Biomimetic immobilization method of multienzyme system

Abstract

The invention relates to a technique for immobilizing a multienzyme system, which comprises the following steps: 1) preparation of precursor solution, namely, adding TMOS (tetramethyl orthosilicate) into a 1 mmol / L HCl solution at room temperature, adding a phosphate buffer of which the pH value is 5-9 after the TMOS is completely dissolved and the solution becomes clear, and evenly mixing; 2) preparation of immobilized enzyme: evenly mixing the multienzyme solution with a PDADMA (polydimethyldiallyl ammonium chloride) solution to obtain an enzyme-containing solution A; and under the 30 DEG C homothermal condition, adding the pretreated precursor solution into the solution A, immediately shaking, standing at room temperature, carrying out centrifugal separation, washing with the phosphate buffer, and carrying out vacuum freeze drying on the residual solid for 24 hours, thereby obtaining the biomimetic silicified multienzyme. The method provided by the invention has the advantages of simple preparation technique, mild conditions, low loss of enzyme activity, and high enzyme activity recovery rate (up to 85.12%); and the carrier is the TMOS which is cheap and accessible.

Description

Technical field

[0001] The invention belongs to the field of immobilized enzymes, in particular to a process method for immobilized multi-enzyme systems. Background technique

[0002] Multi-enzyme system refers to a whole structure and function composed of different enzymes, which can carry out continuous reaction catalysis. The material metabolism of organisms in nature is maintained by various multi-enzyme systems. The multi-enzyme system is Efficient and precise systems often consist of two or more enzymes forming a complex. The product catalyzed by the first enzyme in the complex is directly catalyzed by the second enzyme as the substrate, and the product catalyzed by the second enzyme is the substrate of the next enzyme in the complex. The continuous catalytic reaction is carried out in this way to make the catalytic efficiency significant Improve and organically coordinate the balance between a series of enzyme reactions. The high efficiency and high coordination of the m...

Examples

example 1

[0020] The method of preparing buffers with different pH values ​​used in the experiment is as follows: Mother liquor 1: Weigh NaH 2 PO 4 -2H 2 O solid 0.1mol, dissolved in 1000mL distilled water to obtain 0.1mol / L NaH 2 PO4 solution; mother liquor 2: Weigh Na 2 HPO 4 -12H 2 O solid 0.1mol, dissolved in 1000mL distilled water to obtain 0.1mol / L Na 2 HPO 4 Solution, buffer solutions of different pH are obtained by mixing mother liquor 1 and mother liquor 2 in a certain proportion and calibrating with a pH meter.

[0021] Add 0.6mL of TMOS (analytical grade) to 4.4mL of 1mmol / L HCl solution at room temperature, gently shake to completely dissolve TMOS, add 5mL of phosphate buffer (pH=5) after the solution becomes clear, and mix well , To obtain the pre-treated precursor solution.

[0022] Mix 1mL of a multi-enzyme solution with a ratio of 0.2:4 (referring to the masses of glucose oxidase 0.2mg and α-amylase 4mg in 1mL, pH 7 phosphate buffer) and 1mL PDADMA (6mg / mL) ) Mix evenly to ob...

example 2

[0024] Add 0.6mL of TMOS (analytical grade) to 4.4mL of 1mmol / L HCl solution at room temperature, shake gently to dissolve TMOS completely, add 5mL of phosphate buffer (pH=7) after the solution becomes clear, and mix well .

[0025] Mix 1mL of a multi-enzyme solution with a ratio of 0.3:4 (referring to the masses of glucose oxidase and α-amylase dissolved in 1mL of phosphate buffer at pH 7 of 0.3mg and 4mg respectively) and 1mL PDADMA (6mg / mL) The solution is evenly mixed to obtain enzyme-containing solution A. At a constant temperature of 30°C, take 200μL of the precursor solution pretreated in the previous step and add it to solution A, shake and mix immediately, let stand at room temperature for 20 minutes, centrifuge (5000rpm, 5min), and thoroughly use pH=7 phosphate buffer After washing 3 times, the remaining solid is vacuum freeze-dried for 24 hours to obtain the biomimetic siliconized multi-enzyme. In the experiment, the release rate of hydrogen peroxide is used to expr...

example 3

[0027] Add 0.6mL of TMOS (analytical grade) to 4.4mL of 1mmol / L HCl solution at room temperature, shake gently to dissolve TMOS completely, add 5mL of phosphate buffer (pH=7) after the solution becomes clear, and mix well .

