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7beta-hydroxysteroid dehydrogenase mutant and application thereof

A technology of hydroxysteroids and dehydrogenases, which is applied in the field of biological enzymes, can solve problems such as difficulty in large-scale industrial production, low enzyme activity, and poor thermal stability, so as to avoid short maintenance time, prolong activity time, and heat loss. The effect of stability improvement

Active Publication Date: 2022-03-25
宋建芳
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to overcome the deficiencies in the prior art, the object of the present invention is to provide a 7β-hydroxysteroid dehydrogenase mutant to solve the existing wild-type 7β-HSDH and mutant enzymes with low enzyme activity, poor thermal stability and difficult Problems in application to large-scale industrial production

Method used

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  • 7beta-hydroxysteroid dehydrogenase mutant and application thereof
  • 7beta-hydroxysteroid dehydrogenase mutant and application thereof
  • 7beta-hydroxysteroid dehydrogenase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 Contains the construction of the expression vector of 7β-HSDH (N115V)

[0042] 1. With the 7β-hydroxysteroid dehydrogenase gene (DNA sequence: SEQ ID NO: 2, encoded protein sequence: SEQ ID NO: 1), first use the primer pair (SEQ ID NO: 5) and (SEQ ID NO: 7 ) is amplified by PCR, and then amplified by PCR with primer pairs (SEQ ID NO: 6) and (SEQ ID NO: 8), the PCR product is recovered twice, and then the product of the second PCR is used as a template, and the primer pair (SEQ ID NO: 5) and (SEQ ID NO: 6) were amplified and cloned by PCR, the restriction sites were: NdeI and XhoI, and the primers used were as follows:

[0043]

[0044] The first PCR reaction system and conditions are shown in the table below:

[0045]

[0046] PCR reaction conditions: pre-denaturation at 98°C for 5 minutes; 35 cycles, including: denaturation at 98°C for 10 seconds, annealing at 55°C for 5 seconds, extension at 72°C for 5 seconds; extension at 72°C for 10 minutes afte...

Embodiment 2

[0060] Embodiment 2 The recombinant plasmid containing pET28a-7β-HSDH (N115V) is expressed in Escherichia coli

[0061] 1. Plasmid transformation of BL21(DE3) competent cells:

[0062] Take out 50 μL of BL21(DE3) competent cells at -80°C and place on ice to thaw, add 1 μL of pET28a-7β-HSDH(N115V) plasmid, and place on ice for 30 minutes. Heat shock at 42°C for 90 seconds and place on ice for 3 minutes. Add 450 μL LB medium, 37°C, 180 rpm, 45 minutes. Pipette 200 μL of the culture solution and spread it on the Kana+LB plate medium, and cultivate overnight at 37°C.

[0063] 2. Small-dose expression test: select 5 single clones, inoculate them into 5 mL of LB medium containing kanamycin, culture at 37°C, 220 rpm, when the OD value is 0.6-1.0, add a final concentration of 1mM IPTG to induce 2h, the expression level was detected by SDS-PAGE, and clones with high expression level were selected for strain preservation.

[0064] 3. High-dose expression culture:

[0065] Inoculate...

Embodiment 3

[0066] Example 3 Purification of 7β-HSDH (N115V) Protein

[0067] Weigh 25g of 7β-HSDH (N115V) bacteria and add 250mL Lysis buffer (1XPBS) to resuspend the bacteria, and ultrasonically break the bacteria until the bacteria liquid is clear. 4°C, 10000rpm, centrifuge for 60min to take the supernatant. Pass the supernatant through a nickel column with 5ml at 4°C with a flow rate of 3ml / min, wash the column with 100mL Lysis buffer until the reading of the UV detector remains unchanged, and wash off the bound foreign proteins with 50ml wash buffer (1XPBS, 20mM imidazole) until the UV detection The instrument reading remained unchanged, and the target protein was eluted with 20ml Elution buffer (1XPBS, 300mM imidazole), and stored at -80°C. The resulting sample was subjected to SDS-PAGE to identify its molecular weight and purity, and the BCA kit was used to test the concentration of the purified protein. The results were as follows: figure 2 shown.

[0068] Such as figure 2 A...

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Abstract

The invention discloses a 7beta-hydroxysteroid dehydrogenase mutant, and belongs to the technical field of biological enzymes. According to the 7 beta-hydroxysteroid dehydrogenase, asparagine at the 115th site of the amino acid sequence of the wild 7 beta-hydroxysteroid dehydrogenase is mutated into valine with stronger nonpolarity, so that the hydrophobicity of the molecular surface is changed, and the thermal stability of the 7 beta-hydroxysteroid dehydrogenase is improved on the premise of not influencing the activity; the method prolongs the activity time of the enzyme in a high-temperature reaction system, avoids the embarrassing situation that the enzyme is high in activity and short in maintenance time at a high temperature and low in activity and long in maintenance time at a low temperature, can effectively solve the problem that the low temperature needs to be controlled by industrial production and is difficult to realize, and is suitable for large-scale industrial production.

Description

technical field [0001] The invention relates to the technical field of biological enzymes, in particular to a 7β-hydroxysteroid dehydrogenase mutant and application thereof. Background technique [0002] Ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid (TUDCA) are the main active ingredients of the precious traditional Chinese medicine bear bile, which are clinically used to treat various gallstone diseases, various acute and chronic liver diseases, and have good effects . The yield of extracting UDCA from bear bile of artificially raised bears is low, the source is limited, and it is against animal protection, so artificial synthesis of UDCA is of great significance. At present, the artificial synthesis methods of UDCA and TUDCA usually include chemical synthesis and biotransformation, among which the biotransformation has attracted increasing attention due to its advantages of low cost, simple process, green environmental protection and mild reaction. [0003] T...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P33/06C12R1/19
CPCC12N9/0006C12N15/70C12P33/06C12Y101/01201C12N2800/101C12N2800/22
Inventor 宋建芳
Owner 宋建芳
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