Frozen gel embedding method for enzyme

A technology of cryogel and enzyme liquid, which is applied in the direction of fixing on/in the organic carrier, can solve the problems of leakage and reduce enzyme activity, etc., and achieve the effect of simple process, high catalytic activity and strong resistance to organic solvents

Inactive Publication Date: 2013-07-24
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The embedding method does not require a binding reaction with the amino acid residues of the enzyme protein, and the recovery rate of the enzyme activity is high, but the mass transfer resistance encountered by the substrate when passing through the denser grid structure of the polymer gel will cause the enzyme kinetics Changes in the enzyme activity, and there is a phenomenon of enzyme leakage during recycling

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] First, weigh urease powder and dissolve it in boric acid buffer solution to make 0.2g / L enzyme solution for later use, add 5mL N-isopropylacrylamide with a concentration of 70g / L to 2mL enzyme solution, add 0.035g polyethylene glycol 200 diacrylate, 0.04mL hydrogen peroxide with a concentration of 30%, and then placed in a tetrafluoroethylene tray, placed in a low-temperature constant temperature bath at -20°C for 3 hours; placed in an ultraviolet light (wavelength 365nm) polymerization instrument for 2 minutes, The obtained gel was soaked in distilled water for 2 days, the water was changed every 4 hours, and after 3 times, the water was changed every 12 hours, and then vacuum-dried at 40°C to obtain the cryogel-embedded enzyme. The measured activity yield of the enzyme was 95%, the gel yield was 92%, and the gel swelling rate was 35%. After storage at 4°C for 28 days, the activity of the enzyme retained 94.8% of the original activity.

[0026] Wherein, the activity y...

Embodiment 2

[0028] First, weigh urease powder and dissolve it in boric acid buffer solution to make 1g / L enzyme solution for later use. Add 5mL acrylamide with a concentration of 70g / L to 2mL enzyme solution, add 0.07g polyethylene glycol 400 diacrylate, 0.06 Put hydrogen peroxide with a concentration of 30% in mL, then place it in a tetrafluoroethylene dish, place it in a low-temperature thermostat at -25°C for 2 hours; Soak for 3 days, change the water every 3 hours, and change the water every 12 hours after 4 times, and then vacuum-dry at 40°C to obtain the cryogel-embedded enzyme. The enzyme activity yield was measured to be 89%, the gel yield was 94%, and the gel swelling rate was 34%. After storage at 4°C for 28 days, the activity of the enzyme retained 80% of the original activity.

Embodiment 3

[0030] First, weigh papain powder and dissolve it in phosphate buffer solution to prepare 2.5g / L enzyme solution for later use, add 5mL acrylamide with a concentration of 100g / L to 2mL enzyme solution, add 0.10g polyethylene glycol 200 dimethyl Acrylic acid ester, 0.09mL of hydrogen peroxide with a concentration of 30%, and then placed in a tetrafluoroethylene tray, placed in a low-temperature thermostat at -15°C for 5h; placed in an ultraviolet light (wavelength 365nm) polymerizer for 4min to obtain The gel was soaked in distilled water for 2 days, the water was changed every 4 hours, and the water was changed every 12 hours after 3 times, and then vacuum-dried at 40°C to obtain the cryogel-embedded enzyme. The measured activity yield of the enzyme was 95%, the gel yield was 99%, and the gel swelling rate was 29%. After storage at 4°C for 28 days, the enzyme activity retained 95.7% of its original activity.

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Abstract

The invention discloses a frozen gel embedding method for an enzyme. The frozen gel embedding method comprises the steps of: (1) dissolving enzyme powder in a buffer solution to prepare an enzymic solution with the concentration of 0.2-5g / L; (2) uniformly mixing the enzymic solution with a polymerized monomer water solution, a crosslinking agent and an initiator, placing a mixture in a container,and standing for 1-5h at the temperature of minus 15-minus 40 DEG C to obtain a frozen solid; and (3) irritating the frozen solid in ultraviolet light for 2-6min to obtain gel, soaking obtained gel with distilled water, changing the distilled water for a plurality of times, and carrying out vacuum drying to obtain the frozen gel embedded enzyme. According to the method disclosed by the invention,the frozen gel embedded enzyme achieves the advantages of high catalytic activity, strong stability, small mass transfer resistance, strong organic solvent resistance and the like which cannot be obtained by using other methods; in addition, the method is simple in process and mild in conditions, and the obtained immobilized enzyme is high in recovery rate and wide in applicable environmental range.

Description

technical field [0001] The invention relates to an enzyme embedding method, in particular to an enzyme cryogel embedding method. Background technique [0002] The method of embedding the enzyme in a porous carrier to immobilize the enzyme is called the embedding method. The embedding method does not require a binding reaction with the amino acid residues of the enzyme protein, and the recovery rate of the enzyme activity is high, but the mass transfer resistance encountered by the substrate when passing through the denser grid structure of the polymer gel will cause the enzyme kinetics The change of the enzyme reduces the activity of the enzyme, and there is a phenomenon that the enzyme leaks during the recycling process. [0003] Encapsulation and immobilization of enzymes in cryogel has been greatly developed since the 1990s. The gel network in this embedding method is gradually formed around the enzyme, unlike the ordinary embedding method that has strict restrictions on...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/08C12N11/04
Inventor 宋锡瑾李倩王杰
Owner ZHEJIANG UNIV
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