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Proline-4-hydroxylase and application of recombinant expressing host cells of proline-4-hydroxylase

A technology of proline and hydroxylase, applied in the fields of genetic engineering and microorganisms

Inactive Publication Date: 2017-09-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the use of recombinant Bacillus subtilis to synthesize trans-4-hydroxyproline in China

Method used

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  • Proline-4-hydroxylase and application of recombinant expressing host cells of proline-4-hydroxylase
  • Proline-4-hydroxylase and application of recombinant expressing host cells of proline-4-hydroxylase
  • Proline-4-hydroxylase and application of recombinant expressing host cells of proline-4-hydroxylase

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Experimental program
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Effect test

Embodiment 1

[0030] Codon optimization of proline-4-hydroxylase gene: the original sequence of proline-4-hydroxylase gene sequence is derived from GenBank: KU517670.1, and the gene codon elimination is optimized according to the codon usage frequency of Bacillus Codons with low usage rate, while using the synonymous transformation method to eliminate restriction sites such as BamH I (GGATCC), EcoR I (GAATTC) in the sequence, and remove 3 small hairpins and 2 medium hairpin structures in the sequence, The optimized proline-4-hydroxylase gene sequence is shown in SEQID No.1, and the optimized proline-4-hydroxylase gene sequence was submitted to Suzhou Hongxun Biotechnology Co., Ltd. for artificial synthesis. Considering the secondary structure of mRNA, first of all, it is necessary to ensure that the codon translation pocket consisting of several bases after the ATG start codon is open, so as to reduce the energy potential of ribosomes binding to mRNA, so that ribosomes can Smoothly translat...

Embodiment 2

[0032] Construction of the recombinant expression plasmid: the upstream sequence of the codon-optimized proline-4-hydroxylase gene was added with a BamH I (GGATCC) restriction site, and the downstream sequence was added with an EcoR I (GAATTC) restriction site, Linked to pHY-Bs.xyl ( figure 2 Shown), construct carrier pHY-Bs.xyl-P4H, concrete steps are:

[0033] (1) 6.5 μL of proline-4-hydroxylase gene fragment, 2 μL of pHY-Bs.xyl vector, 0.5 μL of T4 ligase, 1 μL of T4 ligase buffer, made into a ligation system, ligated overnight at 16°C, and prepared ligation liquid;

[0034] (2) Add 10 μL of the connection solution prepared in step (1) to 100 μL of JM109 competent cells, then add 10 μL of 5×KCM buffer, mix well, place on ice for 30 minutes, and heat shock at 42°C After 90s, place on ice for 30min, add 500μL of LB medium, incubate at 37°C, 200rpm for 1h, spread on Amp (ampicillin) resistant plate, culture for 12h, extract plasmid for verification, and then further sequenc...

Embodiment 3

[0036] The construction of recombinant Bacillus subtilis WB600 / pHY-Bs.xyl-P4H, the specific method is:

[0037] (1) Inoculate a single colony of Bacillus subtilis WB600 in 20 mL of LB liquid medium, culture overnight at 37°C and 200 rpm;

[0038] (2) Transfer 1mL of the overnight culture to 50mL growth medium GM, and cultivate to OD at 37°C and 200rpm 600 It is about 0.8~0.9;

[0039] (3) Cells were ice-bathed for 10 minutes, and then centrifuged at 6000 rpm for 10 minutes at 4°C to collect the cells;

[0040] (4) Suspend and wash the cells with ice-bathed EP buffer for 4 times, and finally resuspend the cells in 1 mL EP Buffer to make the cell concentration reach about 1-1.3×10 10 CFU / mL, divide competent cells into 1.5mL EP tubes, 60μL per tube;

[0041](5) Add the ligation product to the competent cells, transfer it to the pre-cooled electroporation cup, and transform with 2.0kv pulse electric shock, quickly add 1mL resuscitation medium RM to the electroporation cup, res...

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Abstract

The invention discloses proline-4-hydroxylase. The gene sequence of the enzyme is encoded as shown by SEQ ID No.1 in the description; and the protein sequence of the enzyme is as shown by SEQ ID No.2 in the description. According to the invention, the proline-4-hydroxylase gene is optimized according to preference of bacillus codon and recombined bacillus subtilis containing the gene is prepared to be used for synthesizing trans-4-hydroxyproline. According to tpralineine-4-hydroxylase, a good prospect of industrial production of the trans-4- hydroxyproline synthesized by utilizing the bacillus subtilis is provided.

Description

technical field [0001] The invention relates to the field of genetic engineering and microorganism technology, in particular to an enzyme expressed by a proline-4-hydroxylase gene optimized by codons in bacillus and its synthesis of trans-4-hydroxyproline in bacillus subtilis acid application. Background technique [0002] Trans-4-hydroxyproline (trans-4-hydroxyproline, HYP), white flaky crystal or powder, molecular formula C 5 h 9 NO 3 , the molecular weight is 131.10. HYP is the product of free L-proline hydroxylated by proline-4-hydroxylase, which has a hydroxyl group on the fourth carbon, which belongs to the derivative of L-proline, and is the same as L-proline Amino acids are also asymmetric. HYP is a small variety of amino acid with high added value, which is widely used in medicine, chemical industry, animal feed and beauty industry. [0003] In medicine, HYP can be used as a raw material to synthesize anti-inflammatory drugs and carbapenems. In chemical indus...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N1/21C12P13/24C12R1/125
CPCC12N9/0071C12N9/12C12N9/93C12P13/24C12Y114/11002C12Y207/02011C12Y603/01002
Inventor 石贵阳朱惠霖吴志勇李由然
Owner JIANGNAN UNIV
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