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7α-hydroxysteroid dehydrogenase gene s1-a-1

A technology of hydroxysteroid and dehydrogenase, which is applied in genetic engineering, plant genetic improvement, enzymes, etc., can solve the problem that the activity and stability of hydroxysteroid dehydrogenase cannot meet industrial production at the same time, and achieve the effect of huge industrial application value

Active Publication Date: 2019-07-26
CHONGQING KINBEAR BIOTECHNOLOGY CO LTD
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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 gene s1-a-1
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  • 7α-hydroxysteroid dehydrogenase gene s1-a-1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 17

[0043] Example 1.7 Isolation of α-HSDH Gene

[0044] 1. Gene discovery

[0045]Using a sterilized medicine spoon, a feces sample from a healthy black bear in the Sichuan Black Bear Conservation and Incubation Base was collected, stored in dry ice and transported back to the laboratory. Using the Qiagen Fecal DNA Genome Extraction Kit, the total DNA of the black bear feces was extracted according to the kit instructions, and submitted to Shanghai Meiji Biomedical Technology Co., Ltd. for metagenomic sequencing. Then, a new 7α-HSDH gene was found by comparing the existing Clostridium sardinia 7α-hydroxysteroid dehydrogenase (7α-HSDH) coding gene sequence (Genebank No. AET80685) with the metagenomic sequencing data , named 7α-HSDHS1-a-1, and its nucleotide sequence is shown in SEQ ID NO.2. Sequence alignment results showed that the identity of this new 7α-HSDH gene with the existing Clostridium sardinia 7α-HSDH gene sequence was 72.34% ( figure 1 ).

[0046] 2. Isolation of G...

Embodiment 27

[0065] Example 2.7 Expression of α-HSDHS1-a-1 gene

[0066] 1. Vector construction

[0067] Using the PCR one-step directional cloning kit purchased from Zuoan Protein Technology Co., Ltd., according to the operation steps in the kit manual, the full-length sequence of the 7α-HSDHS1-a-1 gene was ligated to the vector pGEX-6p-1.

[0068] 2. Transformation of E.coli DH5α competent cells

[0069] 1) Competent cells E.coli DH5α were placed on ice to thaw.

[0070] 2) Add the connection system obtained in step 1 into the melted E.coli DH5α competent, and keep it on ice for 30 minutes.

[0071] 3) Heat treatment at 42°C for 90s.

[0072] 4) Stand on ice for 2 minutes.

[0073] 5) Add 600 μL of LB medium, the temperature of the shaker is 37° C., the shaking speed of the shaker is 150 rpm, and the time is 45 minutes.

[0074] 6) Aspirate 200 μL of bacterial solution and apply it on ampicillin (Amp + ) on resistant LB plate medium.

[0075] 7) Cultivate overnight at 37°C.

[00...

Embodiment 37

[0101] Example 3.7 Functional identification of α-HSDHS1-a-1 gene

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Abstract

The invention relates to HSDH (hydroxysteroid dehydrogenase), in particular to a gene S1-a-1 of novel 7 alpha-HSDH. A nucleotide sequence of the gene is shown as SEQ ID NO.2, the novel 7 alpha-HSDH is encoded, a nucleotide sequence of the novel 7 alpha-HSDH is shown as SEQ ID NO.1, the novel 7 alpha-HSDH can be used for catalyzing CDCA (chenodeoxycholic acid) and TCDCA (taurochenodeoxycholic acid) to generate 7K-LCA (7-ketone lithocholic acid) and T7K-LCA (taurine-7-ketone lithocholic acid), wherein the catalytic activity of the novel 7 alpha-HSDH for CDCA is about 5 times of that of 7 alpha-HSDH of Sardinia clostridium, and the catalytic activity of the novel 7 alpha-HSDH for TCDCA is about over 2.5 times of that of 7 alpha-HSDH of Sardinia clostridium, therefore, the novel 7 alpha-HSDH has a great industrial application value.

Description

technical field [0001] The invention relates to hydroxysteroid dehydrogenase, in particular to a 7α-hydroxysteroid dehydrogenase gene S1-a-1. 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 a high degree of 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 acid (C...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P33/00
CPCC12N9/0006C12P33/00C12Y101/01159
Inventor 祝连彩王伯初宋璨娄德帅季顺林
Owner CHONGQING KINBEAR BIOTECHNOLOGY CO LTD
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