The development of artificial enzymes with similar functions to enzymes has become one of the hot topics in the field of chemistry. Although there are several kinds of molecules which can be used as apoproteinase substitutes, cyclodextrin systems have been widely used and are superior. The cyclodextrin-constructed enzyme model of oxidoreductase is not as much as hydrolase, and their roles are different. Some simulate NADH to reduce ninhydrin [1], some can reversibly bind molecular oxygen, and have a longer half-life [ 2,3], some as a model of iron redox protease [4,5], but as an enzyme model simulating malate dehydrogenase has not been reported. In this paper, the product of b-CD modified by m-carboxybenzenesulfonyl chloride was reacted with ferric chloride to prepare bis(6-oxo-m-carboxyphenylsulfonyl)-b-CD.Fe3+ complex to simulate L-malic acid. Dehydrogenase catalyzes the oxidation of L-malic acid to oxaloacetate. A one-step redox reaction of malic acid in the tricarboxylic acid cycle in vivo was carried out, and the amount of reduced coenzyme I (NADH) and L-malic acid produced in the reaction was obtained by using oxidized coenzyme I (NAD+) as a hydrogen acceptor. There is a stoichiometric relationship between the amounts, and the amount of malic acid consumed or the amount of oxaloacetate produced can be determined by measuring the change in absorbance of NADH at 340 nm.
Under standard thermodynamic conditions, the reaction equilibrium is favorable for the reverse reaction. Under physiological conditions, the reaction product oxaloacetate is continuously synthesized and removed, so that the concentration in the cell is lowered, and the reaction proceeds to a positive reaction [6]. The author added a trapping agent (2,4-dinitrophenylhydrazine) to the catalytic reaction of the simulated enzyme, and converted the oxaloacetate to oxaloacetate in a glycine-NaOH buffer solution at pH 9.00, which promoted the reaction to positive reaction. Carry out [7].
Its reaction formula is as follows:
1 experiment
1.1 Instruments and reagents Shimadzu AA-646 atomic absorption spectrometer, AIC UV-9100 UV-visible spectrophotometer (Shenzhen Aike Analytical Instrument Co., Ltd.), pHS-3C digital acidity meter (Hangzhou Wanda Instrument and Meter Factory), β- CD is the industrial refinery of Suzhou MSG Factory. It has been recrystallized twice, L-malic acid, L-malic acid dehydrogenase, SIGMA CHEMICAL CO., NAD, biochemical reagent (Shanghai Boao Biotechnology Co., Ltd.), 2, 4-Dinitrophenylhydrazine, analytically pure (Shanghai Chemical Reagent Factory). The remaining reagents were of analytical grade.
1.2 Synthesis of simulated enzymes 0.11mol.L-1b-CD-(M)2 (synthesis and structure identification results: nuclear magnetic resonance spectroscopy and elemental analysis data see literature [8]) aqueous solution 30mL, 0.48 mol.L-1 FeCl3 aqueous solution 40 mL, the FeCl3 solution was all added to the b-CD-(M)2 solution under stirring. At this time, the solution gradually changed from colorless to yellow, and an orange-red transparent solution was obtained after the Zui. The solution was placed in a constant temperature water bath, cooled at 70 ° C for 8 h, and filtered. An appropriate amount of acetone was added to the filtrate to precipitate a pale yellow flocculent precipitate. The mixture was allowed to stand for filtration, washed with 95% ethanol until the filtrate was free of Cl-, dried, and placed in a desiccator to obtain a dry pale yellow powdery solid: b-CD-(M)2.Fe3+ weighs 4.6 g. The rate is 88%. The spot of Rf=0.66 (developing agent: ethyl acetate: methanol: glacial acetic acid: water = 12:3:3:2), infrared gmax: 3300 cm-1 (s, OH), 2900cm-1 (w, CH2), 1720cm-1 (w, C=O), 1625, 750cm-1 (mw, benzene ring), 1143cm-1 (w, ArSO2), the amount of Fe3+ measured by atomic absorption spectrophotometry It is proved that the molar ratio of b-CD-(M)2 to Fe3+ in the complex is 1:1, and the M?ssbauer spectrum proves to be the Fe3+ state.
