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7α-Hydroxysteroid Dehydrogenase and Its Encoding Gene and Application

An amino acid and substrate technology, applied in the fields of 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

Active Publication Date: 2021-06-15
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 suitable enzymes. Hydroxysteroid dehydrogenase for industrial mass production

Method used

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  • 7α-Hydroxysteroid Dehydrogenase and Its Encoding Gene and Application
  • 7α-Hydroxysteroid Dehydrogenase and Its Encoding Gene and Application
  • 7α-Hydroxysteroid Dehydrogenase and Its Encoding Gene and Application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1, Discovery of a new 7α-HSDHJ-1-1 gene

[0054] 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. Comparison of the existing Clostridium sardinica 7α-hydroxysteroid dehydrogenase (Clostridium sardinia 7α-HSDH) gene sequence with metagenomic sequencing data revealed a novel 7α-HSDHJ-1-1 The gene has an open reading frame from position 1 to position 786 at the 5' end of its nucleotide sequence, and the part of the open reading frame is 786bp, and the amino acid of the 7α-HSDHJ-1-1 protein encoded by it is shown in SEQ ID No.1 , Sequence 1 consists of 260 amino acid sequences. Sequence alignment results showed that the iden...

Embodiment 2

[0055] Embodiment 2, the cloning of gene

[0056] 1) Primer design: design primers for the new 7α-HSDHJ-1-1 gene sequence obtained by sequencing, and the nucleotide sequences of the primers are as follows:

[0057] J-1-1-BamHI-F: 5'-CGGGATCCATGAGAGTAAAAGATAAAATAG-3' (sequence 2 in the sequence listing),

[0058] J-1-1-XhoI-R: 5'-CGCTCGAGTTATCTTTGAACAAGGTCTCCGTATT-3' (SEQ ID NO: 3 in the Sequence Listing).

[0059] 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.

[0060] PCR system includes: template DNA is 0.5 μL, J-1-1-BamHI-F (10 μM) is 2 μL, J-1-1-XhoI-R (10 μM) is 2 μL, 0.1% BSA is 3 μL, PrimeSTAR HS DNA Polymerase It is 25μL, add ddH 2 O to PCR system is 50 μL.

[0061] The PCR program is as follows:

[0062] a. Pre-denaturation at 94°C for 5 minutes;

[0063] b. Denaturation at 98°C for 10 sec, annealing at 53°C for 10 sec, e...

Embodiment 3

[0070] Embodiment 3, expression, extraction and purification of new gene

[0071] 1. Construction of the connection vector: the full-length sequence of the 7α-HSDHJ-1-1 gene was connected to the vector pGEX-6p-2.

[0072] The ligation system includes: 6 μL for the double-digested 7α-HSDHJ-1-1 gene, 1 μL for T4 DNA Ligase, 2 μL for the vector pGEX-6p-2, 1 μL for 10×T4 Ligase buffer, 10 μL for the system, and 16 °C for 12 hours.

[0073] 2. To transform E.coli DH5α competent cells, the steps are as follows:

[0074] 1) Competent cells E.coli DH5α (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., item number: CB101) were placed on ice to thaw.

[0075] 2) Add the connection system obtained in step 1 into the melted E.coli DH5α competent, and keep it on ice for 30 minutes.

[0076] 3) Heat treatment at 42°C for 90s.

[0077] 4) Stand on ice for 2 minutes.

[0078] 5) Add 600 μL of anti-antibody-free LB medium, shaker temperature 37° C., shaker speed 150 rpm, ...

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Abstract

The invention discloses 7α-hydroxysteroid dehydrogenase, its coding gene and application. The amino acid sequence of the 7α-hydroxysteroid dehydrogenase provided by the present invention is shown in SEQ ID NO.1, and the nucleotide sequence of the gene of the 7α-hydroxysteroid dehydrogenase is also disclosed as shown in SEQ ID NO.2 Show. The 7α-hydroxysteroid dehydrogenase can catalyze the substrate taurocholic acid (TCA) to generate tauro 7-ketocholic acid (T7K-CA), and catalyze the substrate glycocholic acid (GCA) to generate glycine 7-ketocholic acid acid (G7K‑CA), catalytic substrate taurochenodeoxycholic acid (TCDCA) to generate tauro7‑ketolithocholic acid (T7K‑LCA), catalytic substrate glycochenodeoxycholic acid (GCDCA) to generate glycine Ammonia 7-ketolithocholic acid (G7K-LCA), catalytic substrate ethyl benzoylformate (Ethyl benzoylformate, EB) generates benzyl alcohol ethyl formate (Ethyl 2-hydroxy-2-phenylacetate), and has better The catalytic activity to TCDCA, GCDCA, and EB is 10 times, 5 times, and 3 times that of Clostridium sardinia 7α-HSDH, respectively, and has great industrial application value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to 7α-hydroxysteroid dehydrogenase and its coding gene and application. Background technique [0002] The asymmetric reduction of carbonyl groups has always been one of the hotspots in chemical reaction research. Although the current chemical methods have achieved certain results, they often have disadvantages such as limited types and numbers of catalysts, low stereoselectivity, expensive auxiliary reagents, and difficult recovery. The enzymatic reaction not only has high efficiency, chemoselectivity, regioselectivity but also high stereoselectivity. The enzymatic reaction mediated by hydroxysteroid dehydrogenase (Hydroxysteroid dehydrogenase, HSDH) has relatively strict stereoselectivity and "not" strict substrate specificity. For example, as early as the early 1980s, scientists have begun to use 7α-, 7β-HSDH produced by microorganisms to combine epimerization with chenodeoxycholic ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/63C12P33/02C12P7/62
CPCC12N9/0006C12P7/62C12P33/02C12Y101/01159
Inventor 王伯初季顺林唐士金祝连彩娄德帅谭君
Owner CHONGQING UNIV
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