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Microwave-assisted co-immobilization method of aldehyde ketone reductase and glucose dehydrogenase

A technology of glucose dehydrogenase and aldehyde-ketone reductase, which is applied to the multi-enzyme system immobilized on or in an inorganic carrier, can solve the problems of poor stability of free enzymes and cannot be reused, and achieve pH stability improvement, The effect of strengthening the force and reducing the production cost

Active Publication Date: 2015-10-21
HANGZHOU NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to overcome the shortcomings of free enzymes such as poor stability and non-recyclability, the present invention provides a simple and fast covalent immobilization method of aldehyde ketone reductase and glucose dehydrogenase dual enzymes, p-benzoquinone as a cross-linking agent through With the nucleophilic group (such as amino or hydroxyl) on the enzyme and MCFs-NH 2 Amino reaction on the carrier to covalently immobilize the enzyme on the carrier, and finally obtain the co-immobilized enzyme with improved performance

Method used

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

Examples

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Embodiment 1

[0034] Example 1 Preparation of aldehyde and ketone reductase

[0035] 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.

[0036] 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 15mi...

Embodiment 2

[0039] Example 2 Preparation of glucose dehydrogenase

[0040] 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.

[0041] 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, wa...

Embodiment 3

[0044] Example 3 Making double enzymes into co-immobilized enzymes

[0045] (1) Activation of the carrier: 20mgMCFs-NH 2 Dissolve the powder in 3mL of 1.0mM p-quinone solution (the solvent is 50% ethanol aqueous solution), at 25°C and 160rpm, vibrate on a constant temperature water bath shaker for 2h, centrifuge, and take the precipitate with 3mL, pH 7.0, 0.1M Wash with PBS solution, centrifuge, and redisperse the pellet in 2.42mL, pH 7.0, 0.1M PBS solution. According to the mass ratio of 100mg enzyme / g carrier, add 0.26mL of the 7.7mg / mL aldehyde and ketone reductase solution obtained in Example 1 and 0.32 mL of the 6.25mg / mL glucose dehydrogenase solution obtained in Example 2 to the mixed solution successively. mL, mix well;

[0046] (2) Dual-enzyme co-immobilization: take the mixed solution of step (1) and irradiate it under 30W microwave conditions for 4 minutes at 0-10°C, centrifuge, and wash the precipitate with pH 7.0, 0.1M PBS solution to obtain the co-immobilized e...

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Abstract

The invention discloses a microwave-assisted co-immobilization method of aldehyde ketone reductase and glucose dehydrogenase. The method includes the steps that a carrier and a crosslinking agent are mixed and activated, centrifuging is conduced, sediments are taken and washed through a PBS solution, centrifuging is conducted, and the sediments are re-dispersed in the PBS solution; the aldehyde ketone reductase and the glucose dehydrogenase are added to a dispersed solution, the mixed liquid is irradiated at the temperature of 0 DEG C to 10 DEG C under the 20-45 W microwave condition for 1 min to 5 min, centrifuging is conducted, sediments are taken and washed through the PBS solution, and co-immobilized enzyme is obtained; compared with free enzyme, the catalytic activity, heat stability and PH stability of the co-immobilized enzyme prepared through the method are improved; the glucose dehydrogenase serves as a coenzyme regeneration system of the glucose dehydrogenase to provide NAD(P)H required by a reaction, the cost is lowered, and industrial production is facilitated.

Description

(1) Technical field [0001] The invention relates to a microwave-assisted 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. Ba...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/18C12N11/14
Inventor 谢恬殷晓浦马转转谌容庞潇卿
Owner HANGZHOU NORMAL UNIVERSITY
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