1.3 Simulated enzyme catalytic reaction
1.3.1 Catalytic reaction In the cuvette, add 3 mL of Glycine-NaOH buffer (containing 2,4-dinitrophenylhydrazine) at pH 9.00, NAD + 50 mL of 1 mol.L-1, 4.44 mmol.L. -1 simulating enzyme 30mL, 1mol.L-1 malic acid 30mL, mixing, reaction temperature 25 ° C, 30mL buffer instead of substrate L-malic acid solution for the corresponding reference, measured at NADHzui large absorption 340nm A340 value. Comparative experiment: FeCl3 was used instead of the mimetic enzyme, and other conditions were the same as described above.
1.3.2 Effect of Substrate Concentration on the Rate of Simulated Enzyme Catalytic Reaction—Use the double reciprocal plot method to calculate the Michaelis constant of the simulated enzyme. In the cuvette, add 2.7 mL of glycine-NaOH buffer at pH 9.00, 1 mol. L-1 NAD + 200 mL, 0.523 mol.L-1 of 2,4-dinitrophenylhydrazine 30 mL, 4.44 mmol.L-1 mimetic enzyme 30 mL and different amounts of substrate (malic acid) as a measuring cell, Under the reaction temperature of 25 ° C, the same reference solution was used as the corresponding reference (both to 3.010 mL with buffer), the A340 value was measured, and the catalytic rate was determined, namely: V=DC/Dt= DA/ε.Dt, where: e is the molar absorptivity of NADH, 6.3×10-6 L/mmol.cm-1 at 340 nm [9], substrate concentration [S] is known, by 1/V The Michaelis constant Km of the mimetic enzyme was obtained by plotting 1/[S].
1.4 Conditional experiment
1.4.1 Effect of pH on the speed of simulated enzyme reaction In the cuvette, add 2.7 mL of glycine-NaOH buffer with a pH of 8.79, 9.00, 9.50, 10.00 and 10.70, and NAD + 50 mL of 1 mol.L-1. 0.523 mol.L-1 of 2,4-dinitrophenylhydrazine 30 μL, 4.44 mmol.L-1 of the mimetic enzyme 30mL, 1mol.L-1 L-malic acid 30mL, after mixing, corresponding blank (ie corresponding The pH buffer is used as a reference to measure the change in A340.
1.4.2 Effect of simulated enzyme concentration on simulated enzyme reaction rate Add 2.7 mL of pH 9.00 glycine-NaOH buffer, 1 mol.L-1 of NAD + 50 mL, 0.523 mol.L-1 of 2, 4 to the cuvette. - Dinitrophenylhydrazine 30mL, 1 mol.L-1 L-malic acid 20mL, 4.44mmol.L-1 mimic enzymes were taken 10, 15, 30, 40, 50, 60, 70mL, respectively, buffered to volume The total volume was 2.90 mL. After mixing, the corresponding blank (ie, buffer with pH=9.00 instead of substrate) was used as a reference to measure the change of A340.
1.4.3 Effect of 2,4-dinitrophenylhydrazine concentration on the catalytic speed of the simulated enzyme. Add 2.7 mL of pH=9.00 glycine-NaOH buffer to the cuvette, 1 mol.L-1 of NAD+ 50 mL, 4.44 mmol. L-1 mimetic enzyme 30mL, 1mol.L-1 L-malic acid 30mL, 0.523mol.L-1 2,4-dinitrophenylhydrazine respectively take 10,15,20,25,30,40,50 60,70mL, the total volume of the volume was 2.90mL with buffer, and after mixing, the corresponding blank (the buffer with pH=9.00 instead of the substrate) was used as a reference to measure the change of A340.
1.5 Control experiment with 1/100 concentration of SIGMA company after dialysis, L-malate dehydrogenase enzyme solution 30mL instead of mimic enzyme, at 25 ° C, and ensure other experimental conditions are the same, the determination of biological enzymes in this system The value of the Michaelis constant Km.
