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Method for producing trans-4-hydroxyproline from glucose in fermentation manner

A kind of hydroxyproline, trans technology, applied in the field of microbial genetic engineering, can solve problems such as a large number of wastes, and achieve the effect of broad industrial application prospects and important industrial application value

Inactive Publication Date: 2015-09-23
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Trans-4-hydroxyproline is an important precursor of some chemically synthesized drugs. The traditional production method is mainly obtained by hydrolyzing animal collagen. This method requires a complete and complicated set of purification steps and produces a large amount of waste

Method used

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  • Method for producing trans-4-hydroxyproline from glucose in fermentation manner
  • Method for producing trans-4-hydroxyproline from glucose in fermentation manner
  • Method for producing trans-4-hydroxyproline from glucose in fermentation manner

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Obtaining of recombinant plasmid (pET28a-Ptrp2-proBA2) containing proBA2

[0028] 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

[0029] Example 2: Obtaining of recombinant plasmid (pUC19-Ptrp2-hyp) containing hyp

[0030] 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 3

[0031] Embodiment 3: the single expression vector pET28a-PH that contains two kinds of genes and the acquisition of recombinant bacteria

[0032] Treat pET28a-Ptrp2-proBA2 and pUC19-Ptrp2-hyp with two enzymes, EcoRI and BamHI, respectively, and recover the linear pET28a-Ptrp2-proBA2 and Ptrp2-hyp fragments from the gel of the digested products. After connecting overnight at 16°C, transfer 10 μL of the connection solution into Escherichia coli JM109, pick the transformed single colony and culture it to extract the plasmid for enzyme digestion verification, and verify that the correct recombinant plasmid is pET28a-PH.

[0033] The pET28a-PH recombinant plasmid was transformed into Escherichia coli BL21(DE3) to obtain recombinant Escherichia coli BL21(DE3) / pET28a-PH.

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Abstract

The invention discloses a method for producing trans-4-hydroxyproline from glucose in a fermentation manner by virtue of recombinant Escherichia coli without adding exogenous L-proline. A recombinant plasmid carried by the recombinant Escherichia coli has a mutant gene proBA2 and hyp (4-hydroxyproline), wherein proBA2 is mutated on a glutamyl kinase encoding gene proB, and the inhibition effect of L-proline on glutamyl kinase encoded by the mutated gene is remarkably reduced. proBA2 and hyp are co-expressed, and trans-4-hydroxyproline can be produced directly from glucose in the fermentation manner without adding exogenous L-proline. The recombinant plasmid is obtained by recombining proBA2 and hyp onto the same expression plasmid or by co-transforming recombinant plasmids which contain the two genes respectively and have different resistances. The invention further discloses the application of the Escherichia coli to the production of hydroxyproline.

Description

technical field [0001] The invention discloses a method for directly fermenting and producing trans-4-hydroxyproline from glucose by using recombinant Escherichia coli, which belongs to the field of microbial genetic engineering. Background technique [0002] Trans-4-hydroxyproline is an important precursor of some chemically synthesized drugs. The traditional production method is mainly obtained by hydrolyzing animal collagen. This method requires a complete and complicated set of purification steps and produces a large amount of waste. With the development of biotechnology, microbial fermentation is currently used to produce trans-4-hydroxyproline, which can overcome various defects of hydrolysis, improve production efficiency, reduce production costs, and so on. [0003] In microorganisms, proline hydroxylase catalyzes proline to generate trans-4-hydroxyproline, and the synthesis of proline uses glutamic acid as a substrate, through γ-glutamic acid kinase, glutamine Pro...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N1/21C12N1/19C12N1/15C12P13/24
Inventor 张震宇胡丹丹姚动邦范永明
Owner JIANGNAN UNIV
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