7alpha-oxhydryl sterol dehydrogenase and encoding gene and application thereof

A hydroxysteroid and dehydrogenase technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problem that the activity and stability of hydroxysteroid dehydrogenase cannot meet the needs of industrial production at the same time

Inactive Publication Date: 2019-10-22
CHONGQING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Enzyme activity and thermal stability are important parameters to determine whether it can be put into industrial production. The activity and stability of the existing hydroxysteroid dehydrogenase cannot meet the needs of industrial production at the same time. Therefore, it is necessary to further find and develop new enzymes suitable for Hydroxysteroid dehydrogenase for industrial mass production

Method used

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  • 7alpha-oxhydryl sterol dehydrogenase and encoding gene and application thereof
  • 7alpha-oxhydryl sterol dehydrogenase and encoding gene and application thereof
  • 7alpha-oxhydryl sterol dehydrogenase and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1、7

[0050] Example 1, Discovery of 7α-HSDH St-2-2 Gene

[0051] Fecal samples from healthy black bears in the Sichuan Black Bear Conservation and Incubation Base were collected using sterilized medicine spoons, stored on dry ice and transported back to the laboratory. The total DNA of black bear feces was extracted using the Qiagen Fecal DNA Genome Extraction Kit, and submitted to Shanghai Meiji Biomedical Technology Co., Ltd. for metagenomic sequencing. Compared with the existing Clostridium sardinica 7α-hydroxysteroid dehydrogenase (Clostridium sardinia 7α-HSDH) coding gene sequence (Genebank accession number is AET80685) and metagenomic sequencing data, a new 7α-Hydroxysteroid dehydrogenase gene, named 7α-HSDH St-2-2, its nucleotide sequence is shown in sequence 2 in the sequence listing, the open reading frame is 789bp; its encoded 7α-HSDH St-2- 2 The protein consists of 262 amino acids, and the amino acid sequence is shown in SEQ ID NO: 1. Sequence alignment results showed ...

Embodiment 2、7

[0052] Cloning of Example 2, 7α-HSDH St-2-2 Gene

[0053] (1) Primer design: design primers for the new 7α-HSDH St-2-2 gene sequence obtained by sequencing, and the nucleotide sequences of the primers are as follows:

[0054] St-2-2-BamHI-F: 5'-CGCGGATCCATGAAAAGAGTAGAAAATAAAGTAGC-3' (sequence 3 in the sequence listing),

[0055] St-2-2-XhoI-R: 5'-CCGCTCGAGTTATTTTCTTAACAGCATCCCCAT-3' (sequence 4 in the sequence listing).

[0056] (2) PCR amplification: the total DNA of black bear feces was used as a template, and the primers obtained in step (1) were used to amplify according to the following PCR system and procedure.

[0057] The PCR system is: template DNA 0.5 μL, St-2-2-BamHI-F (10 μM) 2 μL, St-2-2-XhoI-R (10 μM) 2 μL, 0.1% BSA 3 μL, PrimeSTAR HS DNA Polymerase 25 μL, ddH 2 0 to 50 μL.

[0058] The PCR program is as follows:

[0059] a. Pre-denaturation at 94°C for 5 minutes;

[0060] b. Denaturation at 98°C for 10 sec, annealing at 53°C for 10 sec, extension at 72°C fo...

Embodiment 3、7

[0067] Example 3, Expression, extraction and purification of 7α-HSDH St-2-2 gene

[0068] 1. Construction of the ligation vector: the full-length sequence of the 7α-HSDH St-2-2 gene was ligated to the vector pGEX-6p-2.

[0069] The ligation system includes: 6 μL of double digested 7α-HSDH St-2-2 gene, 1 μL of T4 DNA Ligase, 2 μL of vector pGEX-6p-2, 1 μL of 10×T4 Ligase buffer, 10 μL of the system, and ligation at 16°C for 12 hours.

[0070] 2. To transform E.coli DH5α competent cells, the steps are as follows:

[0071] 1) Competent cells E.coli DH5α were placed on ice to thaw.

[0072] 2) Add the ligation product obtained in step 1 into the melted E.coli DH5α competent, and keep it on ice for 30 minutes.

[0073] 3) Heat treatment at 42°C for 90s.

[0074] 4) Stand on ice for 2 minutes.

[0075] 5) Add 600 μL of anti-antibody-free LB medium, shaker temperature 37° C., shaker speed 150 rpm, time 45 min.

[0076] 6) Aspirate 200 μL of bacterial solution and apply it on amp...

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Abstract

The invention relates to 7alpha-oxhydryl sterol dehydrogenase and an encoding gene and application thereof, and belongs to the technical field of biology. The 7alpha-oxhydryl sterol dehydrogenase is protein shown in SEQ ID NO.1 or protein with the same functions, obtained through substitution and/or deletion and/or addition of one or more amino acid residues of an amino acid sequence shown in theSEQ ID NO.1. The 7alpha-oxhydryl sterol dehydrogenase can catalyze an asymmetric reduction reaction of carbanyl groups of C7alpha-oxhydryl groups of taurocholic acid, glycocholic acid, taurochenodeoxycholic acid and glycochenodeoxycholic acid and catalyze an asymmetric reduction reaction of carbanyl groups and oxhydryl groups in benzoyl groups of ethyl benzoylformate. Compared with 7alpha-oxhydrylsterol dehydrogenase in clostridium sardiniense, the 7alpha-oxhydryl sterol dehydrogenase has higher catalytic activity and thermostability, and has very high industrial application value.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to 7α-hydroxysteroid dehydrogenase and its encoding gene and application. Background technique [0002] Asymmetric reduction of carbonyl groups has always been one of the hotspots in chemical reaction research. Although chemical methods have achieved certain results, chemical methods often have disadvantages such as limited types and numbers of catalysts, low stereoselectivity, expensive auxiliary reagents and difficult recovery. The enzymatic reaction is not only highly efficient, chemoselective, regioselective but also highly stereoselective. Hydroxysteroid dehydrogenase (Hydroxysteroid dehydrogenase, HSDH)-mediated enzymatic reactions have relatively strict stereoselectivity and "not" strict substrate specificity. For example, as early as the early 1980s, scientists have begun to try to synthesize ursodeoxycholic acid by using 7α- and 7β-HSDH produced by microorganisms comb...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P33/00C12P7/62C12R1/19
CPCC12N9/0006C12N15/70C12P7/62C12P33/00C12Y101/01159
Inventor 潘银平唐士金杨碧玲赵文艳祝连彩王伯初
Owner CHONGQING UNIV
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