2 Results and discussion
2.1 Catalytic rate
Figure 1 Kinetic curve of simulated enzyme catalyzed malic acid
a mimic enzyme (â–²) b FeCl3 (â– )
The results in Figure 1 show that the catalytic rate of the simulated enzyme is much faster than FeCl3, while the curve a shows the catalytic kinetics of the catalytic enzyme to oxidize L-malic acid to oxaloacetate, and the catalytic reaction is very fast before 6 min. It is basically linear, and the reaction rate increases slowly after 6 minutes, and tends to remain unchanged after zui, indicating that the reaction has proceeded completely. Therefore, when calculating the catalytic rate, Dt is selected within 5 min. In order to facilitate the comparison with the biological enzyme in the determination of the Michaelis constant, we determined at the determination of the biological enzyme, the acidity of the acidity, pH=9.00, and the catalytic rate was faster if it was determined at the pH of the simulated enzyme, pH=9.50.
2.2 Reaction conditions In the conditional experiment, it can be seen from Fig. 2 that the pH value of the catalytic reaction of the system has a suitable acidity, that is, pH 9.5, which is greater or less than this value, and the catalytic ability of the system is decreased to some extent, while the biological enzyme zui The pH value is 9.00, which also indicates that the mimetic enzyme is closer to the biological enzyme in this respect. The curve a of Fig. 3 shows that when the concentration of the substrate L-malic acid is constant, the concentration of the mimetic enzyme is proportional to the reaction rate; the curve b indicates that the amount of 2,4-dinitrophenylhydrazine added in the reaction system has one Zui good value (4.46mmol.L-1).
Fig. 2 Effect of acidity on reaction rateFig.3 Effect of simulated enzyme and hydrazine concentration on reaction rate
a mimic enzyme (C × 10-5mol.L-1) (▲)
b 肼(C×10-3mol.L-1)(■)
2.3 The Km value of the simulated enzyme and the biological enzyme under the same catalytic conditions is based on the double reciprocal mapping method [10], and the Mie equation can be written as the following form: V-1=Km.Vmax-1.[S]-1 + Vmax -1.
Through experiments, the author selects different [S] to determine the corresponding V, and finds the reciprocal of the two, plots 1/V versus 1/[S], ​​and gives a straight line (see Figure 4). The linear equations are :Va-1=0.0307[S]-1+3.5142 (mimetic enzyme) and Vb-1=0.0325[S]-1+4.2738 (biological enzyme), therefore, the Michaelis constant (km) of the biological enzyme can be obtained Enzyme = 0.0325 / 4.2738 = 7.60 mmol. L-1, Michaelis constant (km) of the mimetic enzyme mimetic enzyme = 0.0307 / 3.5142 = 8.74 mmol. L-1. The smaller the value of km, the greater the affinity of the enzyme for the substrate [11], and it is clear that the high velocity Vmax of the enzymatic reaction can be easily achieved without the need for a high substrate concentration. Based on the obtained Michaelis constant, it was found that (Km) mimetic enzyme: (Km) bio-enzyme = 1.15:1, which indicates that the mimetic enzyme has a slightly weaker affinity to the substrate than the biological enzyme, but is very close.
Figure 4 Lineweaxer-Burk double reciprocal mapping method
a mimic enzyme (â–²)
b biological enzyme (malate dehydrogenase) (â– )
The size of the enzyme activity can be expressed by the reaction rate of a certain chemical reaction catalyzed by a certain condition, that is, the faster the reaction rate catalyzed by the enzyme, the higher the activity of the enzyme. It can be seen from Fig. 2 that the enzymatic reaction rate zui can be as high as 2.34 mol/L.min at 1.32×10-4 mmol simulated enzyme at 25 ° C and pH=9.00, while using 1/100 pure bio-zymogen. Under the same conditions, 30 mL of the enzyme solution had a large reaction rate of 0.287 mol/L·min for the enzymatic reaction in this system. Obviously, the reaction was catalyzed by 1.332×10-4 mmol of mimic enzyme, and the reaction rate of Zui was 1/. The 30 μmL of the enzyme zymogen solution 30 catalyzed this reaction was 8 times higher.
