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Biosynthesis method of isotope 13C labeling 4-oxyproline

A hydroxyproline and biosynthesis technology, applied in the field of 4-hydroxyproline synthesis, can solve the problems that 4-hydroxyproline is not suitable for the synthesis of 4-hydroxyproline, etc., achieving low cost and rapid effect , The effect of simple purification process

Inactive Publication Date: 2012-05-23
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Abstract
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Problems solved by technology

[0005] The purpose of the invention is to solve the problem that the existing method for preparing 4-hydroxyproline is not suitable for synthetic 13 The problem with C-labeled 4-hydroxyproline, while providing the isotope 13 Biosynthetic method of C-labeled 4-hydroxyproline

Method used

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  • Biosynthesis method of isotope 13C labeling 4-oxyproline
  • Biosynthesis method of isotope 13C labeling 4-oxyproline
  • Biosynthesis method of isotope 13C labeling 4-oxyproline

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specific Embodiment approach 1

[0011] Specific implementation mode one: the isotope of this implementation mode 13 The biosynthetic method of the 4-hydroxyproline of C mark is carried out as follows: one, carry out codon optimization to the original sequence of the gene phy-1 of the 4-hydroxylase of coding L-proline in the cystic fungus , to obtain the optimized phy-1 gene; 2. The optimized phy-1 gene and the modified prokaryotic expression vector pET19 were subjected to double enzyme digestion with NdeI and BamHI, respectively, and the desired target fragment was recovered, and the prokaryotic expression vector pET19-pp was connected and constructed -histag-phy-1, and then RNA polymerase was inserted into Escherichia coli C41 on DE3 for high-efficiency protein expression and purification to obtain purified L-proline 4-hydroxylase; 3. Purified L- The 4-hydroxylase of proline performs an enzymatic synthesis reaction to generate a reaction product, which is then purified to obtain 13 C-labeled 4-hydroxyproli...

specific Embodiment approach 2

[0029] Specific embodiment two: the difference between this embodiment and specific embodiment one is that the enzyme digestion system in step two is as follows:

[0030] Ingredients Dosage

[0031] 10× buffer 10μL

[0032] 10U / μL NdeI 2μL

[0033] 10U / μL BamHI 2μL

[0034] 1 μg / μL optimized phy-1 gene / prokaryotic expression vector pET19 10 μL

[0035] Nuclease-free purified water 76 μL.

[0036] Other steps and parameters are the same as those in Embodiment 1.

specific Embodiment approach 3

[0037] Specific embodiment three: the difference between this embodiment and specific embodiment one is that the connection system in step two is as follows:

[0038] Ingredients Dosage

[0039] 10× buffer 1 μL

[0040] 100ng / μL optimized phy-1 gene after double enzyme digestion 1μL

[0041] 100ng / μL prokaryotic expression vector pET19 after double enzyme digestion 5μL

[0042] 10U / μL T4 ligase 1μL.

[0043] Other steps and parameters are the same as those in Embodiment 1.

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Abstract

A biosynthesis method of isotope 13C labeling 4-oxyproline relates to a synthesis method of 4-oxyproline. The method solves the problem that the current 4-oxyproline preparing method is not applicable to synthesizing the 13C labeling 4-oxyproline. The method comprises the following steps that: 1 the initial sequence codon of the 4-hydroxylase gene phy-1 of coding L-proline in dactylosporangium isoptimized to obtain the optimized phy-1 gene, 2 double-enzyme cleavage is respectively carried out on the optimized phy-1 gene and pET19 and a pronucleus expression vector is constructed which is transferred into colibacillus for protein high efficiency expression and purification, 3 the purified L-proline 4-hydroxylase reacts with enzyme, which can be completed after the purification. The invention realizes the preparation of the13C labeling 4-oxyproline. The purification process is simple and the cost is low. The purity of the obtained13C labeling 4-oxyproline reaches 99.99 percent and the recovery rate is 75.34 percent.

Description

technical field [0001] The present invention relates to the synthetic method of 4-hydroxyproline. Background technique [0002] The most prevalent form of kidney stones are calcium oxalate stones, accounting for 80% of all cases. The precursor of oxalic acid is glyoxylic acid. Glyoxylic acid levels are normally maintained at a balanced level. This level of balance is achieved by the conversion of glyoxylate to glycolate by two important enzymes (alanine:glyoxylate aminotransferase and glyoxylate dehydrogenase). In this way, glyoxylic acid cannot accumulate in the body in large quantities. And when glyoxylic acid accumulates in large quantities, it will increase the chance of forming oxalic acid. Studies have shown that 4-hydroxyproline can be degraded into glyoxylic acid in the body. It has been reported that with the increase of human intake of 4-hydroxyproline, the content of oxalic acid in urine will also increase significantly, and the probability of stone formation...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N15/70C12N9/02C12P13/24
Inventor 姜巨全李士龙徐伟慧王志刚于丁
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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