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Technique method of immobilized candida antarctica lipase B

A Candida Antarctica and a process method, which are applied in directions such as being immobilized on/in an organic carrier, can solve the problems of large amount of free enzyme, low immobilization efficiency, large amount of enzyme, etc., and achieve simple preparation process and immobilization. High chemical efficiency and small amount of free enzyme

Inactive Publication Date: 2009-11-11
HEBEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Water phase adsorption has the following disadvantages: the amount of enzyme used in the immobilization process is large, the immobilization efficiency is low, and most of the enzymes still exist in the buffer and cannot be effectively combined with the carrier, which increases the cost of fixing, and the cost to itself is higher enzymes are not applicable
The above relevant literature only uses CALB as a model enzyme to investigate the ability of the new carrier to carry the enzyme and the influence of the carrier properties or structural characteristics on the immobilization. The immobilization methods used involve aqueous phase adsorption, cross-linking and covalent bonding. The limitations of the method are: the amount of free enzyme in the aqueous phase adsorption method is large, and the binding rate between the enzyme and the carrier is low, and most of the free enzyme still exists in the supernatant; The combination of enzymes depends on chemical reactions and has a great influence on the activity of enzymes. Therefore, it is very important to find a method with high immobilization efficiency and little influence on enzyme activities.
So far, there are no relevant reports in the literature on the immobilization of CALB in organic solvents.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0026] Soak resin S-8 in 95% ethanol by volume percentage for 24h, take it out and rinse it with distilled water; then soak it in 2% NaOH (mass percentage) solution for 4h, take it out and wash with distilled water until it is neutral ; Then soak in 5% HCL (mass percentage concentration) solution for 4 hours, wash with distilled water to neutral, and then dry at 40 ℃ for 15 hours;

[0027] Weigh 60 mg of the pretreated resin S-8 into a 50 mL conical flask with a cork, and add 5 mL of n-hexane to equilibrate for 1 hour to fully swell the carrier. Then add 50μL of zymogen solution (1mg lipase CALB is dissolved in 50μL of phosphate buffer with pH 7.0). After vortex mixer shakes evenly, the bottle mouth is sealed with tetrafluoroethylene sealing tape and placed on a constant temperature water bath shaker. Adsorb and fix for 2h at a temperature of 25℃ and a shaker speed of 100rpm, then decanted the upper layer of n-hexane, and freeze the remaining solid under vacuum (absolute vacuum of...

example 2

[0030] The pretreatment method of resin AB-8 is the same as in Example 1;

[0031] Weigh 80 mg of the pretreated resin AB-8 into a 50 mL conical flask with a cork, and add 5 mL of heptane to equilibrate for 1 hour to fully swell the carrier. Then add 50μL of zymogen solution (1mg lipase CALB is dissolved in 50μL of phosphate buffer with pH 5.0), vortex mixer shakes evenly, tetrafluoroethylene sealing tape seals the bottle mouth and places it in a constant temperature water bath shaker , Adsorb and fix for 2.5h at a temperature of 30°C and a shaker speed of 100rpm, then decanted the upper layer of heptane, and freeze-dry the remaining solid to obtain the immobilized enzyme. The olive oil emulsification method was used to determine the immobilized enzyme activity, and the recovery rate of the enzyme activity was 79.2%.

[0032] The phosphate buffer with pH 5.0 is 0.025mol / L KH 2 PO 4 Solution and 0.025mol / L Na 2 HPO 4 The solution is mixed and prepared according to the volume ratio ...

example 3

[0034] The pretreatment method of resin NKA-9 is the same as in Example 1;

[0035] Weigh 80 mg of the pretreated resin NKA-9 into a 50 mL conical flask with a cork, and add 5 mL of isooctane to equilibrate for 1 hour to fully swell the carrier. Then add 75μL zymogen solution (1mg lipase CALB is dissolved in 75μL phosphate buffer with pH 6.0), vortex mixer shakes evenly, tetrafluoroethylene sealing tape seals the bottle mouth and places it in a constant temperature water bath shaker , Adsorb and fix for 2h at a temperature of 30°C and a shaker speed of 100rpm, then decanted the upper layer of isooctane, and freeze-dry the remaining solid to obtain the immobilized enzyme. The activity of the immobilized enzyme was determined by the olive oil emulsification method, and the recovery rate of the enzyme activity was 83.3%.

[0036] The phosphate buffer with a pH of 6.0 is 0.025mol / L KH 2 PO 4 Solution and 0.025mol / L Na 2 HPO 4 The solution is mixed and prepared in a ratio of 9:1 by vol...

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Abstract

The invention relates to a technique method of immobilized candida antarctica lipase B, which adopts an organic solvent as an immobilizing medium and realizes the immobilization of lipase in microaqueous phase. The technique method comprises the following steps of: (1) pretreatment of resin; (2) preparation of immobilized enzyme: the pretreated resin is added into a reactor and an organic medium is added; and after standing, then zymogen solution is added according to the mass ratio of the candida antarctica lipase B and the resin, which is equal to 1:60-1:100; the reactor is sealed and then arranged in a shaking bath with constant temperature of 25-35 DEG C for 1h-3h; after vacuum freezing and drying, the immobilized candida antarctica lipase B is prepared. The method for preparing the immobilized enzyme has low consumption of resolvase and high immobilization efficiency and avoids the considerable waste of resolvase caused by an aqueous-phase absorption method, thereby reducing immobilization cost; a carrier has low price and is easily obtained; furthermore, the method has simple technique, mild condition, low loss in enzyme activity and enzyme activity recovery rate of 83.3 percent.

Description

Technical field [0001] The invention belongs to the field of immobilized enzymes, in particular to a process method for immobilizing Candida antarctica lipase B. Background technique [0002] Enzyme immobilization is to combine the original water-soluble enzyme with a solid water-insoluble support or be embedded in a carrier through chemical or physical treatment methods. Enzymes confined to a certain area of ​​the space are beneficial to improve the stability of the enzyme and can be recycled and reused, which is convenient for continuous production, so it is widely used. After immobilization, the enzyme has more advantages than the original water-soluble enzyme: (1) It is very easy to separate the immobilized enzyme from the substrate and product; (2) It can carry out repeated batch reactions and continuous packing in a long time. Reaction; (3) in most cases, it can improve the stability of the enzyme; (4) the enzyme reaction process can be strictly controlled; (5) there is no ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/02
Inventor 高静孙江娜
Owner HEBEI UNIV OF TECH
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