[0028] Mix 1mL of a multi-enzyme solution with a ratio of 0.4:4 (referring to 1mL of phosphate buffer with a pH of 7 in which the masses of glucose oxidase and α-amylase are 0.4mg and 4mg respectively) solution and 1mL PDADMA ( 6mg / mL) mixed uniformly to obtain enzyme-containing solution A. At a constant temperature of 30℃, take 200μL of the precursor solution pretreated in the previous step and add it to solution A, shake and mix immediately, let stand at room temperature for 30min, centrifuge (5000rpm, 5min), and thoroughly use pH=7 phosphate buffer After washing 3 times, the remaining solid is vacuum freeze-dried for 24 hours to obtain the biomimetic siliconized multi-enzyme. In the experiment, the release rate of hydrogen peroxide is used to express th...

Technical field

[0001] The invention belongs to the field of immobilized enzymes, in particular to a process method for immobilized multi-enzyme systems. Background technique

[0002] Multi-enzyme system refers to a whole structure and function composed of different enzymes, which can carry out continuous reaction catalysis. The material metabolism of organisms in nature is maintained by various multi-enzyme systems. The multi-enzyme system is Efficient and precise systems often consist of two or more enzymes forming a complex. The product catalyzed by the first enzyme in the complex is directly catalyzed by the second enzyme as the substrate, and the product catalyzed by the second enzyme is the substrate of the next enzyme in the complex. The continuous catalytic reaction is carried out in this way to make the catalytic efficiency significant Improve and organically coordinate the balance between a series of enzyme reactions. The high efficiency and high coordination of the m...

Examples

example 1

[0020] The method of preparing buffers with different pH values ​​used in the experiment is as follows: Mother liquor 1: Weigh NaH 2 PO 4 -2H 2 O solid 0.1mol, dissolved in 1000mL distilled water to obtain 0.1mol / L NaH 2 PO4 solution; mother liquor 2: Weigh Na 2 HPO 4 -12H 2 O solid 0.1mol, dissolved in 1000mL distilled water to obtain 0.1mol / L Na 2 HPO 4 Solution, buffer solutions of different pH are obtained by mixing mother liquor 1 and mother liquor 2 in a certain proportion and calibrating with a pH meter.

[0021] Add 0.6mL of TMOS (analytical grade) to 4.4mL of 1mmol / L HCl solution at room temperature, gently shake to completely dissolve TMOS, add 5mL of phosphate buffer (pH=5) after the solution becomes clear, and mix well , To obtain the pre-treated precursor solution.

[0022] Mix 1mL of a multi-enzyme solution with a ratio of 0.2:4 (referring to the masses of glucose oxidase 0.2mg and α-amylase 4mg in 1mL, pH 7 phosphate buffer) and 1mL PDADMA (6mg / mL) ) Mix evenly to ob...

example 2

[0024] Add 0.6mL of TMOS (analytical grade) to 4.4mL of 1mmol / L HCl solution at room temperature, shake gently to dissolve TMOS completely, add 5mL of phosphate buffer (pH=7) after the solution becomes clear, and mix well .

[0025] Mix 1mL of a multi-enzyme solution with a ratio of 0.3:4 (referring to the masses of glucose oxidase and α-amylase dissolved in 1mL of phosphate buffer at pH 7 of 0.3mg and 4mg respectively) and 1mL PDADMA (6mg / mL) The solution is evenly mixed to obtain enzyme-containing solution A. At a constant temperature of 30°C, take 200μL of the precursor solution pretreated in the previous step and add it to solution A, shake and mix immediately, let stand at room temperature for 20 minutes, centrifuge (5000rpm, 5min), and thoroughly use pH=7 phosphate buffer After washing 3 times, the remaining solid is vacuum freeze-dried for 24 hours to obtain the biomimetic siliconized multi-enzyme. In the experiment, the release rate of hydrogen peroxide is used to expr...

example 3

[0027] Add 0.6mL of TMOS (analytical grade) to 4.4mL of 1mmol / L HCl solution at room temperature, shake gently to dissolve TMOS completely, add 5mL of phosphate buffer (pH=7) after the solution becomes clear, and mix well .

[0028] Mix 1mL of a multi-enzyme solution with a ratio of 0.4:4 (referring to 1mL of phosphate buffer with a pH of 7 in which the masses of glucose oxidase and α-amylase are 0.4mg and 4mg respectively) solution and 1mL PDADMA ( 6mg / mL) mixed uniformly to obtain enzyme-containing solution A. At a constant temperature of 30℃, take 200μL of the precursor solution pretreated in the previous step and add it to solution A, shake and mix immediately, let stand at room temperature for 30min, centrifuge (5000rpm, 5min), and thoroughly use pH=7 phosphate buffer After washing 3 times, the remaining solid is vacuum freeze-dried for 24 hours to obtain the biomimetic siliconized multi-enzyme. In the experiment, the release rate of hydrogen peroxide is used to express th...