2.4 Summary In summary, b-CD-(M)2.Fe3+ successfully simulated the one-step redox reaction of zui in the tricarboxylic acid cycle in vivo. L-malate dehydrogenase was simulated, and L-malic acid was rapidly dehydrogenated at room temperature to form oxaloacetate. Other inorganic catalysts were not available (such as FeCl3), which could achieve the same catalytic performance of biological enzymes in this system. The Michaelis constant of the biological enzyme is 7.60 mmol.L-1, and the Michaelis constant of the mimic enzyme is 8.74 mmol.L-1. A certain amount of an organic solvent such as acetone is added to the reaction product, and the simulated enzyme is precipitated, thereby separating the simulated enzyme from the product, and the recovered simulated enzyme is reacted with FeCl3 to be used for catalytic oxidation of malic acid, and the catalytic performance is not obvious. influences.
The mimetic enzyme is easy to prepare, convenient to use, cheap, non-toxic and harmless, and overcomes the defects that the biological enzyme is greatly affected by the environment, and can be recycled and recycled. It can be said that this is a promising synthetic biomimetic compound.
3 References
[1] Yoon C J, Ikeda H, Kojin R et al. J. Chem. Soc. Chem Commun., 1986, 1080.
[2] Kuroda Y, Hiroshige T, Sera T et al. J. Am. Chem. Soc., 1989, 111:1918.
[3] Kuroda Y, Hiroshige T, Sera T et al. Carbohydr. Res., 1989, 192:347.
[4] Siegel B. J. Inorg. Nucl. Chem., 1979, 41: 609.
[5] Kuroda Y, Sasaki Y, Shiroiwa Y et al. J. Am. Chem. Soc., 1988, 110: 4049.
[6] Shen Tong, Wang Jingyan. Biochemistry (2). Beijing: Higher Education Press, 1995:99.
[7] Townshend A, Vaughan A. Talanta, 1970, 17: 299.
[8] Ding Zhigang. Liu Xuequn et al. Acta Chimica Sinica, 1995, 53:578-582.
[9] Li Shuqi. Modern Enzymatic Analysis. Beijing: Beijing Medical University, China Union Medical University, United Press, 1994: 93.
[10] Lineweaver H, Burk DJ J. Am. Chem. Soc., 1934, 6: 658.
[11] Shen Tong, Wang Jingyan. Biochemistry (Volume 1). Beijing: Higher Education Press, 1995:251
Under standard thermodynamic conditions, the reaction equilibrium is favorable for the reverse reaction. Under physiological conditions, the reaction product oxaloacetate is continuously synthesized and removed, so that the concentration in the cell is lowered, and the reaction proceeds to a positive reaction [6]. The author added a trapping agent (2,4-dinitrophenylhydrazine) to the catalytic reaction of the simulated enzyme, and converted the oxaloacetate to oxaloacetate in a glycine-NaOH buffer solution at pH 9.00, which promoted the reaction to positive reaction. Carry out [7].
Its reaction formula is as follows:
1 experiment
1.1 Instruments and reagents Shimadzu AA-646 atomic absorption spectrometer, AIC UV-9100 UV-visible spectrophotometer (Shenzhen Aike Analytical Instrument Co., Ltd.), pHS-3C digital acidity meter (Hangzhou Wanda Instrument and Meter Factory), β- CD is the industrial refinery of Suzhou MSG Factory. It has been recrystallized twice, L-malic acid, L-malic acid dehydrogenase, SIGMA CHEMICAL CO., NAD, biochemical reagent (Shanghai Boao Biotechnology Co., Ltd.), 2, 4-Dinitrophenylhydrazine, analytically pure (Shanghai Chemical Reagent Factory). The remaining reagents were of analytical grade.
