Soybean epoxy compound hydrolase preparation and immobilization method

An epoxy compound and immobilization method technology, applied in the field of materials, can solve the problems of long immobilization time and low enzyme loading, and achieve the effects of improving the recovery rate of enzyme activity, increasing the enzyme loading, and improving the immobilization effect.

Active Publication Date: 2015-07-29
SOUTH CHINA UNIV OF TECH
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Problems solved by technology

[0003] At present, encapsulation and physical adsorption methods are commonly used to immobilize enzymes on metal-organic frameworks to immobilize enzymes on mesoporous carriers, but the encapsulation and physical adsorption methods require a long time for immobilization (50h, Lykourinou V., Chen Y., Wang X.-S., et al.Immobilization of MP-11into a mesoporous metal–organic framework, MP-11mesoMOF: a new platform for enzymatic catalysis[J].Journal of the American Chemical Society,2011,133:10382-10385 ), low enzyme load (7.0-50.5mg enzyme / g carrier, Chen Y., Lykourinou V., Vetromile C., et al. How can proteins enter the interior of a MOF Investigation of cytochrome c translocation into a MOF consisting of mesoporous cages with microporous windows[J].Journal of the American Chemical Society,2012,134:13188-13191)

Method used

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  • Soybean epoxy compound hydrolase preparation and immobilization method
  • Soybean epoxy compound hydrolase preparation and immobilization method
  • Soybean epoxy compound hydrolase preparation and immobilization method

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Soybean epoxide hydrolase is dispersed in a phosphate buffer solution with a pH of 6.5 to configure an enzyme solution; 50mg UiO-66-NH 2 Mix nanocrystals (substrate) with 1mL 5mg / mL enzyme solution (mass ratio 10:1); add a certain volume of saturated ammonium sulfate solution (saturation is 100%) under stirring at 0°C to make the final saturation of ammonium sulfate The concentration is 80%, stirred at a stirring speed of 200rpm for 30min; a certain amount of 25% glutaraldehyde solution is added to make the final mass of glutaraldehyde 65mg, and the cross-linking reaction is stirred at 0°C and 300rpm for 120min; washed and freeze-dried to obtain Soybean epoxide hydrolase preparation 1.

[0033] The relative enzyme activity recovery rate of enzyme preparation 1 was 82.911%. The soybean epoxide hydrolase and UiO-66-NH in the product 2 The substrate ratio was 89.56 mg / g. Carry out the test of enzyme activity to enzyme preparation 1, test result is as follows Figure 4 ,...

Embodiment 2

[0036] Soybean epoxide hydrolase is dispersed in a phosphate buffer solution with a pH of 6.5 to configure an enzyme solution; 50mg UiO-66-NH 2 Mix with 1mL 5mg / mL enzyme solution (mass ratio 10:1); add a certain volume of saturated ammonium sulfate solution under stirring at 0°C to make the final saturation of ammonium sulfate 80%, stir at 200rpm for 30min; add A certain amount of 25% glutaraldehyde solution was used so that the final mass of glutaraldehyde was 65 mg, and the cross-linking reaction was stirred at 0° C. and 300 rpm for 20 minutes; washed and freeze-dried to obtain soybean epoxy compound hydrolase preparation 7.

[0037] The relative enzyme activity recovery rate of enzyme preparation 7 was 42.676%. The soybean epoxide hydrolase and UiO-66-NH in the product 2 The base material ratio is 82.49mg / g.

[0038]Only change the cross-linking reaction time, other conditions are identical with embodiment 2, obtain following enzyme preparation respectively: cross-linking...

Embodiment 3

[0040] Soybean epoxy compound hydrolase is dispersed in a phosphate buffer solution with a pH of 6.5 to configure an enzyme solution; 25mg UiO-66-NH 2 Mix the nanocrystals (i.e. substrate) with 1mL of 5mg / mL enzyme solution (mass ratio 5:1); add a certain volume of saturated ammonium sulfate solution under stirring at 0°C, so that the final saturation of ammonium sulfate is 80%. Stir at a speed of 200 rpm for 30 min; add a certain amount of 25% glutaraldehyde solution to make the final mass of glutaraldehyde 65 mg, stir at 0°C and 300 rpm for 120 min; wash and freeze-dry to obtain soybean epoxy compound hydrolase preparation 14.

[0041] The relative enzyme activity recovery rate of enzyme preparation 14 was 64.191%. The soybean epoxide hydrolase and UiO-66-NH in the product 2 The substrate ratio was 173.58 mg / g.

[0042] Change only UiO-66-NH 2 The mass ratio of nanocrystal (being substrate) and enzyme, other conditions are identical with embodiment 3, obtain following enzy...

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Abstract

The invention belongs to the technical field of materials and discloses a soybean epoxy compound hydrolase immobilization method and an enzyme preparation. The soybean epoxy compound hydrolase immobilization method comprises the following steps: dispersing soybean epoxy compound hydrolase in a buffer solution with the pH of 5-9 to prepare an enzyme solution, mixing the enzyme solution with UiO-66-NH2 nano crystals to obtain a mixture, adding a saturate ammonium sulfate solution in the mixture at 0-35 DEG C to enable the final saturability of ammonium sulfate to be 80%, stirring and mixing for 1-60 min to obtain a mixed turbid liquid, mixing the mixed turbid liquid with a glutaraldehyde solution, reacting for 1-200 min in a stirring manner, and freeze-drying to obtain a soybean epoxy compound hydrolase preparation. According to the invention, a simple sedimentation crosslinking technology is adopted, the cost is lower, the reaction is moderate, the enzyme activity can be kept to a greater degree, the enzyme loading amount of a carrier can be increased, the enzyme activity recovery rate can be increased, and the immobilization effect is excellent.

Description

technical field [0001] The invention belongs to the technical field of materials, and relates to a soybean epoxy compound hydrolase immobilization method and an enzyme preparation. Background technique [0002] Enzyme immobilization on metal-organic frameworks is a research hotspot at home and abroad because of its unique and diverse topology, porosity, and physical properties. Metal-organic framework materials have many properties such as porosity, large specific surface area, and multiple metal sites, and have good application prospects in the fields of gas storage, molecular separation, catalysis, and drug sustained release. [0003] At present, encapsulation and physical adsorption methods are commonly used to immobilize enzymes on metal-organic frameworks to immobilize enzymes on mesoporous carriers, but the encapsulation and physical adsorption methods require a long time for immobilization (50h, Lykourinou V., Chen Y., Wang X.-S., et al.Immobilization of MP-11into a ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/02
Inventor 娄文勇夏高辉宗敏华曹诗林岳东梅
Owner SOUTH CHINA UNIV OF TECH
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