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Cross-linked enzyme aggregate prepared in water-in-oil emulsion and preparation method thereof

A technology of water-in-oil emulsion and enzyme aggregates, applied in the field of biochemical industry, can solve the problems of affecting the activity of cross-linked enzyme aggregates, not easy to disperse, large in size, etc. High enzymatic activity

Inactive Publication Date: 2012-01-04
天津市林业果树研究所 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the method established by the Sheldon group is to construct cross-linked enzyme aggregates in the solution. The obtained cross-linked enzyme aggregates form clusters, which are not easy to disperse. They are amorphous clusters, large in size, and have obvious mass transfer problems. Affect the full activity of cross-linked enzyme aggregates

Method used

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  • Cross-linked enzyme aggregate prepared in water-in-oil emulsion and preparation method thereof
  • Cross-linked enzyme aggregate prepared in water-in-oil emulsion and preparation method thereof
  • Cross-linked enzyme aggregate prepared in water-in-oil emulsion and preparation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1: the selection of precipitation agent

[0043] Prepare trehalose synthase solution with 50mmol / L, pH7.0 phosphate buffer solution, take 6 parts of the trehalose synthase solution, each 1mL, add saturated ammonium sulfate, PEG6000 with a concentration of 30%, isopropanol, 9 mL each of n-propanol, acetone and ethanol, carry out aggregation and precipitation, regularly sample and centrifuge, measure the protein content of the supernatant until about 90% of the protein is precipitated, centrifuge to collect the precipitated enzyme aggregates, and wash the enzyme aggregates with phosphate buffer solution After redissolving, the enzyme activity was measured, and the recovery rate of the enzyme activity was calculated. The results are shown in Table 1.

[0044] Table 1 Effect of precipitant on enzyme activity

[0045]

[0046] It can be seen from Table 1 that using PEG6000 as the precipitating agent has little effect on the activity of trehalose synthase, so P...

Embodiment 2

[0050] Example 2: Preparation of cross-linked enzyme aggregates in water-in-oil emulsion

[0051] Step 1: Prepare trehalose synthase solution with 50mmol / L, pH7.0 phosphate buffer;

[0052] Step 2: Take 10mL of trehalose synthase solution, gradually add 3g of PEG6000, stir or oscillate, after PEG6000 dissolves, put it in a shaker at 25°C and 150r / min, and the mixture obtained after 30min is used as the dispersed phase containing enzyme;

[0053] Step 3: Add 2 mL of 25% glutaraldehyde to the enzyme-containing dispersed phase obtained in Step 2, then add 50 mL of mineral oil, and emulsify with a homogeneous emulsifier at 10,000 r / min for 1 min to form a water-in-oil emulsion;

[0054] Step 4: Stir the water-in-oil emulsion formed in Step 3 at 25°C and 1000r / min for 1h;

[0055] Step 5: After stirring, the emulsion was centrifuged at 10,000 r / min for 10 min, and the precipitate was recovered to obtain cross-linked enzyme aggregates.

Embodiment 3

[0056] Embodiment 3: add emulsifier

[0057] Step 1: Prepare trehalose synthase solution with 50mmol / L, pH7.0 phosphate buffer;

[0058] Step 2: Take 1mL of trehalose synthase solution, gradually add 0.25g of PEG6000, stir or oscillate to dissolve and mix the PEG6000, and the resulting mixture is used as the dispersed phase containing the enzyme;

[0059] Step 3: Add 0.2 g of Span60 to 20 mL of soybean oil, mix well, add it to the dispersed phase containing enzyme obtained in Step 2, stir and emulsify at 1500 r / min for 1 min, and form a water-in-oil emulsion;

[0060] Step 4: Add 20 μL of 25% glutaraldehyde to the water-in-oil emulsion formed in step 3, stir at 1500 r / min for 5 minutes, and then stand at 25°C for 2 hours for cross-linking;

[0061] Step 5: After cross-linking, the emulsion was centrifuged at 3000 r / min for 20 min, and the obtained precipitate was centrifuged and washed 3 times with 10 mmol / L, pH 7.0 phosphate buffer to obtain cross-linked enzyme aggregates. ...

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Abstract

The invention discloses a method for preparing a cross-linked enzyme aggregate prepared in water-in-oil emulsion, which comprises the following steps: (1) preparing enzyme solution; (2) adding a precipitator capable of aggregating and precipitating enzyme in enzyme solution, and preparing an enzyme-containing dispersion phase; (3) dispersing the enzyme-containing dispersion in hydrophobic liquid to form water-in-oil emulsion; (4) cross-linking the aggregate in the water-in-oil emulsion by using a cross-linking agent; (5) collecting the cross-linked enzyme aggregate; and the like. The cross-linked enzyme aggregate prepared by the method is carrier-free immobilized enzyme, the unit enzyme activity of the cross-linked enzyme aggregate is high, and the cross-linked enzyme aggregate can be used in corresponding enzymatic reactions.

Description

technical field [0001] The invention belongs to the field of biochemical industry, and relates to a cross-linked enzyme aggregate prepared in a water-in-oil emulsion and a preparation method thereof. Background technique [0002] Compared with free enzymes, immobilized enzymes have high stability, are easy to separate from the reaction system, can be reused, and are conducive to automatic production. The immobilized enzymes with excellent performance can realize the efficient utilization of enzymes in industrial environments and promote the development of the biocatalysis industry. Rapid development is of great significance. [0003] The enzyme immobilization method can be divided into carrier immobilization method and carrier-free immobilization method according to whether a carrier is needed. The carrier immobilization method "dilutes" the activity of the enzyme due to the introduction of the inert carrier (in the weight of the entire immobilized enzyme, the carrier gener...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00
Inventor 张峻陈颖陈晓云唐柳齐欣杨丽维李明春邢来君王宇凡
Owner 天津市林业果树研究所
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