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7β-hydroxysteroid dehydrogenase gene y1-b-1

A hydroxysteroid and dehydrogenase technology, applied in genetic engineering, plant genetic improvement, enzymes and other directions, can solve the problem that the activity and stability of hydroxysteroid dehydrogenase cannot meet industrial production at the same time, and achieve large industrial application prospects, good The effect of thermal stability

Active Publication Date: 2019-07-26
CHONGQING KINBEAR BIOTECHNOLOGY CO LTD
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  • Summary
  • 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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  • 7β-hydroxysteroid dehydrogenase gene y1-b-1
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  • 7β-hydroxysteroid dehydrogenase gene y1-b-1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1. Isolation of 7β-HSDH Gene

[0049] 1. Gene discovery

[0050] Using a sterilized medicine spoon, a fecal sample of a healthy black bear in the Sichuan Black Bear Conservation and Incubation Base was collected, stored on dry ice and transported back to the laboratory. The total DNA of the 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 sardinia 7β-hydroxysteroid dehydrogenase (7β-HSDH) coding gene sequence (Genebank No. AET80684) and metagenomic sequencing data, a new 7β-HSDH gene was found, Named as 7β-HSDHY1-b-1, its nucleotide sequence is shown in SEQ ID NO.2. Sequence alignment results showed that the identity of this new 7β-HSDH gene with the existing Clostridium sardinia 7β-HSDH gene sequence was 56.40% ( figure 1 ).

[0051] 2. Isolation of Genes

[0052] (1) Primer design: design primers ...

Embodiment 27

[0070] Example 2.7 Expression of β-HSDHY1-b-1 gene

[0071] 1. Vector construction:

[0072] Using the PCR one-step directional cloning kit purchased by Zuoan Protein Technology Co., Ltd., the full-length sequence of the 7β-HSDHY1-b-1 gene was ligated to the vector pGEX-6p-1 according to the operation steps in the kit manual.

[0073] 2. Transformation of E.coli DH5α competent cells

[0074] 1) Competent cells E.coli DH5α 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 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.

[0079] 6) Aspirate 200 μL of bacterial solution and apply it on ampicillin (Amp + ) on resistant LB plate medium.

[0080] 7) Cultivate overnight at 37°C.

[0081...

Embodiment 37

[0106] Example 3.7 Functional identification of β-HSDHY1-b-1 gene

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Abstract

The invention relates to a hydroxysteroid dehydrogenase and particularly relates to a novel 7beta-hydroxysteroid dehydrogenase gene Y1-b-1. A nucleotide sequence of the gene is as shown in SEQ ID NO.2; a novel 7beta-hydroxysteroid dehydrogenase is encoded and an amino acid sequence thereof is as shown in SEQ ID NO.1. Epimerization of 7-hydroxy of ursodesoxycholic acid (UDCA) and tauroursodeoxycholic acid (TUDCA) can be catalyzed to generate intermediates 7-ketone lithocholic acid (7K-LCA) and taurine 7-ketone lithocholic acid (T7K-LCA) of chenodeoxycholic acid (CDCA) and taurochenodeoxycholic acid (TCDCA). The specific activity of the novel 7beta-hydroxysteroid dehydrogenase gene Y1-b-1 on the UDCA and the TUDCA is equivalent to that of existing clostridium sardiniense 7beta-HSDH, but the novel 7beta-hydroxysteroid dehydrogenase gene Y1-b-1 has better heat stability and a great industrial application prospect.

Description

technical field [0001] The invention relates to hydroxysteroid dehydrogenase, in particular to a 7β-hydroxysteroid dehydrogenase gene Y1-b-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 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 acid (Chenodeoxychol...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P33/02
CPCC12N9/0006C12P33/02C12Y101/01201
Inventor 王伯初宋璨祝连彩娄德帅张智
Owner CHONGQING KINBEAR BIOTECHNOLOGY CO LTD
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