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Method for regeneration of oxidized coenzyme I using whole cell biotransformation

An oxidized coenzyme and coenzyme technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of large molecular weight and difficulty, and achieve the effect of improving conversion efficiency

Inactive Publication Date: 2012-02-15
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because NADH is a charged molecule with a large molecular weight (709.4Da), it is usually difficult to enter the cell for reaction

Method used

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  • Method for regeneration of oxidized coenzyme I using whole cell biotransformation
  • Method for regeneration of oxidized coenzyme I using whole cell biotransformation
  • Method for regeneration of oxidized coenzyme I using whole cell biotransformation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] Example 1. Enterobacter aerogenes converts NADH into NAD +

[0013] 1) Cultivation of Enterobacter aerogenes

[0014] Enterobacter aerogenes (Enterobacter aerogenes) IAM1183 is connected to the culture medium (glucose 15g / L, peptone 5g / L, K 2 HPO 4 14g / L, KH 2 PO 4 6g / L, (NH 4 ) 2 SO 4 2g / L, MgSO 4 ·7H 2 O0.2g / L), 37°C, 170rpm air bath shaker culture.

[0015] Take 60 ml of Enterobacter aerogenes IAM1183 cultured in aerobic shake flask for 12 hours, centrifuge at 10,200×g for 5 minutes at 4°C, and collect Enterobacter aerogenes bacteria.

[0016] 2) Convert NADH to NAD +

[0017] Wash the Enterobacter aerogenes cells in step 1) with 50mM triethanolamine buffer (TEA) pH 6.80 three times, then resuspend in 50mM triethanolamine buffer (TEA) pH 6.80, adjust the cell concentration to OD 600 It was 0.8, NADH was added to make the final concentration of NADH 210μM, 37°C, 170rpm culture, as the experimental group.

[0018] Wash the Enterobacter aerogenes cells in step 1) with 50mM trietha...

Embodiment 2

[0037] Example 2: Enterobacter aerogenes converts NADH into NAD +

[0038] 1) Cultivation of Enterobacter aerogenes

[0039] Enterobacter aerogenes IAM1183 is connected to the culture medium (glucose 15g / L, peptone 5g / L, K 2 HPO 4 14g / L, KH 2 PO 4 6g / L, (NH 4 ) 2 SO 4 2g / L, MgSO 4 ·7H 2 00.2g / L), 37°C, 170rpm air bath shaker culture.

[0040] Take 60 ml of Enterobacter aerogenes IAM1183 cultured in aerobic shake flask for 12 hours, centrifuge at 10,200×g for 5 min at 4°C, and collect Enterobacter aerogenes bacteria.

[0041] 2) Convert NADH to NAD +

[0042] Wash the Enterobacter aerogenes cells in step 1) with pH 9 50 mM triethanolamine buffer (TEA) three times, and then resuspend in 50 mM triethanolamine buffer (TEA) pH 9 to adjust the cell concentration to OD 600 It was 1.0, NADH was added to make the final concentration of NADH 10mM, 50℃, 170rpm culture, as the experimental group.

[0043] NADH was added with 50 mM triethanolamine buffer (TEA) at pH 9 to make the final concentration ...

Embodiment 3

[0050] Example 3. Enterobacter aerogenes converts NADH into NAD +

[0051] 1) Cultivation of Enterobacter aerogenes

[0052] Enterobacter aerogenes IAM1183 is connected to the culture medium (glucose 15g / L, peptone 5g / L, K 2 HPO 4 14g / L, KH 2 PO 4 6g / L, (NH 4 ) 2 SO 4 2g / L, MgSO 4 ·7H 2 O0.2g / L), 37°C, 170rpm air bath shaker culture.

[0053] Take 60 ml of Enterobacter aerogenes IAM1183 cultured in aerobic shake flask for 12 hours, 10,200×g, centrifuge at 4°C for 5 minutes, and collect Enterobacter aerogenes bacteria.

[0054] 2) Convert NADH to NAD +

[0055] Wash the Enterobacter aerogenes cells in step 1) with pH 4 50 mM triethanolamine buffer (TEA) three times, and then resuspend in 50 mM triethanolamine buffer (TEA) pH 4 to adjust the cell concentration to OD 600 It was 0.8, NADH was added to make the final concentration of NADH 20μM, 4°C, 170rpm culture, as the experimental group.

[0056] NADH was added with 50 mM triethanolamine buffer (TEA) at pH 4 to make the final concentratio...

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PUM

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Abstract

The invention discloses a method for regenerating oxidized coenzyme I through whole cell biotransformation. The method for regenerating oxidized coenzyme I of the present invention is to use the reduced coenzyme I as a substrate to convert the reduced coenzyme I into oxidized coenzyme I by using Enterobacter aerogenes in a buffer solution. The pH of the buffer solution is 4.0-9.0. The concentration of the reduced coenzyme I in the buffer solution is 0.02mM-10mM. The method for regenerating oxidized coenzyme I of the present invention can convert NADH into NAD+, and the conversion rate reaches 72%.

Description

Technical field [0001] The invention relates to a method for regenerating oxidized coenzyme I by using whole cell biological transformation. Background technique [0002] Among oxidoreductases, approximately 80% of oxidoreductases require nicotinamide adenine dinucleotide (NAD + , NADH) as a coenzyme, and 10% of the oxidoreductase uses nicotinamide adenine dinucleotide phosphate (NADP + , NADPH) is a coenzyme, and only a few of them use flavin (FMN, FAD) and coenzyme Q (PQQ) as coenzymes. When oxidoreductase is used for biocatalysis, a certain amount of coenzyme will be consumed while synthesizing the product. The efficient supply of coenzymes is one of the key technologies for developing oxidoreductase catalyzed reactions. Coenzyme is expensive, and every mole of oxidized coenzyme I (NAD + The price of) is about 1,300 Euros, while the price of PQQ exceeds 2,700,000 Euros per mole, and the stability of the coenzyme is low. From a technical and economic point of view, it is not f...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/32C12P7/24C12R1/01
Inventor 邢新会张翀吴希
Owner TSINGHUA UNIV
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