1.2 Synthesis of simulated enzymes 0.11mol.L-1b-CD-(M)2 (synthesis and structure identification results: nuclear magnetic resonance spectroscopy and elemental analysis data see literature [8]) aqueous solution 30mL, 0.48 mol.L-1 FeCl3 aqueous solution 40 mL, the FeCl3 solution was all added to the b-CD-(M)2 solution under stirring. At this time, the solution gradually changed from colorless to yellow, and an orange-red transparent solution was obtained after the Zui. The solution was placed in a constant temperature water bath, cooled at 70 ° C for 8 h, and filtered. An appropriate amount of acetone was added to the filtrate to precipitate a pale yellow flocculent precipitate. The mixture was allowed to stand for filtration, washed with 95% ethanol until the filtrate was free of Cl-, dried, and placed in a desiccator to obtain a dry pale yellow powdery solid: b-CD-(M)2.Fe3+ weighs 4.6 g. The rate is 88%. The spot of Rf=0.66 (developing agent: ethyl acetate: methanol: glacial acetic acid: water = 12:3:3:2), infrared gmax: 3300 cm-1 (s, OH), 2900cm-1 (w, CH2), 1720cm-1 (w, C=O), 1625, 750cm-1 (mw, benzene ring), 1143cm-1 (w, ArSO2), the amount of Fe3+ measured by atomic absorption spectrophotometry It is proved that the molar ratio of b-CD-(M)2 to Fe3+ in the complex is 1:1, and the M?ssbauer spectrum proves to be the Fe3+ state.
1.3 Simulated enzyme catalytic reaction
1.3.1 Catalytic reaction In the cuvette, add 3 mL of Glycine-NaOH buffer (containing 2,4-dinitrophenylhydrazine) at pH 9.00, NAD + 50 mL of 1 mol.L-1, 4.44 mmol.L. -1 simulating enzyme 30mL, 1mol.L-1 malic acid 30mL, mixing, reaction temperature 25 ° C, 30mL buffer instead of substrate L-malic acid solution for the corresponding reference, measured at NADHzui large absorption 340nm A340 value. Comparative experiment: FeCl3 was used instead of the mimetic enzyme, and other conditions were the same as described above.
1.3.2 Effect of Substrate Concentration on the Rate of Simulated Enzyme Catalytic Reaction—Use the double reciprocal plot method to calculate the Michaelis constant of the simulated enzyme. In the cuvette, add 2.7 mL of glycine-NaOH buffer at pH 9.00, 1 mol. L-1 NAD + 200 mL, 0.523 mol.L-1 of 2,4-dinitrophenylhydrazine 30 mL, 4.44 mmol.L-1 mimetic enzyme 30 mL and different amounts of substrate (malic acid) as a measuring cell, Under the reaction temperature of 25 ° C, the same reference solution was used as the corresponding reference (both to 3.010 mL with buffer), the A340 value was measured, and the catalytic rate was determined, namely: V=DC/Dt= DA/ε.Dt, where: e is the molar absorptivity of NADH, 6.3×10-6 L/mmol.cm-1 at 340 nm [9], substrate concentration [S] is known, by 1/V The Michaelis constant Km of the mimetic enzyme was obtained by plotting 1/[S].
1.4 Conditional experiment
1.4.1 Effect of pH on the speed of simulated enzyme reaction In the cuvette, add 2.7 mL of glycine-NaOH buffer with a pH of 8.79, 9.00, 9.50, 10.00 and 10.70, and NAD + 50 mL of 1 mol.L-1. 0.523 mol.L-1 of 2,4-dinitrophenylhydrazine 30 μL, 4.44 mmol.L-1 of the mimetic enzyme 30mL, 1mol.L-1 L-malic acid 30mL, after mixing, corresponding blank (ie corresponding The pH buffer is used as a reference to measure the change in A340.
