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Gene, enzyme, carrier, engineering bacterium of trans-L-proline-4-hydroxylase and application thereof

A technology of genetically engineered bacteria and proline, applied in the field of genetic engineering, can solve the problems of high equipment requirements, high cost, long chemical synthesis route, etc., and achieve the effect of great application potential

Active Publication Date: 2017-04-26
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The yield of this method is only 4%-7%, and the safety of the process is poor, the equipment requirements are high, and it will cause serious pollution to the environment
At the same time, the outbreak of animal diseases (mad cow disease, foot-and-mouth disease, etc.) severely limited the source of collagen, making this method unable to meet market demand.
The chemical synthesis method has a long route (protection / deprotection), serious environmental pollution, high cost, and is not suitable for industrial production

Method used

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  • Gene, enzyme, carrier, engineering bacterium of trans-L-proline-4-hydroxylase and application thereof
  • Gene, enzyme, carrier, engineering bacterium of trans-L-proline-4-hydroxylase and application thereof
  • Gene, enzyme, carrier, engineering bacterium of trans-L-proline-4-hydroxylase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Gene synthesis of trans-P4H

[0046] According to the sequence information (GenBank No.BAL06808.1) of the gene of trans-P4H from bradyrhizobium japonicum USDA 6 disclosed in the NCBI database, it is optimized with reference to the codon bias of Escherichia coli, so that Codons with a frequency below 10% were adjusted and replaced, and the GC content of the entire sequence was as close to 50% as possible. At the same time, the commonly used restriction endonucleases Bgl II, BamH I, Nco I and Xho I and other recognition sites were removed, and the new The designed trans-P4H gene sequence (p4hBJ) is shown in SEQ ID NO.1. The gene synthesis work was entrusted to Shanghai Xuguan Biotechnology Development Co., Ltd., and the synthesized gene fragment was connected to the cloning vector pES.

[0047] The same method was used to synthesize the trans-P4H gene p4hD with the highest enzyme activity reported so far, and the sequence was optimized according to the literatu...

Embodiment 2

[0048] Embodiment 2: Construction of trans-P4H recombinant Escherichia coli

[0049] On the basis of Example 1, using PCR technology, using the cloning plasmid with the p4hBJ gene sequence as a template, p4hBJ-F (5'-AGG CCATGG GTAAACTGTCTGAAGCGCAGC-3′, the underlined part is the Nco I enzyme digestion recognition site) and p4hBJ-R (5′-AAT CTCGAG TTACTCAGCA GCCTGACGCGG-3', the underlined part is the Xho I restriction restriction site) as primers to amplify the p4hBJ gene. Similarly, using the cloning plasmid with the p4hD gene sequence as a template, p4hD-F (5'-AGG CCATGG GTACTCCAACCGAACTGAAAC-3′, the underlined part is the Nco I enzyme digestion recognition site) and p4hD-R (5′-AAT CTCGAG CACCGGTTGAGCCAGTGCGAA-3', the underlined part is the Xho I digestion recognition site) were used as primers to amplify the p4hD gene.

[0050] The PCR reaction system (50 μL) was: 5 μL of 10×Pfu PCR buffer, 8 μL of dNTP Mixture; 1 μL of template DNA; 2 μL of upstream and downstream pri...

Embodiment 3

[0053] Example 3: Protein electrophoresis and enzyme activity assay of recombinant Escherichia coli

[0054] The recombinant Escherichia coli E.coli BL21(DE3) / pET-28b-p4hBJ and E.coliBL21(DE3) / pET-28b-p4hD constructed in Example 2 were inoculated into 50mL LB liquid medium containing 40 μg / mL Kan , 37°C, 150rpm shaking culture to OD 600 =0.8~1.0; the culture solution was inoculated into fresh 100mL LB liquid medium containing 40μg / mL Kan with 2% (v / v) inoculum amount, cultured with shaking at 37°C and 150rpm until OD 600 =0.4~0.8, add IPTG with a final concentration of 0.5mM, induce culture at 28°C and 150rpm for 10h. Take the bacterial culture solution, centrifuge at 8000rpm, 4°C for 10min, wash the bacterial pellet twice with 80mMMES buffer (pH6.5), then resuspend in MES buffer, take out 20μL of the whole cell bacterial suspension for use; the remaining bacteria The solution was crushed by ultrasonic waves, centrifuged at 12000 rpm for 1 min, and 20 μL of the supernatant o...

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Abstract

The invention discloses a gene, an enzyme, a carrier, an engineering bacterium of trans-L-proline-4-hydroxylase and application thereof in synthesis of trans-4-hydroxyl-L-proline by catalyzing L-proline. The enzyme activity of the trans-L-proline-4-hydroxylase reaches 1654.08U / L, and the specific enzyme activity is 82.74U / mg; thallus cells containing enzymes are collected directly as an enzyme source to perform biotransformation, and the amount of the trans-4-hydroxyl-L-proline is 24.8mg / L; after a 0.8% surfactant Nymeen-S215 is added into a reaction system, the output reaches 176.0mg / L, and is improved by 6.36 times under the same condition without adding the Nymeen-S215; and on the basis, glucose (5g / L) is added into the reaction system, and then the product amount is 329.2mg / L and is 1.87 times of that without adding the glucose.

Description

[0001] (1) Technical field [0002] The invention relates to a genetically engineered bacterium producing trans-L-proline-4-hydroxylase (trans-L-Proline-4-hydroxylase, referred to as trans-P4H) and its construction method and application, belonging to genetic engineering field. [0003] (2) Technical background [0004] Trans-L-Proline-4-hydroxylase (trans-L-Proline-4-hydroxylase, referred to as trans-P4H) is dependent on α-ketoglutarate and Fe 2+ One of the members of the dioxygenase superfamily, it can be used as a biocatalyst in the biosynthesis of trans-4-hydroxy-L-proline (trans-4-hydroxy-L-proline, referred to as t-4Hyp) Key role. trans-P4H cofactors α-ketoglutarate (α-KG), Fe 2+ and O 2 The free L-proline can be converted into t-4Hyp under the condition of existence (sometimes need to use L-ascorbic acid as reducing agent). [0005] t-4Hyp is an important unnatural amino acid, which is an isomer of L-hydroxyproline. Its structure contains a tetrahydropyrrole ring an...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12N1/21C12P13/24C12R1/19
CPCC12N9/0071C12P13/24C12Y114/11002
Inventor 郑裕国周海岩王培李会帅柳志强
Owner ZHEJIANG UNIV OF TECH
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