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Gelatin hydrogel-based method for immobilizing diacetylchitobiose deacetylase

A technology of deacetylase and hexanebiose, which is applied in the field of immobilization of hexanbiose deacetylase, can solve problems such as inability to withstand high temperature, and achieves the effects of good stability, good high temperature resistance and improved catalytic activity.

Active Publication Date: 2021-08-31
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when the existing gels are used for the immobilization of hexanobiose deacetylase, they often cannot withstand high temperatures, and the effect can only be relatively long at room temperature.

Method used

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  • Gelatin hydrogel-based method for immobilizing diacetylchitobiose deacetylase
  • Gelatin hydrogel-based method for immobilizing diacetylchitobiose deacetylase
  • Gelatin hydrogel-based method for immobilizing diacetylchitobiose deacetylase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Prepare immobilized hexanobiose deacetylase according to the following method: take the crude enzyme solution or concentrated enzyme solution containing hexbutose deacetylase, add 10% (w / v) type A gelatin; heat to 50°C and Shake well to fully dissolve the gelatin; add 1% Genipin (Genipin) with a concentration of 50mg / mL, and immediately add HFE-7500 fluorinated solution with 0.5% surfactant in a ratio of 1:1; shake while hot For 3 minutes, mix the water phase and oil phase thoroughly and place it at 4°C to cool; shake and incubate at room temperature for 1 day; add 10% volume of 1H, 1H, 2H, 2H-perfluorooctyl alcohol, and add 3 times the volume of buffer After mixing, let it stand for 5 minutes to separate the water and oil, remove the upper water phase, and the hydrogel will form a precipitate in the water phase after standing; use 3 times the volume of buffer to wash the hydrogel in the water phase 3 After that, 3 times the volume of buffer solution was added, and 1% g...

Embodiment 2

[0028] In order to verify the effectiveness of this method for immobilizing succinate deacetylase, in this example, the hydrogel containing succinate deacetylase prepared in Example 1 was used to catalyze the hydrolysis of N-acetylglucosamine and recover , The content of glucosamine in the solution is detected by detecting the fluorescence intensity with a microplate reader to judge the catalytic activity of the hexbutadiose deacetylase after recovery after each use. Take 2mL crude enzyme solution to prepare hydrogel, due to the volume expansion in the preparation process, a total of 4mL hydrogel ( figure 1 ). The hydrogel and substrate N-acetylglucosamine (concentration: 100g / L) were preheated at 40°C, 4mL of hydrogel and 4mL of substrate were mixed, and incubated with shaking at 40°C for 10min. The solution after the reaction was diluted 20 times with a buffer, and the yield of the product was detected using a glucosamine detection reagent. For the detection method of gluc...

Embodiment 3

[0031] Example 3: Changes in Enzyme Activity of Hexobutadiose Deacetylase After Hydrogel Immobilization After High Temperature Treatment

[0032] In this example, we first made 4 mL of hydrogel according to the method in Example 1. In order to verify the stability of the hydrogel at high temperature, we heated the hydrogel at 80°C for 10 min, and washed the hydrogel 3 times with buffer. At this time, due to the heating effect, the actual volume of the hydrogel is less than 4mL, about 2.5mL; in order to verify the change of the enzyme activity of the enzyme immobilized in the hydrogel by heating, this experiment still maintains the hydrogel by adding buffer. The volume is 4 mL. Afterwards, the hydrogel after the heating and cleaning steps will be used to catalyze the substrate to produce glucosamine, and the experimental process is the same as in Example 1. The results of hydrogel repeated reaction 8 times are as follows: Figure three As shown in Table 2, the results show t...

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Abstract

The invention discloses a gelatin hydrogel-based method for immobilizing a diacetylchitobiose deacetylase. The method comprises the following steps: adding gelatin into enzyme liquid containing a diacetylchitobiose deacetylase and heating to dissolve the gelatin; adding genipin, adding a fluorinated solution containing a surfactant according to a volume ratio of 1:(0.8-1.2), shaking and uniformly mixing, cooling, and shaking and culturing for 0.5-2 days; adding 5-15% of 1H, 1H, 2H, 2H-perfluorooctanol according to a volume ratio, adding a buffer solution, taking a water phase after layering of water and oil, and standing to obtain a first hydrogel; and washing the first hydrogel, adding a buffer solution, adding genipin, shaking and culturing for 1-5 days to obtain a second hydrogel, namely the immobilized diacetylchitobiose deacetylase. A two-step gel forming mode is adopted, the prepared immobilized diacetylchitobiose deacetylase has good high-temperature resistance, can tolerate 80 DEG C high-temperature treatment, is remarkably helpful for improving catalytic activity of an enzyme and has good stability, and the catalytic activity of the hydrogel is not obviously changed after the hydrogel is repeatedly used for 8 times.

Description

technical field [0001] The invention relates to a gelatin hydrogel-based immobilization method of hexobutadiose deacetylase, which belongs to the technical field of enzyme immobilization. Background technique [0002] Glucosamine is a small molecular monosaccharide that widely exists in nature and is widely used in the fields of medicine and health care products. At present, the industrial production of glucosamine mainly adopts chemical hydrolysis. This method uses chitin in shrimp and crab shells as a substrate and uses strong acid for hydrolysis. The reaction conditions are harsh, it is easy to cause environmental pollution, and the production efficiency is limited. With the promotion of green chemistry and environmentally friendly manufacturing, more research has begun to focus on green and environmentally friendly microbial fermentation technology, and use cheap products such as chitin and glucose as substrates to produce glucosamine through hydrolysis or synthesis. Am...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/02C12N9/80C12P19/26
CPCC12N11/02C12N9/80C12P19/26
Inventor 刘龙陈坚吕雪芹堵国成李江华刘延峰孙国运
Owner JIANGNAN UNIV
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