1.4.2 Effect of simulated enzyme concentration on simulated enzyme reaction rate Add 2.7 mL of pH 9.00 glycine-NaOH buffer, 1 mol.L-1 of NAD + 50 mL, 0.523 mol.L-1 of 2, 4 to the cuvette. - Dinitrophenylhydrazine 30mL, 1 mol.L-1 L-malic acid 20mL, 4.44mmol.L-1 mimic enzymes were taken 10, 15, 30, 40, 50, 60, 70mL, respectively, buffered to volume The total volume was 2.90 mL. After mixing, the corresponding blank (ie, buffer with pH=9.00 instead of substrate) was used as a reference to measure the change of A340.
1.4.3 Effect of 2,4-dinitrophenylhydrazine concentration on the catalytic speed of the simulated enzyme. Add 2.7 mL of pH=9.00 glycine-NaOH buffer to the cuvette, 1 mol.L-1 of NAD+ 50 mL, 4.44 mmol. L-1 mimetic enzyme 30mL, 1mol.L-1 L-malic acid 30mL, 0.523mol.L-1 2,4-dinitrophenylhydrazine respectively take 10,15,20,25,30,40,50 60,70mL, the total volume of the volume was 2.90mL with buffer, and after mixing, the corresponding blank (the buffer with pH=9.00 instead of the substrate) was used as a reference to measure the change of A340.
1.5 Control experiment with 1/100 concentration of SIGMA company after dialysis, L-malate dehydrogenase enzyme solution 30mL instead of mimic enzyme, at 25 ° C, and ensure other experimental conditions are the same, the determination of biological enzymes in this system The value of the Michaelis constant Km.
2 Results and discussion
2.1 Catalytic rate
Figure 1 Kinetic curve of simulated enzyme catalyzed malic acid
a mimic enzyme (â–²) b FeCl3 (â– )
The results in Figure 1 show that the catalytic rate of the simulated enzyme is much faster than FeCl3, while the curve a shows the catalytic kinetics of the catalytic enzyme to oxidize L-malic acid to oxaloacetate, and the catalytic reaction is very fast before 6 min. It is basically linear, and the reaction rate increases slowly after 6 minutes, and tends to remain unchanged after zui, indicating that the reaction has proceeded completely. Therefore, when calculating the catalytic rate, Dt is selected within 5 min. In order to facilitate the comparison with the biological enzyme in the determination of the Michaelis constant, we determined at the determination of the biological enzyme, the acidity of the acidity, pH=9.00, and the catalytic rate was faster if it was determined at the pH of the simulated enzyme, pH=9.50.
2.2 Reaction conditions In the conditional experiment, it can be seen from Fig. 2 that the pH value of the catalytic reaction of the system has a suitable acidity, that is, pH 9.5, which is greater or less than this value, and the catalytic ability of the system is decreased to some extent, while the biological enzyme zui The pH value is 9.00, which also indicates that the mimetic enzyme is closer to the biological enzyme in this respect. The curve a of Fig. 3 shows that when the concentration of the substrate L-malic acid is constant, the concentration of the mimetic enzyme is proportional to the reaction rate; the curve b indicates that the amount of 2,4-dinitrophenylhydrazine added in the reaction system has one Zui good value (4.46mmol.L-1).
Fig. 2 Effect of acidity on reaction rateFig.3 Effect of simulated enzyme and hydrazine concentration on reaction rate
a mimic enzyme (C × 10-5mol.L-1) (▲)
b 肼(C×10-3mol.L-1)(■)
2.3 The Km value of the simulated enzyme and the biological enzyme under the same catalytic conditions is based on the double reciprocal mapping method [10], and the Mie equation can be written as the following form: V-1=Km.Vmax-1.[S]-1 + Vmax -1.
