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Hydroxysteroid dehydrogenase and application thereof in synthesis of ursodeoxycholic acid precursor

A technology of hydroxysteroids and dehydrogenases, applied in the direction of application, microbial-based methods, enzymes, etc., can solve the problem of high cost of coenzyme application

Active Publication Date: 2019-10-29
EAST CHINA UNIV OF SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The technical problem to be solved by the present invention is to provide a kind of Rhodococcus (Rhodococcus ruber) source of high catalytic activity, strong specificity, NAD + coenzyme-dependent hydroxysteroid dehydrogenase

Method used

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  • Hydroxysteroid dehydrogenase and application thereof in synthesis of ursodeoxycholic acid precursor
  • Hydroxysteroid dehydrogenase and application thereof in synthesis of ursodeoxycholic acid precursor
  • Hydroxysteroid dehydrogenase and application thereof in synthesis of ursodeoxycholic acid precursor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Gene cloning and transformation expression of hydroxysteroid dehydrogenase Rr12α-HSDH

[0058] The complete genome of Rhodococcus ruber with the preservation number of CGMCC No.1.10360 was obtained by the high-salt method. Inoculate Rhodococcus ruber with the preservation number of CGMCC No.1.10360 into LB medium, incubate at 37°C for 24 hours, take 100 mL of the bacterial solution, centrifuge at 8,000 rpm for 10 minutes, collect the bacteria, wash with 20 mL of saline, and repeat Twice, then resuspend the bacteria in 20 mL of saline. Add 1mL of lysozyme solution (50mg / mL) to the suspension bacteria, and keep the temperature in a 37℃ water bath for 1h; add 1.6mL of sodium lauryl sulfonate solution (10%, w / v), 160μL of proteinase K (20mg / mL) , Keep the temperature in 55℃ water bath until the suspension becomes clear. Add 1 / 3 volume of saturated NaCl solution, shake and mix until the solution becomes turbid, then centrifuge at high speed for 10 minutes to discard...

Embodiment 2

[0060] Example 2 Random mutation of hydroxysteroid dehydrogenase Rr12α-HSDH

[0061] Based on the gene mining of the enzyme in Example 1, error-prone PCR technology was used for random mutation to further improve the activity of the enzyme.

[0062] According to the open reading frame of Rr12α-HSDH, the upstream and downstream primers are designed as follows:

[0063] Upstream primer, as shown in SEQ ID No. 3:

[0064] 5’-CCG GAATTC ATGAAACTGCGCGGGAAGA-3’

[0065] Downstream primer, as shown in SEQ ID No. 4:

[0066] 5’-CCC AAGCTT TCACCGCAGCTTGATGCTGC-3’

[0067] The sequence underlined in the upstream primer is the restriction site of EcoRI, and the sequence underlined in the downstream primer is the restriction site of Hind III.

[0068] Using pET28a-12α-HSDH as a template, error-prone PCR was performed with rTaq DNA polymerase to construct a random mutation library.

[0069] The PCR system (50μL) contains the following components:

[0070] Taq DNA polymerase 0.5μl, 10×PCR buffer(Mg 2+ P...

Embodiment 3

[0091] Example 3 Hydroxysteroid dehydrogenase Rr12α-HSDH M8 Gene cloning

[0092] On the basis of the gene cloning described in Example 1, according to the hydroxysteroid dehydrogenase Rr12α-HSDH M8 Design the upstream and downstream primers, and use the nucleic acid sequence numbered as SEQ ID No. 6 as the template for PCR amplification.

[0093] Upstream primer, as shown in SEQ ID No. 3:

[0094] 5’-CCG GAATTC ATGAAACTGCGCGGGAAGA-3’

[0095] Downstream primer, as shown in SEQ ID No. 4:

[0096] 5’-CCC AAGCTT TCACCGCAGCTTGATGCTGC-3’

[0097] The underlined sequence of the upstream primer is the restriction site of EcoRI, and the underlined sequence of the downstream primer is the restriction site of Hind III.

[0098] The PCR system is: 2×Taq PCR MasterMix 25μL, upstream primer and downstream primer (10ng / μL) each 2.5μL, 1μL plasmid containing the nucleic acid sequence numbered SEQ ID No.5 gene (150ng / μL), and 19μL ddH 2 O. The PCR amplification program is: pre-denaturation at 95°C for...

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Abstract

The present invention relates to a hydroxysteroid dehydrogenase and an application thereof in synthesis of an ursodeoxycholic acid precursor, and specifically discloses a 12-alpha-hydroxysteroid dehydrogenase derived from rhodococcus ruber and a mutant thereof, encoding gene and amino acid sequences, a recombinant expression vector containing the gene, a recombinant expression transformant, and anapplication of the hydroxysteroid dehydrogenase catalyzing oxidation of 12-alpha-hydroxysteroids to form 12-carbonyl steroids. Compared with the prior art, a used recombinant steroid dehydrogenase-catalyzed oxidation reaction is NAD+coenzyme-dependent type. The hydroxysteroid dehydrogenase has advantages of low application cost, simple operation, mild reaction conditions, environmental friendliness, high yield, etc., and thus has a very good prospect in an application of cholic acid as a raw material for production and preparation of the ursodeoxycholic acid precursor.

Description

Technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a hydroxysteroid dehydrogenase derived from Rhodococcusruber, its gene, a recombinant expression vector and a recombinant expression transformant containing the gene, and the use of the recombinant hydroxysteroid dehydrogenase Enzymes or recombinant expression transformants are used as biocatalysts and specifically catalyze the application of sterol compounds 12α-hydroxyl to 12-ketone. Background technique [0002] Cholic Acid (CA) exists in the bile of many animals, and its chemical name is 3α, 7α, 12α-trihydroxy-5β-cholestane-24-acid. Ursodeoxycholic Acid (UDCA) is the active ingredient of the precious Chinese medicinal material, bear bile. Its chemical name is 3α,7β-dihydroxy-5β-cholestane-24-acid, also known as ursodeoxycholic acid. In 1902, Swedish chemist Hammarsten first discovered UDCA from polar bear bile. In 1927, Shoda of Okayama University in Japan separ...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P33/02C12R1/01C12R1/19
CPCC12N9/0006C12Y101/01176C12N15/70C12P33/02
Inventor 李春秀石守城游智能许建和潘江钱小龙
Owner EAST CHINA UNIV OF SCI & TECH
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