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12-Hydroxycholic acid dehydrogenase and its application

A technology for hydroxycholic acid dehydrogenase and cholic acid, which is applied to 12-hydroxycholic acid dehydrogenase and its application field, and achieves the effects of high activity, environmental friendliness, and simple and convenient preparation method.

Active Publication Date: 2022-02-15
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Up to now, no natural NAD(H)-dependent 12-hydroxysteroid dehydrogenase sequence has been reported, because in the reaction process, it is often necessary to add the cofactor NAD(P) + Increase the reaction rate to achieve the purpose of increasing the substrate concentration, but NADP + The price is NAD + Therefore, the discovery and use of NAD(H)-dependent 12-hydroxysteroid dehydrogenase can significantly reduce the cost of cofactors in the reaction. The awareness of environmental protection issues has improved, so it is necessary to be able to establish a green catalytic process with high substrate concentration and high conversion rate

Method used

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  • 12-Hydroxycholic acid dehydrogenase and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Example 1: Synthesis of 12-hydroxycholic acid dehydrogenase gene and construction of genetically engineered bacteria

[0014] 1.1 Synthesis of 12-hydroxycholic acid dehydrogenase gene

[0015] The possible 12-hydroxycholic acid dehydrogenase sequence from Eggerthella lenta was mined by gene mining method, and the gene was synthesized by codon optimization according to the protein sequence, and constructed into pET21a expression vector, the gene insertion sites were NdeI and XhoI.

[0016] 1.2 Transformation of recombinant plasmids

[0017] Competent Escherichia coli cells were prepared by calcium chloride method.

[0018] (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.

[0019] (2) Heat-shock in a water bath at 42°C for 45 seconds, and quickly place on ice for 1-2 minutes.

[0020] (3) Add 600 μL of fresh LB liquid medium, shake and culture at 37°C for 45-60min.

[0021] (4) Take ...

Embodiment 2

[0022] Embodiment 2: the construction of co-expression bacteria

[0023] Recover the pRSFDuet-flavin reductase vector already in the laboratory with NdeI / XhoI double enzyme digestion, and recover the gene fragment after pET-12-steroid dehydrogenase double enzyme digestion with NdeI / XhoI, and use T4 DNA for the fragment and linear vector After ligation with ligase, BL21(DE3) was transformed into competent cells, single clones were picked for verification and activity detection, and pRSFDuet-flavin reductase-12-hydroxysteroid dehydrogenase co-expression strains were obtained.

[0024] Recover the pRSFDuet-lactate dehydrogenase vector already in the laboratory with NdeI / XhoI double enzyme digestion, pET-12-steroid dehydrogenase with NdeI / XhoI double enzyme digestion and recover gene fragments, and T4 DNA for fragments and linear vectors After ligase ligation, BL21(DE3) was transformed into competent cells, single clones were picked for verification and activity detection, and pRS...

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 the fermentation medium (industrial medium) at an inoculum size of 1%, and cultured at 32° C. and 200 rpm for 20 h. After 1 mL of the fermentation liquid was ultrasonically disrupted, the activities of 12-hydroxycholic acid dehydrogenase and flavin reductase were detected. The definition of 12-hydroxycholic acid dehydrogenase enzyme activity: the amount of enzyme required to generate 1 μmol NADH per minute is 1 enzyme activity unit (U); the definition of enzyme activity of flavin: the amount of enzyme required to consume 1 μmol NADH per minute is 1 enzyme activity u...

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Abstract

The invention discloses a novel 12-hydroxycholic acid dehydrogenase derived from Eggerthella lenta and its encoding gene, and uses the enzyme as a biocatalyst to catalyze the oxidation of cholic acid and its derivative 12-hydroxyl to a carbonyl substrate. The gene encoding this enzyme has low homology with the currently known 12-hydroxysteroid dehydrogenase, and has high enzyme activity to 12-hydroxycholic acid and its derivatives, and in the process of cultivating the Escherichia coli expressing the gene, no Antibiotics are required, and efficient expression can be achieved without traditional isopropyl‑β‑D‑thiogalactopyranoside (IPTG) induction. In the conversion process, after concentrating the bacteria, the substrate concentration can reach 200 g / L, and the conversion rate is >95%, and no organic solvent is used in the reaction process, and the conversion method is green and environmentally friendly.

Description

technical field [0001] The present invention relates to a 12-hydroxycholic acid dehydrogenase from Eggerthella lenta, a sequence encoding the enzyme, a method for obtaining the enzyme, and a method for oxidizing the 12-hydroxyl in cholic acid and its derivatives. Background technique [0002] Ursodeoxycholic acid can be used in the treatment of fatty liver with hyperlipidemia (the effect of ursodeoxycholic acid combined with Lycox in the treatment of fatty liver with hyperlipidemia, Wei Hong, 2017, 30, 133), ursodeoxycholic acid Acid monotherapy for hepatitis in primary biliary cirrhosis. Most of the current methods for synthesizing ursodeoxycholic acid 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 catalysis widely used in ursodeoxycholic acid ( The enzymic and chemical synthesis of ursodeoxycholic and chenodeoxycholic acid from cholic acid, Sutherland, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/04C12P33/02C12R1/19
CPCC12N9/0006C12P33/02
Inventor 张红榴陈曦刘祥涛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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