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Method for producing trans-4-Hyp (trans-4-hydroxy-L-proline) through transformation of recombinant escherichia coli

A proline, proline hydroxylase technology, applied in the field of recombinant Escherichia coli transformation to produce trans-4-hydroxy-L-proline, can solve the problems of poor stability, low catalytic efficiency and the like, and achieves conversion rate High, high utilization rate of raw materials, easy to control effect

Pending Publication Date: 2021-08-06
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, due to the catalytic activity of trans-P4H double oxygen addition, it faces the problems of low catalytic efficiency and poor stability.

Method used

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  • Method for producing trans-4-Hyp (trans-4-hydroxy-L-proline) through transformation of recombinant escherichia coli
  • Method for producing trans-4-Hyp (trans-4-hydroxy-L-proline) through transformation of recombinant escherichia coli
  • Method for producing trans-4-Hyp (trans-4-hydroxy-L-proline) through transformation of recombinant escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: the construction of mutant

[0032] Construction of recombinant strains of E.coli BL21 / pET28a-UB2 and its mutants

[0033] 1. Synthesis and transformation of the full sequence of the P4H enzyme gene

[0034] According to the uncultured bacteria esnapd2 (GeneID: AGS49339.1) P4H sequence published by GENBANK, the codon was optimized to obtain the gene encoding L-proline hydroxylase whose sequence is shown in SEQ ID NO.1. The target gene and expression plasmid pET28a were digested with restriction endonuclease EcoR I / Hind III, and the recombinant plasmid pET28a-UB2 was obtained after the target gene was connected with the vector. The recombinant plasmid pET28a-UB2 was transformed into Escherichia coli E.coli BL21 by chemical transformation method, the colony grown on LB and ampicillin pressure plate was picked, cultured in shake flask, and the plasmid was extracted for single and double enzyme digestion verification to obtain Recombinant bacteria E.coli BL2...

Embodiment 2

[0050] Example 2: Detection of whole cell transformation performance of recombinant strains

[0051] The recombinant bacteria E.coli BL21 / pET28a-UB2 and its mutant recombinant strains constructed in Example 1 were activated and transferred into the self-inducing medium, and the cells were collected for whole cell transformation. The transformation system was 10 mL, and the temperature was 40 ℃, the conversion pH is 7.0, cell wall permeabilization agent Triton 10 μL, add 20 mM L-proline, 20 mM α-ketoglutarate, 1 mM FeSO 4 -7H 2 O and 5 mM ascorbic acid for transformation. The product Hyp in the transformation solution was detected by HPLC. The product Hyp was detected, indicating that the recombinant Escherichia coli strain with transformed L-proline into Hyp was successfully constructed.

[0052] Blank control: The plasmid pET28a was transformed into E.coli BL21 for expression to obtain the recombinant strain E.coliBL21 / pET28a. Pick the colonies grown on LB and kanamycin p...

Embodiment 3

[0053] Embodiment 3: biotransformation method produces trans-4-hydroxyl-L-proline

[0054] The recombinant bacteria E.coli BL21 / pET28a-UB2 and its mutant recombinant strains constructed in Example 1 were inoculated as a single colony in 10 mL of LB medium, and cultured for 12 hours. Inoculate 10% of the inoculum into a 1L baffled Erlenmeyer flask filled with 250mL autoinduction medium, and culture at 25°C for about 24h. Collect the bacteria, and use 100mL, 100mM, pH 7.0 Na 2 HPO 4 -Resuspend the recombinant E. coli cells in citric acid buffer to a wet weight of 250g / L, add 100 μL of the cell wall permeabilizer Triton, add 40mML-proline, 40mMα-ketoglutarate, 1mM FeSO 4 -7H 2 0, 5mM ascorbic acid for transformation, the transformation temperature is 30°C, and the transformation pH is 7.0. After 24 hours of whole-cell transformation, the Hyp in the whole-cell transformation solution of the recombinant bacteria was determined by HPLC. After 24 hours of whole cell transformati...

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Abstract

The invention discloses a method for producing trans-4-Hyp (trans-4-hydroxy-L-proline) through transformation of recombinant escherichia coli, and belongs to the technical field of biological engineering. A P4H enzyme derived from unulturated bacterium esnapd2 is cloned, related modification is performed on the basis of the P4H enzyme to obtain a mutant, and the recombinant Escherichia coli expressing the L-proline hydroxylase or the mutant thereof is constructed. The recombinant escherichia coli expressing the L-proline hydroxylase or the mutant of the L-proline hydroxylase is subjected to whole-cell transformation to produce Hyp, the yield of trans-4-Hyp after 24h of transformation is 5.38 g / L, and the transformation rate can reach 94.5%. A foundation is provided for industrial production of Hyp by microorganisms.

Description

technical field [0001] The invention relates to a method for transforming and producing trans-4-hydroxyl-L-proline by recombinant Escherichia coli, belonging to the technical field of bioengineering. Background technique [0002] Trans-4-hydroxy-L-proline (Hyp) is an important amino acid, which is the main component of collagen. It plays a key role in collagen stability by allowing the sharp twisting of the collagen helix with proline. Hyp can be produced by hydroxylation of the amino acid proline by several prolyl hydroxylases, resulting in different hydroxyprolines. Foremost among these is Hyp, which is also the most abundant component of collagen. Hyp has been widely used in biochemistry, food, cosmetics and other fields. In microorganisms, Hyp is also involved in the synthesis of secondary metabolites such as actinomycin, echinocandin, etamycin and pneumocandine. In addition, N-acetyl trans-4-hydroxyproline, one of the derivatives of Hyp, acts as an inflammation inhi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12P13/24C12R1/19
CPCC12N9/0071C12P13/24C12Y114/11002
Inventor 饶志明龙梦飞乔郅钠徐美娟杨套伟张显邵明龙
Owner JIANGNAN UNIV
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