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Prolyl hydroxylase high-activity self-induced expression method and high-efficiency conversion method for producing trans-4-hydroxy-L-proline by prolyl hydroxylase high-activity self-induced expression method

A technology of proline hydroxylase and expression method, which is applied in the field of high-efficiency transformation, can solve the problems of low substrate input, low transformation efficiency, high price, and unsuitability for industrial production, and achieve good industrial application prospects, low cost, The effect of short conversion reaction time

Active Publication Date: 2014-07-09
HEBEI BOLUNTE PHARMA +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In 2011, German scientist Christian Klein carried out transformation conditions for the transformation of Escherichia coli engineering bacteria containing cofactors to produce trans-4-hydroxyl-L-proline Optimization, at 37°C, 140 rpm to culture recombinant engineered bacteria to OD600=1.1–1.4, add 0.2mM IPTG to induce expression, then add 6.25mM L-proline, 0.5 mM ferrous sulfate, 8 mM α- Ketoglutaric acid, under the condition of 28 ℃, after 72 hours of culture at 140rpm, the measured conversion rate of proline into trans-4-L-proline is only 61%, and the input amount of substrate and the conversion efficiency are low. Relatively low, not suitable for industrial production of trans-4-L-proline
Among them, the Nonidet P-40 additive also has the disadvantage of being expensive and not suitable for industrial production

Method used

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  • Prolyl hydroxylase high-activity self-induced expression method and high-efficiency conversion method for producing trans-4-hydroxy-L-proline by prolyl hydroxylase high-activity self-induced expression method
  • Prolyl hydroxylase high-activity self-induced expression method and high-efficiency conversion method for producing trans-4-hydroxy-L-proline by prolyl hydroxylase high-activity self-induced expression method
  • Prolyl hydroxylase high-activity self-induced expression method and high-efficiency conversion method for producing trans-4-hydroxy-L-proline by prolyl hydroxylase high-activity self-induced expression method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1a

[0030] Example 1a Self-inducible expression method of high activity proline hydroxylase in recombinant Escherichia coli engineering bacteria

[0031] Inoculate the single clone of the engineering strain containing the recombinant plasmid into the fermentation medium: glucose 20g / L, yeast extract powder 5 g / L, ammonium sulfate 5 g / L, potassium dihydrogen phosphate 1 g / L, sodium chloride 2 g / L, Magnesium Sulfate Heptahydrate 0.2 g / L, Ferrous Sulfate Heptahydrate 0.1 g / L and Lactose 8 g / L, after self-induction culture directly at 28°C and 140rpm shaker for 24 hours, when the concentration of the bacterial solution is OD 600 When reaching 2-2.5, centrifuge to collect the bacteria.

Embodiment 1b

[0032] Example 1b Self-inducible expression method of high activity proline hydroxylase in recombinant Escherichia coli engineering bacteria

[0033] Inoculate the single clone of the engineering strain containing the recombinant plasmid into the fermentation medium: glucose 20g / L, yeast extract powder 5 g / L, ammonium sulfate 5 g / L, potassium dihydrogen phosphate 1 g / L, sodium chloride 2 g / L, magnesium sulfate heptahydrate 0.2 g / L, ferrous sulfate heptahydrate 0.1 g / L and lactose 8 g / L, after self-induction culture directly at 24°C and 220rpm shaking table for 20 hours, when the concentration of bacteria solution is OD 600 When reaching 2-2.5, centrifuge to collect the bacteria.

Embodiment 1c

[0034] Example 1cSelf-inducible expression method of high activity proline hydroxylase in recombinant Escherichia coli engineering bacteria

[0035] Inoculate the single clone of the engineering strain containing the recombinant plasmid into the fermentation medium: glucose 20g / L, yeast extract powder 5 g / L, ammonium sulfate 5 g / L, potassium dihydrogen phosphate 1 g / L, sodium chloride 2 g / L, Magnesium Sulfate Heptahydrate 0.2 g / L, Ferrous Sulfate Heptahydrate 0.1 g / L and Lactose 8 g / L, after self-induction culture directly at 37°C and 180rpm shaker for 22 hours, when the concentration of bacteria solution is OD 600 When reaching 2-2.5, centrifuge to collect the bacteria.

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Abstract

The invention discloses a high-efficiency conversion method for producing trans-4-hydroxy-L-proline by a bioconversion technology. The high-efficiency conversion method comprises a high-activity self-induced expression method of L-prolyl hydroxylase in recombinant engineered escherichia coli, types and concentrations of assistants used in an L-proline-to-trans-4-hydroxy-L-proline efficient conversion system, and conversion conditions of the conversion system. The high-efficiency conversion method provides an important scientific basis for establishing bioconversion-based industrial production of trans-4-hydroxy-L-proline.

Description

technical field [0001] The invention belongs to the field of enzyme engineering and biocatalysis, and specifically relates to a method for self-inducing expression of proline hydroxylase with high activity in recombinant Escherichia coli engineering bacteria and using proline hydroxylase to convert L-proline into trans Efficient conversion method of formula-4-hydroxyl-L-proline. Background technique [0002] Hydroxyproline (Hyp) is not one of the 20 common amino acids. It is the result of hydroxylation modification of proline. The hydroxyl group is usually added to the 4th or 3rd carbon. Due to two asymmetric carbon atoms, hydroxyproline has 4 stereoisomers. trans-4-hydroxy-L-proline, cis-4-hydroxy-L-proline, trans-3-hydroxy-L-proline and cis-3-hydroxy-L-proline acid. [0003] Trans-4-hydroxy-L-proline mainly exists in collagen in mammalian cells, and plays a very important role in the formation of stable triple-helix collagen, which can strengthen the elasticity and toug...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/70C12N1/21C12P13/24C12R1/19
CPCC12N9/0071C12N15/70C12P13/24C12Y114/11002
Inventor 李玮张金秀薛张伟王立安李存会鞠建松李天云张琳琳陈娇娇张庆
Owner HEBEI BOLUNTE PHARMA
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