Technique method of immobilized candida antarctica lipase B

A Candida Antarctica and a process method, which are applied in directions such as being fixed on/in an organic carrier, can solve the problems of large amount of free enzyme, increase fixed cost, low immobilization efficiency, etc., and achieve simple preparation process and avoidance of A large amount of waste, the effect of high immobilization efficiency

Inactive Publication Date: 2011-07-20
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 for 24 hours, rinse it with distilled water after taking it out; then soak it in 2% NaOH (mass percent concentration) solution for 4 hours, take it out and wash it with distilled water until neutral ; Soak in 5% HCL (mass percent concentration) solution for 4 hours, wash with distilled water until neutral, and then dry at 40°C for 15 hours;

[0027] Weigh 60mg of the pretreated resin S-8 into a 50mL Erlenmeyer flask with a ground stopper, add 5mL of n-hexane to balance for 1h, so that the carrier is fully swollen. Then add 50 μL zymogen solution (1 mg lipase CALB was dissolved in 50 μL, pH 7.0 phosphate buffer solution), after vortex mixer shakes evenly, tetrafluoroethylene sealing tape seals the bottle mouth and places it in a constant temperature water bath shaker In the process, at a temperature of 25°C and a shaker speed of 100rpm, it was adsorbed and fixed for 2h, then the upper layer of n-hexane was decanted, and the remaini...

example 2

[0030] The pretreatment method of resin AB-8 is with example 1;

[0031] Weigh 80mg of the pretreated resin AB-8 into a 50mL Erlenmeyer flask with a ground stopper, add 5mL of heptane to balance for 1 hour, so that the carrier is fully swollen. Then add 50 μL zymogen solution (1 mg lipase CALB was dissolved in 50 μL, pH 5.0 phosphate buffer solution), vortex mixer shakes evenly, and tetrafluoroethylene sealing tape seals the bottle mouth and places it in a constant temperature water bath shaker , at a temperature of 30° C. and a shaking table with a rotational speed of 100 rpm for 2.5 h, then decanted the upper layer of heptane, and vacuum freeze-dried 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 79.2%.

[0032] Said pH is that the phosphate buffer of 5.0 is the KH of 0.025mol / L 2 PO 4 solution with 0.025mol / L Na 2 HPO 4 T...

example 3

[0034] The pretreatment method of resin NKA-9 is with example 1;

[0035] Weigh 80 mg of the pretreated resin NKA-9 into a 50 mL Erlenmeyer flask with a ground stopper and add 5 mL of isooctane to balance for 1 hour to fully swell the carrier. Then add 75 μL zymogen solution (1 mg lipase CALB was dissolved in 75 μL, pH 6.0 phosphate buffer to prepare), vortex mixer shakes evenly, tetrafluoroethylene sealing tape seals the bottle mouth and places it in a constant temperature water bath shaker , at a temperature of 30° C. and a shaking table with a rotational speed of 100 rpm for 2 h, then decant the upper layer of isooctane, and vacuum 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] Said pH is that the phosphate buffer of 6.0 is the KH of 0.025mol / L 2 PO 4 solution with 0.025mol / L Na 2 HPO 4 The solu...

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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 for immobilizing Candida antarctica lipase B. Background technique [0002] Enzyme immobilization is to combine the original water-soluble enzyme with a solid non-water-soluble support or be embedded by a carrier by chemical or physical treatment. Enzymes confined to a certain area of ​​space are conducive to improving 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 perform repeated batch reactions and continuous column packing for a long time (3) in most cases, the stability of the enzyme can be improved; (4) the enzyme reaction process can be strictly controlled; (5) there is no enzyme residue in t...

Claims

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Application Information

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