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Method for producing trans-4-hydroxyl-L-proline by means of fermentation by aid of recombinant corynebacterium acetoacidophilum

A technology of Corynebacterium acetic acid and hydroxyproline, applied in the directions of biochemical equipment and methods, fermentation, recombinant DNA technology, etc., can solve the problems of high synthetic cost, high cost, serious waste pollution and other problems of chemical synthesis methods, and achieves important The effect of industrial application value

Inactive Publication Date: 2016-05-25
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has long purification steps, high cost, and serious waste pollution; while the chemical synthesis method has high synthesis cost and is not suitable for industrial production

Method used

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  • Method for producing trans-4-hydroxyl-L-proline by means of fermentation by aid of recombinant corynebacterium acetoacidophilum
  • Method for producing trans-4-hydroxyl-L-proline by means of fermentation by aid of recombinant corynebacterium acetoacidophilum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Obtaining of recombinant plasmid (PUC19-Ptrp2-HYP) containing H4P

[0026] Take the strain containing the target plasmid stored in the laboratory for activation, and cultivate it at 37°C and 220rpm for 12-16 hours. The cultured bacterial liquid was taken to extract the plasmid, and all operations were carried out in strict accordance with the instructions.

Embodiment 2

[0027] Example 2: Acquisition of expression vector pk19mobsacB containing H4P gene

[0028] PUC19-Ptrp2-HYP and pk19mobsacB were treated with two enzymes, EcoRI and BamHI, respectively, and the digested products were gel-recovered to obtain linear Ptrp2-HYP and pk19mobsacB fragments, which were ligated overnight at 16°C with T4 DNA ligase. Transfer 10 μL of the connection solution into Escherichia coli JM109, pick the transformed single colony culture and extract the plasmid for enzyme digestion verification, and verify that the correct recombinant plasmid is pk19mobsacB-Ptrp2-HYP.

Embodiment 3

[0029] Example 3: Obtaining of recombinant strain (pk19mobsacB-Ptrp2-HYP) containing H4P gene

[0030] The pk19mobsacB-Ptrp2-HYP recombinant plasmid was transformed into Corynebacterium acetoacidophilum C. acetoacidophilum NO.45 to obtain the recombinant strain C. acetoacidophilum NO.45 (pk19mobsacB-Ptrp2-HYP).

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Abstract

The invention discloses a method for producing trans-4-hydroxyl-L-proline by means of fermentation by the aid of recombinant corynebacterium acetoacidophilum. Recombinant plasmids carried by the recombinant corynebacterium acetoacidophilum have proline hydroxylase genes (P4H) capable of catalyzing proline to generate trans-4-hydroxyproline. The method has the advantage that the free L-proline can be converted into the trans-4-hydroxyl-L-proline by the aid of proline-4-hydroxylase in cells of the recombinant corynebacterium acetoacidophilum under the condition of existence of alpha-ketoglutaric acid and ferrous ions. The invention further discloses application of corynebacterium acetoacidophilum to producing the trans-4-hydroxyl-L-proline.

Description

technical field [0001] The invention discloses a method for fermenting and producing trans-4-hydroxy-L-proline by recombinant Corynebacterium acetophilus, which belongs to the field of microbial genetic engineering. Background technique [0002] Trans-4-hydroxyproline is widely used in medicine, chemical industry, animal feed and beauty industry. In medicine, trans-4-hydroxyproline can be used as a raw material to synthesize anti-inflammatory drugs and carbapenems. In chemical industry, trans-4-hydroxyproline can be used as chiral raw material to synthesize various polymers. In animal feed applications, adding trans-4-hydroxyproline can prevent malnutrition caused by insufficient glycine synthesis. Trans-4-hydroxyproline is used in advanced cosmetics to eliminate oxidants and adjust the redox state of cells. [0003] At present, the main method of domestic production of trans-4-hydroxyproline is the biological extraction method, which uses animal protein sources, such as ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/24C12N15/63
CPCC12P13/24C12N9/0071C12Y114/11002
Inventor 张震宇王晓姣姚动邦魏照辉黄建华
Owner JIANGNAN UNIV
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