3   95.9        97.6   100                                    
14       74.4   89.2        90.6   100                                    
21       66.6   76.2        80.0   93.4                                   
32       55.3   69.7        70.5   85.8                                   
______________________________________                                    
 
    
     As is clear from the results of Table 23, the storage stability in the solution of the modified AODs of the present invention is remarkably improved compared with the intact AOD. 
     As mentioned above, the modified enzyme of the present invention has almost the same fundamental properties for enzymatic reaction such as Km values, optimum pH range, etc. as the starting enzyme, and is remarkably improved in resistance to heat, proteases, denaturants and in storage stability in an aqueous solution compared with the intact enzyme. It is very advantageous in this art that hydrogen peroxide generating enzymes, AOD, POD, URS, CAT and GK can be modified to provide the above-mentioned properties.