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Bacteria for L-hydroxyproline production and application of bacteria for L-hydroxyproline production

A technology of hydroxyproline and Escherichia coli, applied in the field of microorganisms, can solve problems such as high price, long fermentation period, polluted environment, etc., and achieve the effects of improving product yield and quality, reducing the use of inducers, and reducing environmental pollution.

Active Publication Date: 2016-01-13
FUJIAN MINDONG REJUVENATION PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, the chemical synthesis method has many synthesis steps, the source of raw materials is limited, the cost is high, the environment is polluted, and it is difficult to realize industrial production.
In 2011, German scientist Christian Klein optimized the conditions for the production of L-hydroxyproline by fermentation and transformation of Escherichia coli engineering bacteria containing cofactors, and the conversion rate of proline into L-hydroxyproline was measured to be 61%. Its fermentation cycle is long, substrate input amount and conversion efficiency are all low, it is not suitable for industrialized production of L-hydroxyproline, and for the enzyme expression of Escherichia coli engineering bacteria, isopropylthiogalactopyranoside (IPTG) is mostly used As an inducer, although its induction effect is good, it is expensive and not suitable for industrial production
At the same time, in the process of direct fermentation and production of L-hydroxyproline with L-proline as the substrate, the utilization rate of L-proline becomes low because the host bacteria itself will consume the substrate L-proline

Method used

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  • Bacteria for L-hydroxyproline production and application of bacteria for L-hydroxyproline production
  • Bacteria for L-hydroxyproline production and application of bacteria for L-hydroxyproline production
  • Bacteria for L-hydroxyproline production and application of bacteria for L-hydroxyproline production

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preparation example Construction

[0033] The preparation method of chloramine T solution: 1.41g of chloramine T (product of Shanghai Aladdin Biochemical Technology Co., Ltd., product catalog number is 104060) was dissolved in 10mL of water, then 10mL of n-propanol and 80mL of buffer solution were added in sequence, and mixed evenly. Chloramine T solution was obtained. Preparation method of the buffer solution: 50 g of citric acid, 26.3 g of NaOH and 146.1 g of crystalline sodium acetate were dissolved in deionized water, and the volume was adjusted to 1 L, and then mixed with 200 mL of water and 300 mL of n-propanol to obtain a buffer solution.

[0034] The preparation method of the developer: fully dissolve 10g of p-dimethylaminobenzaldehyde with 35mL of perchloric acid, then slowly add 65mL of isopropanol and mix well.

[0035] The medium involved in the following examples is as follows:

[0036]LB slant medium: solute and its concentration are: tryptone 1g / 100mL, yeast extract 0.5g / 100mL, sodium chloride 1...

Embodiment 1

[0041] Embodiment 1, construction of recombinant plasmid pBR322-tac-Hyp-proC

[0042] 1. Artificially synthesize the double-stranded DNA molecule shown in Sequence 1 of the Sequence Listing. In this double-stranded DNA molecule, the 1st to 8th positions from the 5' end of Sequence Listing Sequence 1 are the restriction endonuclease AatII restriction endonuclease recognition sites, the 9th to 62nd nucleotides are the tac promoter, and the 63rd nucleotides are tac promoters. The 878th nucleotide is the coding gene of proline hydroxylase, the 895th to 1701st nucleotide is the coding gene of dihydropyrrole-5-carboxylate reductase, and the 1705th to 1723rd nucleotide is T7 As for the terminator, positions 1724 to 1731 are the recognition sites for restriction endonuclease HindⅢ.

[0043] 2. Double-digest the artificially synthesized double-stranded DNA molecule in step 1 with restriction endonucleases AatII and HindIII, recover the digested products, and obtain fragments.

[0044...

Embodiment 2

[0048] Embodiment 2, knockout of putA gene and construction of recombinant Escherichia coli

[0049] The nucleotide sequence of the putA gene in the genomic DNA of Escherichia coli BL21(DE3) plysS is shown in sequence 4 of the sequence listing.

[0050] 1. Obtaining target DNA homologous recombination fragments

[0051] Follow the steps below to obtain targeted DNA homologous recombination fragments:

[0052] 1. According to the nucleotide sequence of the putA gene and the gene sequence encoding kanamycin in the plasmid pKD13, a pair of homology arm primers was synthesized by Treasure Bioengineering (Dalian) Co., Ltd. The homology arm primer pair contains two primers, named respectively for putac1 and putac2.

[0053] The nucleotide sequences of putac1 and putac2 are shown in Table 1. Wherein the single underline is the upstream segment of the putA gene, the dotted line is the downstream segment of the putA gene, the wavy line is the upstream segment of the nucleotide seque...

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Abstract

The invention discloses a strain of bacteria for L-hydroxyproline production and application of the bacteria for L-hydroxyproline production and provides Escherichia coli.HYP035 with a preservation number being CCTCC NO:M 2015401 in China Center For Type Culture Collection. According to experiments, the Escherichia coli.HYP035 can be used for L-hydroxyproline production, yield of L-hydroxyproline in fermentation broth is 45g / L, the proline conversion rate is 85%, a fermentation period is 40h, and high production value is achieved.

Description

technical field [0001] The invention relates to the field of microorganisms, in particular to a bacterium producing L-hydroxyproline and its application. Background technique [0002] L-Hydroxyproline is an imino acid, which does not belong to the 20 common amino acids. It is the product of hydroxylation of proline and the main component of collagen. It is mainly used in medicine, chemical industry, animal feed, nutrition and beauty industries, etc. aspect. In recent years, the research and development of L-hydroxyproline has attracted widespread attention: L-hydroxyproline has anti-oxidation and anti-radiation effects, and can be used as a cosmetic additive in the beauty industry; L-hydroxyproline has the function of reducing weight. It can become an ideal weight-loss drug in the future; L-hydroxyproline has a variety of physiological functions and unique biological activities, and can be used as a drug for various soft tissue diseases, such as damaged connective tissue, r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P13/24C12R1/19
Inventor 黄建忠吴伟斌郭小雷黄平吴松刚
Owner FUJIAN MINDONG REJUVENATION PHARMA
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