7 beta-hydroxycholic acid dehydrogenase and application thereof

A technology of hydroxycholic acid dehydrogenase and dehydrogenase, which is applied in the field of synthesizing ursodeoxycholic acid, can solve the problems of inactivation, low purity, reduced yield and the like, and achieves mild conditions, no by-products, and conversion time. short effect

Active Publication Date: 2019-05-07
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the current industrial production, it is attempted to prepare ursodeoxycholic acid by regioselective oxidation of 7-OH and subsequent reduction of chenodeoxycholic acid, which is relatively cheap and easy to obtain, but the reaction often involves the compatibility of multiple enzymes. Sexual problems, agent coenzyme regeneration, so that the 7-OH in the first step is oxidized to carbonylation, and the reaction system is heated to promote the inactivation of the enzyme
(Two-step enzymatic synthesis of ursod

Method used

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  • 7 beta-hydroxycholic acid dehydrogenase and application thereof
  • 7 beta-hydroxycholic acid dehydrogenase and application thereof
  • 7 beta-hydroxycholic acid dehydrogenase and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0014] Example 1: Synthesis of 7β-hydroxycholic acid dehydrogenase (7β-HSDHCM) gene and construction of genetically engineered bacteria

[0015] 1. Synthesis of 17β-hydroxycholic acid dehydrogenase gene

[0016] The possible 7β-hydroxycholic acid dehydrogenase sequence from Clostridium sp.Marseille was discovered by gene mining method, and the gene was synthesized by codon optimization according to the protein sequence, and constructed into the pET21a expression vector, and the gene insertion site was NdeI and XhoI.

[0017] 1.2 Transformation of recombinant plasmids

[0018] Competent Escherichia coli cells were prepared by calcium chloride method.

[0019] (1) Put 10 μL of the recombinant plasmid in 50 μL of Escherichia coli BL21(DE3) competent cells, and place in ice bath for 30 minutes.

[0020] (2) Heat-shock in a water bath at 42°C for 45 seconds, and quickly place on ice for 1-2 minutes.

[0021] (3) Add 600 μL of fresh LB liquid medium, shake and culture at 37°C fo...

Embodiment 2

[0023] Embodiment 2: the construction of co-expression bacteria

[0024] The pRSFDuet-GDH vector already in the laboratory was digested with NdeI / XhoI and then recovered. The pET-7β-HSDHCM was digested with NdeI / XhoI to recover the gene fragment. The fragment and the linear vector were ligated with T4 DNA ligase and then transformed into BL21 (DE3 ) competence, single clones were selected for verification and activity detection, and pRSFDuet-GDH-7β-HSDHCM co-expression bacteria were obtained

Embodiment 3

[0025] Example 3: Induced expression of single-expression bacteria and co-expression bacteria

[0026] Prepare 50 mL of seed liquid, and the medium is LB liquid medium (peptone 10g / L, yeast powder 5g / L, NaCl 10g / L), pick a single colony of genetically engineered bacteria with an inoculation loop and insert it into the medium, 37°C, 200rpm Incubate overnight. The overnight cultured seed solution was transferred to a fermentation medium (industrial medium) at an inoculum size of 1%, and cultured at 32° C. and 200 rpm for 20 h.

[0027] After 1 mL of the fermentation liquid was ultrasonically disrupted, the activities of 7β-hydroxycholic acid dehydrogenase and glucose dehydrogenase were detected. The definition of 7β-hydroxycholic acid dehydrogenase enzyme activity: the amount of enzyme required to consume 1 μmol NADPH per minute is 1 enzyme activity unit (U); the enzyme activity definition of glucose dehydrogenase: the enzyme required to generate 1 μmol NADPH per minute The am...

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Abstract

The invention discloses novel 7 beta-hydroxycholic acid dehydrogenase (7 beta-HSDHCM) derived from Clostridium sp. Marseille, and encoding gene of 7 beta-HSDHCM. Ursodesoxycholic acid is synthesized by using 7 beta-HSDHCM as a biocatalyst to catalyze a substrate, namely 7-oxocholic acid. The gene encoding 7 beta-HSDHCM and currently known 7 beta-hydroxylsteride dehydrogenase have low homology; 7 beta-HSDHCM has high enzyme activity for 7-oxocholic acid; antibiotics are not required to be added in a culture process of escherichia coli expressing the gene; and high-efficiency expression can be achieved without induction by traditional isopropyl-beta-D-thiogalactoside (IPTG). In a conversion process, the concentration of the substrate is 10-60g/L; after fermentation liquid is concentrated, the concentration of the substrate reaches 100g/L; a conversion rate greater than 98% is achieved; any organic solvent is not used in a reaction process; and the conversion method is green and environmentally friendly.

Description

[0001] The present invention relates to the acquisition of a new 7β-sequence encoding this enzyme from Clostridium sp. Marseille, a method for obtaining this enzyme, and a new method for the synthesis of ursodeoxycholic acid (UDCA) in the enzymatic conversion of cholic acid compounds. Background of the invention: [0002] The active substance ursodeoxycholic acid (UDCA) has been used for many years in the medical treatment of gallstone problems. Ursodeoxycholic acid can be used to treat fatty liver complicated with hyperlipidemia (the effect of ursodeoxycholic acid combined with Lycox in the treatment of patients with fatty liver complicated with hyperlipidemia, Wei Hong, 2017, 30, 133), Xiong Qu Oxycholic acid monotherapy for hepatitis in primary biliary cirrhosis. Most of the current synthesis methods are obtained by using cholic acid as a substrate through chemical oxidation-reduction methods. Only in recent years, the development of biocatalysis has made enzymatic catalysi...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/04C12P33/06
Inventor 陈曦崔云凤王玉黄清东冯进辉吴洽庆朱敦明马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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