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Method for regenerating oxidation coenzyme I using intact cell bioconversion

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: 2009-03-18
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 regenerating oxidation coenzyme I using intact cell bioconversion
  • Method for regenerating oxidation coenzyme I using intact cell bioconversion
  • Method for regenerating oxidation coenzyme I using intact cell bioconversion

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0013] 1) Culture Enterobacter aerogenes

[0014] Enterobacter aerogenes (Enterobacteraerogenes) IAM1183 was inserted into 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 O 0.2g / L), 37°C, 170rpm air bath shaker culture.

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

[0016] 2) NADH is converted to NAD +

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

[0018] Wash the Enterobacter ...

Embodiment 2

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

[0038] 1) Culture Enterobacter aerogenes

[0039] Enterobacter aerogenes (Enterobacter aerogenes) IAM1183 was inserted into 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 O 0.2g / L), 37°C, 170rpm air bath shaker culture.

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

[0041] 2) NADH is converted to NAD +

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

[0043] NADH was added with 50 mM triethanolamine buffer...

Embodiment 3

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

[0051] 1) Culture Enterobacter aerogenes

[0052] Enterobacter aerogenes (Enterobacter aerogenes) IAM1183 was inserted into 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 O 0.2g / L), 37°C, 170rpm air bath shaker culture.

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

[0054] 2) NADH is converted to NAD +

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

[0056] Add NADH with pH 4 50 mM trietha...

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PUM

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Abstract

The invention discloses a method for regenerating oxidized coenzyme I by utilizing the transformation of intact cell organisms. The method for regenerating the oxidized coenzyme I comprises the following steps: in a buffer solution, reduced coenzyme I is taken as a substrate and transformed into the oxidized coenzyme I by utilizing Enterobacter aerogenes. The pH value of the buffer solution is between 4.0 and 9.0. The concentration of the reduced coenzyme I in the buffer solution is between 0.02 and 10mM. The method for regenerating the oxidized coenzyme I can transform NADH into NAD<+>, and the transformation rate reaches 72 percent.

Description

technical field [0001] The present invention relates to a method for regenerating oxidized coenzyme I by whole cell biotransformation. Background technique [0002] Among oxidoreductases, about 80% of oxidoreductases require nicotinamide adenine dinucleotide (NAD + , NADH) as a coenzyme, 10% of the oxidoreductase is nicotinamide adenine dinucleotide phosphate (NADP + , NADPH) as coenzyme, only a small part of flavin (FMN, FAD) and coenzyme Q (PQQ) as coenzyme. When oxidoreductase is used for biocatalysis, a certain amount of coenzyme will be consumed while synthesizing the product. Efficient supply of coenzymes is one of the key technologies for developing oxidoreductase-catalyzed reactions. Coenzymes are expensive, and each mole of oxidized coenzyme I (NAD + ) is about 1,300 euros, and the price of PQQ is more than 2,700,000 euros per mole, and the coenzyme has low stability. From a technical and economic point of view, it is not feasible to add a large amount of coenzy...

Claims

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Application Information

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