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Magnetic immobilized cross-linked lipase aggregate and preparation method and application thereof

A technology of lipase and aggregates, which is applied in the direction of immobilization on/in the organic carrier, can solve the problems of unreported enzyme immobilization technology, and achieve the effect of improving poor operability, good precipitation effect and high activity

Active Publication Date: 2012-06-20
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the limitations of the two technologies, the enzyme immobilization technology combining the two technologies has not been reported.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] (1) Preparation of cross-linked lipase aggregates

[0049] Weigh lipase freeze-dried enzyme powder, dissolve it in 50 mL phosphate buffer solution with pH=5.8 and molar concentration of 0.1 mol / L, to prepare 10 mg / mL enzyme liquid. Take 2 mL of the above enzyme solution in a test tube, and add 6 mL of tert-butanol-acetonitrile mixture (100% tert-butanol and 100% acetonitrile by volume ratio of 1:1) drop by drop, at 4°C Stand still for 4.0 hours to precipitate lipase, then add glutaraldehyde solution to make the final volume concentration of glutaraldehyde 0.1%, stir at 4°C for 5 hours to carry out cross-linking reaction, and dissolve the resulting suspension at 3000rpm / min Centrifuge for 10 minutes, wash the precipitate obtained by centrifugation with pH = 7.0, molar concentration of 0.2mol / L phosphate buffer, until no lipase can be detected in the supernatant, and vacuum freeze-dry to obtain cross-linked lipase aggregates .

[0050] (2) Preparation of magnetic chitos...

Embodiment 2

[0060] (1) Preparation of cross-linked lipase aggregates

[0061] Weigh lipase freeze-dried enzyme powder, dissolve it in 50 mL phosphate buffer solution with pH = 9.0 and molar concentration of 1.0 mol / L to prepare 100 mg / mL enzyme solution. Take 2 mL of the above enzyme solution in a test tube, and add 60 mL of tert-butanol and acetone mixture dropwise (obtained by mixing 50% tert-butanol and 50% acetone at a volume ratio of 1:1), and store at 10°C Let it stand for 0.5 hours to precipitate the lipase, then add glutaraldehyde solution to make the final volume concentration of glutaraldehyde 5.0%, stir at 10°C for 2 hours to carry out cross-linking reaction, and mix the resulting suspension at 2000rpm / min Centrifuge for 10 minutes under the conditions of centrifugation, and wash the precipitate obtained by centrifugation with pH = 7.0, molar concentration of 1.0mol / L phosphate buffer, until no lipase can be detected in the supernatant, and vacuum freeze-dry to obtain cross-lin...

Embodiment 3

[0072] (1) Preparation of cross-linked lipase aggregates

[0073] Weigh lipase freeze-dried enzyme powder, dissolve it in 50 mL phosphate buffer solution with pH=6.0 and molar concentration of 0.8 mol / L, to prepare 40 mg / mL enzyme liquid. Take 2mL of the above enzyme solution in a test tube, and add 20mL of acetonitrile-acetone mixture (90% mass concentration of acetonitrile and 90% mass concentration of acetone at a volume ratio of 1:1) dropwise, and stand at 6°C for 3.0 hours, make the lipase precipitation, then add glutaraldehyde solution, make the final volume concentration of glutaraldehyde be the mass concentration 0.09%, stir at 6 ℃ for 4 hours to carry out cross-linking reaction, the gained suspension is in 3000rpm / min Centrifuge for 10 minutes under the same conditions, wash the precipitate obtained by centrifugation with pH = 7.0, molar concentration of 0.4mol / L phosphate buffer, until no lipase can be detected in the supernatant, and vacuum freeze-dry to obtain cros...

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Abstract

The invention discloses a magnetic immobilized cross-linked lipase aggregate and a preparation method and application thereof. The preparation method comprises the following steps of: putting a cross-linked lipase aggregate into phosphate buffer, and fully oscillating to ensure that the cross-linked lipase aggregate is uniformly distributed in the phosphate buffer; adding a glutaraldehyde activated magnetic immobilized cross-linked lipase aggregate, and cross-linking; and separating liquid from a solid, washing the solid, and performing vacuum freeze drying to obtain the magnetic immobilized cross-linked lipase aggregate with the relative enzyme activity of over 80 percent. The preparation method is low in cost and easy to operate. A magnetic immobilized enzyme technology is organically combined with a cross-linked enzyme aggregate (CLEA) technology, so that the activity of a magnetic immobilized enzyme is improved, the problem of low operating performance of CLEAs is solved, and the obtained magnetic immobilized cross-linked lipase aggregate with high enzyme activity and operating performance is stable in property, can be used as a bio-enzyme catalyst to be widely applied, and is particularly suitable for a large-scale enzyme reactor.

Description

technical field [0001] The invention relates to the field of biochemical industry, in particular to a magnetically immobilized cross-linked lipase aggregate and its preparation method and application. Background technique [0002] Lipase is triacylglycerol acyl hydrolase, its function is to catalyze the hydrolysis of natural substrate oil to generate fatty acid, glycerol and monoglyceride or diester. The basic unit of lipase is only amino acids, usually only one polypeptide chain, and its catalytic activity depends on its protein structure. Natural lipase has poor stability, is easily inactivated, and cannot be reused. After the reaction, it is mixed into the product, causing problems such as difficult separation of the product, low purity, and difficulty in purification. There are still great difficulties in its industrial production. Studies have found that magnetically immobilized lipase has the advantages of recyclability, reusability, good operation performance, easy s...

Claims

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Application Information

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IPC IPC(8): C12N11/10C08L5/08C08K3/22
Inventor 李冰董守利李琳钟南京李晓玺陈玲苏健裕
Owner SOUTH CHINA UNIV OF TECH
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