Method of preparing immobilized lipase

A technology for immobilizing lipase and lipase, which is applied in the direction of immobilization on/in organic carriers, fermentation, etc., to achieve the effects of reducing high cost, increasing the number of repeated recycling and increasing thermal stability

Active Publication Date: 2013-07-31
INNOBIO CORP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Patent US5232843 immobilizes lipase on hydrophobic or ion exchange resin, and then embeds it with sodium caseinate. The e...

Method used

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  • Method of preparing immobilized lipase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] (1) Weigh 10g of macroporous ion exchange resin (Amberlite XAD761), add 10ml of absolute ethanol to soak for 2 hours, filter and discard the absolute ethanol, and rinse the remaining absolute ethanol in the resin with pure water to obtain activated macroporous ions Exchange resin (moisture content 70%);

[0053] (2) Add 20g of liquid lipase (10000U / g) from Pseudoalcaligenes (P.pseudoalcaligenes), add 2.7g of dipotassium hydrogen phosphate, stir and dissolve until completely, and adjust the pH to 8.0 with 10% NaOH aqueous solution ±0.5, dilute to 100g with pure water, then add 100mg of glucose, stir and dissolve to obtain lipase enzyme liquid;

[0054] (3) Preparation of 1% cross-linking agent: 10ml of glutaraldehyde solution (50%) was diluted to 500ml with purified water;

[0055] (4) Add 10 g of the solvent-treated macroporous resin obtained in step (1), 10 ml of the 1% crosslinking agent solution obtained in step (3) and 100 g of lipase enzyme solution obtained in st...

Embodiment 2

[0059] (1) According to the method of embodiment 1 step (1), the activated macroporous ion exchange resin is obtained;

[0060] (2) Add 10g of liquid lipase (10000U / g) from P.pseudoalcaligenes to 10g of 0.02% sodium caseinate protein aqueous solution, stir well, add 2.7g of dipotassium hydrogen phosphate, Stir and dissolve until complete, adjust the pH to 8.0±0.5 with 10% NaOH aqueous solution, dilute to 100g with pure water, add 100mg of glucose, stir and dissolve to obtain lipase enzyme liquid;

[0061] (3) 1% cross-linking agent: add 10ml formaldehyde solution (50%) and dilute to 500ml with purified water.

[0062] (4) Add 10 g of the solvent-treated macroporous resin obtained in step (1), 10 ml of the 0.1% crosslinking agent solution obtained in step (3) and 100 g of lipase enzyme solution obtained in step (2) into the continuous mixer. Stir at 25°C and 120rpm for 12h, filter, discard the filtrate to obtain a solid product;

[0063] (5) Add 50ml of 0.1% sodium caseinate ...

Embodiment 3

[0066] (1) The treatment method of the ion exchange resin (Amberlite XAD7HP) is the same as in Example 1.

[0067] (2) Add 10g of liquid lipase (10000U / g) from C.Antarctica to 10g of 0.02% bovine serum albumin aqueous solution, stir evenly, add 2.7g of dipotassium hydrogen phosphate, stir to dissolve Completely, use 10% NaOH aqueous solution to adjust the pH to 8.0±0.5, dilute to 100g with pure water, add 100mg trehalose, stir and dissolve to obtain lipase enzyme liquid;

[0068] (3) 1% crosslinking agent solution: add 10g polycarbodiimide solution (50%) and dilute to 500ml with purified water;

[0069] (4) Add 10 g of the activated ion exchange resin obtained in step (1), 10 ml of the 1% crosslinking agent solution obtained in step (3) and 100 g of lipase enzyme solution obtained in step (2) into the continuous mixer. Stir at 120 rpm for 12 hours at °C, filter, discard the filtrate to obtain a solid product;

[0070] (5) Add 100ml of 0.3% polyvinyl alcohol aqueous solution ...

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Abstract

The invention provides a method of preparing an immobilized lipase. The method utilizes a coupling immobilization technology, that is a method of combination of adsorpting, crosslinking and embedding, to immobilize the lipase. Application of the coupling technology overcomes disadvantages of easily fell lipase and poor recycling rate caused by an adsorption method; the coupling technology has advantages of high bonding capacity of a crosslinking method and a high enzyme activity recovery of an embedding method, enables the lipase combined with a carrier to firmly immobilize on the carrier, and improves a repetition cyclic utilization rate and an enzyme activity. The immobilized lipase prepared by the technology can be used for esterification, transesterification, and ester hydrolysis, so that a conversion rate can reach more 90 %, a reaction time is shortened, a repetition utilization rate reaches more than 100 times, and an enzyme activity is reduced by less than or equal to 5 %. Therefore, the immobilized lipase has an industrial application prospect.

Description

technical field [0001] The invention relates to a method for preparing immobilized lipase, in particular to a method for immobilizing lipase by using coupling immobilization technology, that is, three combined methods of adsorption, cross-linking and embedding. Background technique [0002] Lipase (Lipase El3.1, 1.3) is an enzyme that hydrolyzes fat. It can hydrolyze natural oils and produce fatty acids and glycerol. Because lipase hydrolyzes fat, it has the advantages of high efficiency, mild reaction conditions, non-toxicity, etc., and has a wide range of applications in the fields of detergent, tanning, food, textile and light industry. As a new type of enzyme preparation, lipase has become an important variety in the world enzyme preparation market. [0003] The liquid lipase hydrolyzes the oil, and the insufficient contact with the substrate leads to incomplete hydrolysis and long reaction time. At the same time, the lipase after use cannot be recycled, which increases...

Claims

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Application Information

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IPC IPC(8): C12N11/10C12N11/08C12N11/04C12N11/02C12P7/62C12P7/64
Inventor 吴文忠杨大苹伏萃翠
Owner INNOBIO CORP LTD
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