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Embedding and co-immobilization method of aldehyde ketone reductase and glucose dehydrogenase

A technology of glucose dehydrogenase and aldehyde and ketone reductase, which is applied to the direction of multi-enzyme system fixed on/in it, can solve the problems of non-reusable and poor stability of free enzymes, and achieve the reduction of repeated use, Effects of property improvement and catalytic activity improvement

Active Publication Date: 2015-10-21
HANGZHOU NORMAL UNIVERSITY
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

In order to overcome the shortcomings of free enzymes such as poor stability and non-recyclability, the present invention provides a simple and fast method for embedding and immobilizing aldehyde ketone reductase and glucose dehydrogenase dual enzymes. Sodium alginate is used as a commonly used porous Carrier, mix it with the enzyme solution and drop it into a certain concentration of CaCl 2 In solution, the Na of sodium alginate + partly covered by Ca 2+ Replacement, forming gel beads, and finally obtaining co-immobilized enzymes with improved performance by optimizing the immobilization conditions

Method used

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  • Embedding and co-immobilization method of aldehyde ketone reductase and glucose dehydrogenase
  • Embedding and co-immobilization method of aldehyde ketone reductase and glucose dehydrogenase
  • Embedding and co-immobilization method of aldehyde ketone reductase and glucose dehydrogenase

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Experimental program
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Effect test

Embodiment 1

[0036] Embodiment 1 prepares aldehyde and ketone reductase

[0037] The nucleotide sequence shown in SEQ ID NO.1 was ligated with the vector pET28a, the ligated product was transformed into E.coli DH5α competent cells, and a single colony was picked for PCR verification to obtain positive clones, and the pET28a-Lek plasmid was obtained using a plasmid extraction kit. Then the recombinant plasmid was introduced into E.coli BL21(DE3) to obtain pET28a-Lek-E.coli BL21(DE3) strain.

[0038] The pET28a-Lek-E.coli BL21(DE3) strain was inoculated in LB liquid medium and cultivated overnight at 37°C. Transfer the bacterial solution to 1L of LB medium containing kanamycin (100 μg / mL) according to the volume concentration of 1% inoculum, culture at 37 ° C and 200 rpm until the OD600 reaches 0.6, and add IPTG to the culture solution to make it final The concentration was 0.1 mM, and the expression was induced for 12-16 hours at 25°C and 150 rpm. Centrifuge at 4°C and 8000rpm for 15min, ...

Embodiment 2

[0041] Embodiment 2 prepares glucose dehydrogenase

[0042] The nucleotide sequence shown in SEQ ID NO.2 was ligated with the vector pET22b, and the ligated product was transformed into E.coli DH5α competent cells, and a single colony was picked for PCR verification to obtain positive clones, and the pET22b-GDH104 plasmid was obtained using a plasmid extraction kit. Then the recombinant plasmid was introduced into E.coli BL21(DE3) to obtain pET22b-GDH104-E.coli BL21(DE3) strain.

[0043] The successfully constructed pET22b-GDH104-E.coli BL21(DE3) strain was inoculated in liquid LB medium containing ampicillin (100 μg / mL), and cultured overnight at 37° C. and 220 rpm. Take 10 mL of the bacterial liquid and transfer it to 1 L of LB medium, shake and culture at 37°C, 200rpm until the OD600 reaches 0.6, add IPTG to the culture medium to make the final concentration 0.1mM, and induce expression at 25°C, 150rpm for 12-16h. The bacterial cells were collected by centrifugation, washe...

Embodiment 3

[0046] Embodiment 3 makes co-immobilized enzyme with free double enzyme

[0047] Mix 0.04ml of the 7.7mg / mL aldehyde and ketone reductase solution obtained in Example 1 and 0.06ml of the 6.25mg / mL glucose dehydrogenase solution obtained in Example 2, and add 1mL of 3% ketone reductase solution to the mixed solution. Sodium alginate aqueous solution (the total weight of aldehyde and ketone reductase and glucose dehydrogenase is 0.683 mg / ml based on the volume of sodium alginate aqueous solution), stir well to mix the double enzyme solution and sodium alginate aqueous solution evenly, and draw the mixed solution slowly into the syringe Into 20ml mass concentration 2% CaCl 2 In the aqueous solution, stir continuously during the dropwise addition process. After the end, let it stand in a refrigerator at 4°C for 2 hours, wash with distilled water 2-3 times, and dry it with a vacuum pump to obtain immobilized gel particles, that is, co-immobilized enzymes.

[0048] Table 1 Co-immob...

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Abstract

The invention discloses an embedding and co-immobilization method of aldehyde ketone reductase and glucose dehydrogenase. The method includes the steps that the aldehyde ketone reductase and the glucose dehydrogenase are mixed and then added to a sodium alginate aqueous solution to be stirred and mixed evenly, then the mixture is dropwise added to a Cacl2 aqueous solution, the mixture is made to stand in a refrigerator at the temperature of 4 DEG C, washing is conducted through distilled water, vacuum filtration is conducted, and immobilized particles are obtained. Compared with free aldehyde ketone reductase, the catalytic activity of the co-immobilized enzyme prepared through the method is increased by 1.32 times, the heat stability, the PH stability and other performance of the co-immobilized enzyme prepared through the method are improved as well, and meanwhile, the cost is lowered due to reuse of the co-immobilized enzyme.

Description

(1) Technical field [0001] The invention relates to an embedding and co-immobilization method of aldehyde and ketone reductase and glucose dehydrogenase dual enzymes. (2) Background technology [0002] As a kind of biocatalyst, enzyme is widely used in medicine, food and chemical industry because of its strong specificity, high catalytic efficiency and mild action conditions. However, the stability of natural enzymes is poor. Except for some enzymes resistant to high temperature and a few enzymes that can tolerate lower pH conditions, most enzymes are easily denatured and inactivated under high temperature, strong acid, and strong alkali conditions; after the reaction, the enzyme is mixed with the product , cannot be reused, and it is difficult to separate and purify the product; at the same time, the one-time use of the enzyme greatly increases the production cost, is not conducive to continuous production, and limits the wider application of the enzyme in industry. Based ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/18C12N11/10C12N11/04
Inventor 谢恬殷晓浦马转转谌容庞潇卿
Owner HANGZHOU NORMAL UNIVERSITY
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