Immobilized enzyme for oriented catalysis of esterification reaction and preparation method and application of immobilized enzyme

A technology for catalyzing esterification and reaction, applied in biochemical equipment and methods, immobilized on/in organic carriers, enzymes, etc., can solve the problems such as inability to recycle and reuse, difficulty in lipase, difficulty in separation, etc. The method is simple, the enzyme activity is high, and the reusability is good.

Pending Publication Date: 2020-07-10
GUANGDONG PHARMA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are many problems in free lipase, including poor stability, easy inactivation, inability to recycle and reuse, and mixing into products after reaction, causing separation difficulties, etc. Therefore, there are still great difficulties in the industrial application of lipase.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] (1) Preparation of HEPES buffer solution: Weigh 2.0g HEPES and dissolve in water, adjust the pH to 6.5, dilute to 25ml, and store at low temperature;

[0027] (2) Preparation of HEPES-lipase solution: Dissolve 100 mg of Candida antarctica lipase B in the above (1) buffer, and store at low temperature;

[0028] (3) Preparation of AMP mother liquor: Weigh 0.2g AMP and dissolve in water, dilute to 10ml, and store at low temperature;

[0029] (4)Cu 2+ Preparation of mother liquor: Weigh 0.16g of anhydrous copper chloride, dissolve and set the volume to 10ml, and store at low temperature;

[0030] (5) Preparation of immobilized enzyme: Mix 0.4ml HEPES-lipase solution with 1.0ml AMP solution, then add 1.2ml Cu 2+ Mother liquor mixed. Stand still, centrifuge, discard the supernatant, and freeze-dry to obtain the immobilized enzyme.

[0031] (6) Apply the obtained immobilized enzyme to the esterification reaction of glycerol and oleic acid. The reaction conditions are: 0.46...

Embodiment 2

[0034] (1) Preparation of HEPES buffer solution: Weigh 3.0g HEPES and dissolve in water, adjust the pH to 7.3, dilute to 40ml, and store at low temperature;

[0035] (2) Preparation of HEPES-lipase solution: take 200 mg of Candida antarctica lipase B and dissolve it in the buffer solution of (1) above, and store at low temperature;

[0036] (3) Preparation of GMP mother liquor: Weigh 0.3g GMP and dissolve in water, dilute to 10ml, and store at low temperature;

[0037] (4)Tb 3+ Preparation of mother liquor: Weigh 0.37g of terbium chloride hexahydrate, dissolve and set the volume to 10ml, and store at low temperature;

[0038] (5) Preparation of immobilized enzyme: Take 0.6ml HEPES-lipase solution and mix with 1.0ml GMP solution, then add 1.0ml Tb 3+ Mother liquor mixed. Stand still, centrifuge, discard the supernatant, and freeze-dry to obtain the immobilized enzyme.

[0039] (6) Apply the obtained immobilized enzyme to the esterification reaction of glycerol and oleic aci...

Embodiment 3

[0042] (1) Preparation of HEPES buffer solution: Weigh 5.0g HEPES and dissolve in water, adjust the pH to 8.0, set the volume to 50ml, and store at low temperature;

[0043] (2) Preparation of HEPES-lipase solution: 300 mg of Candida antarctica lipase A was dissolved in the above (1) buffer, and stored at low temperature;

[0044](3) Preparation of GMP mother liquor: Weigh 0.3g AMP and dissolve it in water, dilute to 10ml, and store at low temperature;

[0045] (4)Zn 2+ Preparation of mother liquor: Weigh 0.15g of anhydrous zinc chloride, dissolve in 10ml, and store at low temperature;

[0046] (5) Preparation of immobilized enzyme: Mix 0.5ml HEPES-lipase solution with 1.0ml GMP solution, then add 1.2ml Zn 2+ Mother liquor mixed. Stand still, centrifuge, discard the supernatant, and freeze-dry to obtain the immobilized enzyme.

[0047] (6) Apply the obtained immobilized enzyme to the esterification reaction of glycerol and oleic acid. The reaction conditions are: 0.4605 g ...

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Abstract

The invention belongs to the technical field of biochemical engineering and in particular discloses an immobilized enzyme for oriented catalysis of an esterification reaction and a preparation methodand application of the immobilized enzyme. The method comprises the following steps: mixing candida antarctica lipase with an HEPES (hydroxyethyl piperazin ethanesulfonic) buffer so as to obtain an HEPES-lipase liquid; and mixing the obtained HEPES-lipase liquid with GMP (guanine nucleotide) or AMP (adenine nucleotide) mother liquor so as to obtain a mixed solution, further uniformly mixing the mixed solution with metal ions, performing filtering, and performing freeze drying, so as to obtain the immobilized enzyme. The immobilized enzyme preparation method disclosed by the invention is simple, and the obtained immobilized enzyme is high in enzyme activity, is capable of effectively catalyzing the esterification reaction, in addition, is good in repeated utilization, but cannot catalyze alcoholysis reactions, has an oriented catalysis property, that is, is capable of catalyzing the esterification reaction but not alcoholysis reactions, and thus has very good application prospects.

Description

technical field [0001] The invention belongs to the technical field of biochemical industry, and in particular relates to an immobilized enzyme for directional catalyzing esterification reaction and its preparation method and application. Background technique [0002] Lipase (lipase, E.C.3.1.1.3) is a triacylglyceride hydrolase, whose function is to catalyze the hydrolysis of natural oils to produce diglycerides, monoglycerides, free fatty acids and glycerol, and completely hydrolyze oils to obtain free fatty acids and glycerol . In addition to catalyzing hydrolysis, lipase can also catalyze other reactions, including esterification, transesterification and alcoholysis. These properties of lipase make it widely used in food, chemical, pharmaceutical and other industries. However, there are many problems in free lipase, including poor stability, easy inactivation, inability to recycle and reuse, and mixing into products after reaction to cause separation difficulties, etc. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/02C12N9/20
CPCC12N11/02C12N9/20C12Y301/01003
Inventor 钟南京陈文艺
Owner GUANGDONG PHARMA UNIV
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