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Highly active self-inducible expression method of proline hydroxylase and its transformation method for producing trans 4-hydroxyl-l-proline

A proline hydroxylase and expression method technology, applied in the field of high-efficiency transformation, can solve the problems of low substrate input and transformation efficiency, unsuitable for industrial production, high price, etc., and achieve good industrial application prospects and transformation reaction time. Short, low-cost effects

Active Publication Date: 2016-06-22
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 optimized the transformation conditions for the production of trans-4-hydroxy-L-proline by transforming Escherichia coli engineering bacteria containing concomitant factors. At 37°C and 140rpm, the recombinant engineered bacteria were cultivated to OD600=1.1 At –1.4, add 0.2mMIPTG to induce expression, then add 6.25mML-proline, 0.5mM ferrous sulfate, 8mMα-ketoglutaric acid, and culture at 140rpm for 72h at 28°C. The conversion rate of acid into trans-4-L-proline is only 61%, and the substrate input amount and conversion efficiency are relatively low, so it is not suitable for industrialized production of trans-4-L-proline
Among them, the NonidetP-40 additive also has the disadvantage of being expensive and not suitable for industrial production

Method used

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  • Highly active self-inducible expression method of proline hydroxylase and its transformation method for producing trans 4-hydroxyl-l-proline
  • Highly active self-inducible expression method of proline hydroxylase and its transformation method for producing trans 4-hydroxyl-l-proline
  • Highly active self-inducible expression method of proline hydroxylase and its transformation method for producing trans 4-hydroxyl-l-proline

Examples

Experimental program
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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 5g / L, ammonium sulfate 5g / L, potassium dihydrogen phosphate 1g / L, sodium chloride 2g / L, seven Magnesium sulfate water 0.2g / L, ferrous sulfate heptahydrate 0.1g / L and lactose 8g / L, after self-induction culture directly at 28°C and 140rpm shaker for 24 hours, when the concentration of bacteria 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 5g / L, ammonium sulfate 5g / L, potassium dihydrogen phosphate 1g / L, sodium chloride 2g / L, seven Magnesium sulfate water 0.2g / L, ferrous sulfate heptahydrate 0.1g / L and lactose 8g / L, after self-induction culture directly at 24°C and 220rpm shaker for 20 hours, when the concentration of the bacteria solution is OD 600 When reaching 2-2.5, centrifuge to collect the bacteria.

Embodiment 1c

[0034] Example 1c Self-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 5g / L, ammonium sulfate 5g / L, potassium dihydrogen phosphate 1g / L, sodium chloride 2g / L, seven Magnesium sulfate water 0.2g / L, ferrous sulfate heptahydrate 0.1g / L and lactose 8g / 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, the cells were collected by centrifugation.

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Abstract

The invention discloses a high-efficiency transformation method for producing trans-4-hydroxy-L-proline by a biotransformation method, which specifically includes a high-activity self-induced expression method of L-proline hydroxylase in recombinant E. The type, concentration and conversion conditions of the auxiliary agent in the high-efficiency conversion system of converting -proline into trans-4-hydroxy-L-proline, for the establishment of a biotransformation method for the industrial production of trans-4-hydroxy-L-proline Amino acid technology provides an important scientific basis.

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) does not belong to the common 20 kinds of 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 elast...

Claims

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Application Information

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Patent Type & Authority Patents(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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