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Preparation method of frozen gel glutamine transaminase

A glutamine and cryogel technology, applied in the field of enzyme treatment, can solve problems such as leakage and reduce enzyme activity, and achieve the effects of high catalytic efficiency, high catalytic activity and low mass transfer resistance

Pending Publication Date: 2019-10-22
TAIXING DONGSHENG FOOD TECH +1
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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

[0027] First, weigh transglutaminase enzyme powder and dissolve it in boric acid buffer solution to prepare 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 of 30% hydrogen peroxide, then placed in a tetrafluoroethylene tray, placed in a low-temperature constant temperature bath at -20°C for 3h; placed in an ultraviolet light (wavelength 365nm) polymerization instrument After irradiating under low temperature for 2 minutes, the obtained gel was soaked in distilled water for 2 days, and 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 cryogel-embedded transglutaminase. 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 act...

Embodiment 2

[0030] First, weigh transglutaminase enzyme 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 Acrylate, 0.06mL hydrogen peroxide with a concentration of 30%, and then placed in a tetrafluoroethylene tray, placed in a low-temperature thermostat at -25°C for 2h; placed in an ultraviolet light (wavelength 365nm) polymerization instrument for 5min, the The gel was soaked in distilled water for 3 days, the water was changed every 3 hours, and after 4 times, the water was changed every 12 hours, and then vacuum-dried 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

[0032]First, weigh transglutaminase enzyme 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 dimethacrylate, 0.09mL of 30% hydrogen peroxide, then placed in a tetrafluoroethylene tray, placed in a low-temperature constant temperature bath at -15°C for 5h; placed under ultraviolet light (wavelength 365nm) polymerizer for irradiation For 4 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 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 preparation method of frozen gel glutamine transaminase. According to the method, firstly, a glutamine transaminase solution, a polymerization monomer N-isopropylacrylamide or acrylamide or N-vinylcaprolactam, a cross-linking agent and an initiator are mixed to form a stable and uniform solution, a polymerization reaction is initiated by ultraviolet light irradiation after low-temperature freezing treatment to obtain macroporous gel, and enzyme embedding is conducted to obtain the target product frozen gel embedded glutamine transaminase. By means of the method, the frozen gel embedded glutamine transaminase has the advantages of high catalytic activity, high stability, small mass transfer resistance, high organic solvent resistance and the like which glutamine transaminase prepared by means of other methods does not have; the process is simple, the conditions are mild, and the obtained immobilized enzyme is high in recovery rate and wide in application environment range.

Description

technical field [0001] The invention relates to the technical field of enzyme treatment, in particular to a preparation method of cryogel transglutaminase. Background technique [0002] The method of immobilizing the enzyme by embedding it in a porous carrier 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 s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/08C12N11/04
CPCC12N11/08C12N11/04C12N9/1044C12Y203/02013
Inventor 步国建刘艳甘李张玉如寇雪蕊和超楠贺晓洁
Owner TAIXING DONGSHENG FOOD TECH
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