Through experiments, the author selects different [S] to determine the corresponding V, and finds the reciprocal of the two, plots 1/V versus 1/[S], ​​and gives a straight line (see Figure 4). The linear equations are :Va-1=0.0307[S]-1+3.5142 (mimetic enzyme) and Vb-1=0.0325[S]-1+4.2738 (biological enzyme), therefore, the Michaelis constant (km) of the biological enzyme can be obtained Enzyme = 0.0325 / 4.2738 = 7.60 mmol. L-1, Michaelis constant (km) of the mimetic enzyme mimetic enzyme = 0.0307 / 3.5142 = 8.74 mmol. L-1. The smaller the value of km, the greater the affinity of the enzyme for the substrate [11], and it is clear that the high velocity Vmax of the enzymatic reaction can be easily achieved without the need for a high substrate concentration. Based on the obtained Michaelis constant, it was found that (Km) mimetic enzyme: (Km) bio-enzyme = 1.15:1, which indicates that the mimetic enzyme has a slightly weaker affinity to the substrate than the biological enzyme, but is very close.
Figure 4 Lineweaxer-Burk double reciprocal mapping method
a mimic enzyme (â–²)
b biological enzyme (malate dehydrogenase) (â– )
The size of the enzyme activity can be expressed by the reaction rate of a certain chemical reaction catalyzed by a certain condition, that is, the faster the reaction rate catalyzed by the enzyme, the higher the activity of the enzyme. It can be seen from Fig. 2 that the enzymatic reaction rate zui can be as high as 2.34 mol/L.min at 1.32×10-4 mmol simulated enzyme at 25 ° C and pH=9.00, while using 1/100 pure bio-zymogen. Under the same conditions, 30 mL of the enzyme solution had a large reaction rate of 0.287 mol/L·min for the enzymatic reaction in this system. Obviously, the reaction was catalyzed by 1.332×10-4 mmol of mimic enzyme, and the reaction rate of Zui was 1/. The 30 μmL of the enzyme zymogen solution 30 catalyzed this reaction was 8 times higher.
2.4 Summary In summary, b-CD-(M)2.Fe3+ successfully simulated the one-step redox reaction of zui in the tricarboxylic acid cycle in vivo. L-malate dehydrogenase was simulated, and L-malic acid was rapidly dehydrogenated at room temperature to form oxaloacetate. Other inorganic catalysts were not available (such as FeCl3), which could achieve the same catalytic performance of biological enzymes in this system. The Michaelis constant of the biological enzyme is 7.60 mmol.L-1, and the Michaelis constant of the mimic enzyme is 8.74 mmol.L-1. A certain amount of an organic solvent such as acetone is added to the reaction product, and the simulated enzyme is precipitated, thereby separating the simulated enzyme from the product, and the recovered simulated enzyme is reacted with FeCl3 to be used for catalytic oxidation of malic acid, and the catalytic performance is not obvious. influences.
The mimetic enzyme is easy to prepare, convenient to use, cheap, non-toxic and harmless, and overcomes the defects that the biological enzyme is greatly affected by the environment, and can be recycled and recycled. It can be said that this is a promising synthetic biomimetic compound.
3 References
[1] Yoon C J, Ikeda H, Kojin R et al. J. Chem. Soc. Chem Commun., 1986, 1080.
[2] Kuroda Y, Hiroshige T, Sera T et al. J. Am. Chem. Soc., 1989, 111:1918.
[3] Kuroda Y, Hiroshige T, Sera T et al. Carbohydr. Res., 1989, 192:347.
[4] Siegel B. J. Inorg. Nucl. Chem., 1979, 41: 609.
[5] Kuroda Y, Sasaki Y, Shiroiwa Y et al. J. Am. Chem. Soc., 1988, 110: 4049.
[6] Shen Tong, Wang Jingyan. Biochemistry (2). Beijing: Higher Education Press, 1995:99.
[7] Townshend A, Vaughan A. Talanta, 1970, 17: 299.
[8] Ding Zhigang. Liu Xuequn et al. Acta Chimica Sinica, 1995, 53:578-582.
[9] Li Shuqi. Modern Enzymatic Analysis. Beijing: Beijing Medical University, China Union Medical University, United Press, 1994: 93.
[10] Lineweaver H, Burk DJ J. Am. Chem. Soc., 1934, 6: 658.
[11] Shen Tong, Wang Jingyan. Biochemistry (Volume 1). Beijing: Higher Education Press, 1995:251
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