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Oriented immobilization method of flavin enzymes

A technology of flavinase and flavin adenine, which is applied in the field of directional immobilization of flavinase, can solve the problem of decreased activity of immobilized flavinase, and achieve the effects of retaining biological activity, realizing directional adsorption, and efficient directional adsorption

Inactive Publication Date: 2016-04-27
ZHEJIANG SCI-TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the structure of the immobilized flavinase obtained by the traditional flavoenzyme immobilization method is that the flavoenzyme is connected to the carrier at any position. a sharp drop

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] (1) 10 mg of graphene oxide (GO) was ultrasonically dispersed in 10 mL of pure water for 30 min to prepare a 1 mg / mL GO suspension. Weigh 500mgNaOH and 500mgClCH 2 COONa was added into the GO suspension, ultrasonically reacted for 2 h, and the obtained product was neutralized with dilute HCl. Repeat washing with deionized water and centrifugation (10000rpm, 10min) until the product is well dispersed in deionized water. Finally, use a dialysis bag in deionized water, dialyze (molecular weight cut off: 8000-10000) for 48 hours to remove ions, and dilute the obtained GO-COOH suspension to 10 mL.

[0020] (2) Take 1 mL of GO-COOH solution to adjust the pH to 5.2, add 0.287g EDC (0.15mM) and 0.115g NHS (0.45mM), stir for 30min, and centrifuge (10000rpm, 5min) for three times to wash off unreacted EDC and NHS; re-dissolve the activated GO-COOH in buffer solution (PBS, pH7.4), sonicate for 5 minutes to make it completely dispersed; add and weigh 30 mg of flavin adenine dinuc...

Embodiment 2

[0024] (1) 15 mg of carbon nanotubes were ultrasonically dispersed in 10 mL of pure water for 30 min to prepare a carbon nanotube suspension. Weigh 300mgNaOH and 300mgClCH 2 COONa was added to the carbon nanotube suspension, and the ultrasonic reaction was performed for 2 h, and the obtained product was neutralized with dilute HCl. Washing with deionized water and centrifugation (10000 rpm, 10 min) were repeated until the product was well dispersed in deionized water to obtain a suspension of carboxylated carbon nanotubes.

[0025] (2) Take 1 mL of carboxylated carbon nanotube suspension and adjust the pH to 5.2, add 0.287g EDC (0.15mM) and 0.115g NHS (0.45mM), stir for 30min, centrifuge (10000rpm, 5min) for three times, wash away Reactive EDC and NHS; re-add the activated carboxylated carbon nanotubes into the buffer solution (PBS, pH 7.4), sonicate for 5 minutes to make them completely dispersed; add 5 mg of flavin adenine dinucleotide (FAD), stir React overnight, centrifu...

Embodiment 3

[0029] (1) 15 mg of graphene oxide was ultrasonically dispersed in 10 mL of pure water for 30 min to prepare a graphene oxide suspension. Weigh 300mgNaOH and 300mgClCH 2 COONa was added to the graphene oxide suspension, ultrasonically reacted for 2 h, and the obtained product was neutralized with dilute HCl. Washing with deionized water and centrifugation (10000rpm, 10min) were repeated until the product was well dispersed in deionized water to obtain a suspension of carboxylated graphene oxide.

[0030] (2) Take 1 mL of carboxylated graphene oxide suspension and adjust the pH to 5.2, add 0.287g EDC (0.15mM) and 0.115g NHS (0.45mM), stir for 30min, centrifuge (10000rpm, 5min) for three times, wash away Reactive EDC and NHS; re-add the activated carboxylated graphene oxide into the buffer solution (PBS, pH 7.4), sonicate for 5 minutes to make it completely dispersed; add 5 mg of flavin adenine dinucleotide (FAD), stir React overnight, centrifuge, and dry in a vacuum oven at 4...

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PUM

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Abstract

The invention discloses an oriented immobilization method of flavin enzymes. According to the method, oriented immobilization flavin enzymes are built by using the biological affinity effect between prosthetic group FAD (Flavin Adenine Dinucleotide) and apo flavin enzymes. The oriented immobilization method has the main advantages that high orientation of the flavin enzymes is realized; the active center electron transmission is facilitated; the activity of the oriented affinity effect immobilized enzymes is obviously improved through being compared with the activity of common adsorption method immobilized enzymes; and a feasible method is provided for further building a high-efficiency and low-cost biological catalysis system.

Description

technical field [0001] The invention relates to a method for directional immobilization of flavin enzymes, belonging to the field of protein immobilization. Background technique [0002] Flavinase (flavinenzymes), also known as flavin protein. It is a group of redox enzymes with flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) as coenzyme (electron donor). In 1932, O. Warburg and W. Chr-istian isolated flavoenase from yeast and found that it was combined with FMN as its prosthetic group. Subsequently, in 1938, the coenzyme of D-amino acid oxidase was isolated, and it was clarified that the coenzyme was FAD. Electron acceptor oxygen, coenzyme Q, cytochrome, etc. vary by enzyme. Most flavin enzymes have a strong binding force with coenzymes, and the oxidized type has the maximum absorption near 380 and 450 nanometers, while the reduced type absorption disappears. Fluorescence is also weaker than riboflavin. There are many kinds of flavoenzymes or flavoprot...

Claims

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Application Information

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IPC IPC(8): C12N11/14
CPCC12N11/14
Inventor 叶鹏许永娟
Owner ZHEJIANG SCI-TECH UNIV
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