Trans-4-hydroxy-L-proline hydroxylase modifying gene with high transformation rate and application thereof

A technology of proline hydroxylase and high conversion rate, which is applied in the fields of genetic engineering and enzyme engineering, can solve the problems of low conversion rate, rising separation cost and high production cost, achieves improved conversion rate, good industrial application prospect, and is beneficial to The effect of purification and separation

Active Publication Date: 2014-12-03
HEBEI BOLUNTE PHARMA +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

From the above results, it can be seen that there are still three problems in the production of trans-4-hydroxyl-L-hydroxyproline by using the gene biotransformation method: 1) It is necessary to add cofactors, which will lead to complex production processes; 2) The conversion rate is still relatively low, resulting in more components in the product, especially the large amount of L-proline, which brings difficulties to product separation; 3) The concentration of the target product in the conversion reaction is low, resulting in an increase in separation costs
The existence of these three problems will inevitably lead to high production costs, which is not conducive to industrial production

Method used

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  • Trans-4-hydroxy-L-proline hydroxylase modifying gene with high transformation rate and application thereof
  • Trans-4-hydroxy-L-proline hydroxylase modifying gene with high transformation rate and application thereof
  • Trans-4-hydroxy-L-proline hydroxylase modifying gene with high transformation rate and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033]Example 1 Trans-4-hydroxyl-L-proline hydroxylase gene optimization and construction of engineering strains

[0034] (1) Gene optimization

[0035] According to published in NCBI database from Dactylosporangium trans-4-hydroxy-L-proline hydroxylase gene of sp.RH1 phy -1 Sequence information (GenBank ID: D78338.1), gene optimization is carried out according to the following principles: ① Adjust and replace codons whose frequency is below 10% with reference to the codon frequency of the expressed species, so that the codon usage frequency of the gene sequence Try to be consistent with the optimal codon frequency of the species; ②Reduce the base repeat structure in the sequence as much as possible, so that the RNA secondary structure is relatively simple and stable; ③Make the GC content of the entire DNA sequence as close to 50% as possible .

[0036] (2) Plasmid construction

[0037] The optimized trans-4-hydroxyl-L-proline hydroxylase gene phy -2 were connected with...

Embodiment 2

[0038] Example 2 Different expression vectors in the host E. coli Transformation experiment of BL21(DE3)

[0039] Transform the above three expression plasmids into Escherichia coli BL21 (DE3), shake the bacteria at 140rpm at 28°C for about 2-3h (OD 600 reach 0.5-1.0), add 0.1-0.5mM IPTG, induce expression at 20°C for 8-12h, collect the bacteria by centrifugation, weigh 1g of the bacteria and resuspend in 10mL transformation reaction solution (containing 200mM L-proline, 200mM α-ketoglutaric acid, 6mM ferrous sulfate, 6mM L-ascorbic acid, 80mM pH 6.5 MES buffer, and 1% Nonidet P-40), shaken at 35°C and 140rpm for 72h, centrifuged to remove bacteria, and detected by HPLC The generation of trans-4-hydroxyl-L-proline in the serum, and its conversion rate are shown in Table 1. Among them, the transformation rate of the strain containing the expression vector pET-28a-Phy-1 was the lowest, only 62.0%. phy The transformation rate of strains -2 was significantly improved, and th...

Embodiment 3

[0042] Example 3 with E. coli BL21-CodonPlus (DE3) as the host transformation experiment

[0043] Transform the expression vectors pET28a-Phy-1 and pET28a-Phy-2 into Escherichia coli BL21-CodonPlus (DE3) respectively, and shake the bacteria at 140rpm at 28°C for about 2-3h (OD 600 reach 0.5-1.0), add 0.1-0.5mM IPTG, induce expression at 20°C for 8-12h, collect the bacteria by centrifugation, weigh 1g of the bacteria and suspend in 10mL transformation reaction solution (containing 200mM L-proline, 200mM α -ketoglutaric acid, 6mM ferrous sulfate, 6mM L-ascorbic acid, 80mM pH 6.5 MES buffer and 1% Nonidet P-40), shake and react at 35℃140rpm for 80h, centrifuge to remove bacteria, and detect the supernatant by HPLC The production of trans-4-hydroxy-L-proline, the transformation rate of the strain containing pET28a-Phy-1 is only 64.3%, while the transformation rate of the strain containing the expression vector pET28a-Phy-2 is as high as 91% ( Figure 3A , Figure 3B ).

[0...

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Abstract

The invention discloses a trans-4-hydroxy-L-proline hydroxylase modifying gene with high transformation rate and application thereof. An expression carrier of an optimization gene is constructed by optimizing nucleotide sequence of trans-4-hydroxy-L-proline hydroxylase (GenBankID:D78338.1) in Dactylosporangiumsp.RH1; the thallus is collected as the enzyme source of the transformation reaction through the prokaryotic expression. The experiment result expresses that the transformation rate of generating the trans-4-hydroxy-L-proline by using L-proline as a substrate is as high as 91%. The gene can be applied to biologically generating trans-4-hydroxy-L-hydroxyproline and has good industrial application value.

Description

technical field [0001] The invention relates to a trans-4-hydroxy-L-proline hydroxylase modified gene with high conversion rate and its application, belonging to the fields of genetic engineering and enzyme engineering. Background technique [0002] Trans-4-hydroxy-L-proline (L-hydroxyproline, trans-4-hydroxy-L-proline, Hyp) does not belong to the 20 common amino acids, it is the hydroxylation of proline by hydroxylase As a result of modification, mainly in collagen, its function is to strengthen the elasticity and toughness of connective tissue. The materials most abundant in collagen are bone glue and gelatin. The content of hydroxyproline in bone glue and gelatin is 10-10.5%, and the content of proline is 12-13%. [0003] In recent years, the research and development of Hyp has attracted extensive attention in the fields of medicine, biochemistry, food and beauty. In the field of medicine, Hyp has a variety of physiological functions and unique biological activities. I...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N15/70C12N1/21C12P13/24C12R1/19C12R1/01
Inventor 李玮鞠建松薛张伟张金秀王立安张琳琳李天云张庆陈娇娇
Owner HEBEI BOLUNTE PHARMA
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