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A 7β-hydroxysteroid dehydrogenase mutant, coding sequence, recombinant expression vector, genetic engineering bacteria and application

A technology of hydroxysteroids and mutants, which is applied in genetic engineering, application, plant gene improvement, etc., can solve the problems of small reduction-oxidation activity ratio, product feedback inhibition, and low reduction activity, and achieve a large reduction-oxidation activity ratio and reduction activity High, fast response effect

Active Publication Date: 2020-12-04
HUNAN FLAG BIOTECHNOLOGY CO LTD
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  • Description
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AI Technical Summary

Problems solved by technology

[0008] The primary purpose of the present invention is to provide a 7β-hydroxysteroid dehydrogenase mutant to solve the problem of low reducing activity and high oxidation activity of the wild-type enzyme when preparing ursodeoxycholic acid or tauroursodeoxycholic acid. Technical problems such as small reduction-oxidation activity ratio, severe product feedback inhibition, and long reaction time

Method used

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  • A 7β-hydroxysteroid dehydrogenase mutant, coding sequence, recombinant expression vector, genetic engineering bacteria and application
  • A 7β-hydroxysteroid dehydrogenase mutant, coding sequence, recombinant expression vector, genetic engineering bacteria and application
  • A 7β-hydroxysteroid dehydrogenase mutant, coding sequence, recombinant expression vector, genetic engineering bacteria and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Construction, expression and purification of recombinant protein of wild-type 7β-HSDH recombinant genetically engineered bacteria

[0045] 1-1 Construction and expression of wild-type 7β-HSDH recombinant genetic engineering bacteria

[0046] In order to obtain mutant 7β-HSDH with high reducing activity, low oxidation activity and weak product inhibition, the wild-type 7β-HSDH gene and amino acid sequence used in the present invention are derived from Collinsella aerofaciens (GeneBank accession number: WP_006236005 ), the coding gene was optimized through Escherichia coli codon bias and the whole gene synthesis was carried out, and the wild-type 7β-hydroxysteroid dehydrogenase was named Ca7β-WT, and its coding gene was named 7β-HSDH-wt, See SEQ ID NO:1 and SEQ ID NO:2 for its nucleotide sequence and amino acid sequence.

[0047] refer to figure 2 , the wild-type 7β-HSDH coding gene 7β-HSDH-wt synthesized by the whole gene and the prokaryotic expression vect...

Embodiment 2

[0053] Embodiment 2: Preparation of 7β-HSDH mutant

[0054] 2-1 Mutant library construction and high-throughput screening method

[0055] 2-1-1 Construction of 7β-HSDH mutant library

[0056] In order to improve the activity of wild-type 7β-HSDH, the inventors used the recombinant expression vector pET-7β-HSDH-wt as a DNA template, wherein the primers were T7 universal primers (SEQ ID NO: 7 and 8), and the error-prone PCR method was used to Construct a library of random mutants and adjust the error-prone PCR reaction system by Mg 2+ and Mn 2+ Concentration and concentration of dCTP and dTTP oligonucleotides, so that the base mismatch rate of the mutant library is 5 / 1000, that is, to ensure that a mutant has 1 to 3 amino acid mutations, the specific process of constructing the mutant library is as follows .

[0057] Error-prone PCR reaction system:

[0058] 10×Buffer 5μL 2mmol / L dNTPS 5μL 100mmol / L dCTP 0.5μL 100mmol / L dTTP 0.5μL 10mmol...

Embodiment 3

[0079] Example 3: Comparison of wild-type and mutant 7β-HSDH product inhibition

[0080] The above-mentioned wild-type and mutant 7β-HSDH were purified according to the method of Example 1, and the obtained pure enzyme solution was placed in 10mM, 25mM, 50mM and 100mM UDCA solutions with the same enzyme amount (1000U) respectively ( Since the results of the T-UDCA product inhibition experiment are similar to those of UDCA, here only UDCA is used as an example), after incubation at 30° C. for 1 hour, the residual activity was measured, and the specific data are shown in Table 3.

[0081] The results showed that with the increase of UDCA concentration, the activity of wild-type Ca7β-WT decreased sharply, and at a concentration of 100mM, its activity decreased by nearly 90%; while the Ca7β-2 mutant had better activity than wild-type Ca7β-WT UDCA tolerance, its feedback inhibition by UDCA weakened very much, at a concentration of 100mM, its activity decreased by only 8.4%.

[008...

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Abstract

The invention provides a 7 Beta-Hydroxysteroid dehydrogenase mutant, a coding sequence, a recombinant expression vector, a genetically engineered bacterium and an application thereof. Mutation is carried out on the 7Beta-hydoxy alcohol dehydrogenase from Collins aerogenes, and the reductive activity and the ratio of reducing and oxidizing activity of the obtained mutant Ca7Beta-2 are respectivelyincreased by 7.6 times and 4 times; when incubated in 100mM UDCA for 1 hour, the activity is decreased by 8.4%; at 30 DEG C and Ph 8.0, UDCA and T-UDCA are catalyzed and synthesized, and the conversion time is shortened from 24 hours to 2 hours, and the molar conversion of the substrate 7-KLCA is 100%, and the molar conversion of the substrate T-7-KLCA is 99.5% and the 7Beta-HSDH after mutation greatly reduces the production cost, improves the production efficiency, and is more suitable for industrial application.

Description

technical field [0001] The invention belongs to the field of genetic engineering and enzyme engineering, and specifically relates to a 7β-hydroxysteroid dehydrogenase mutant, a coding sequence, a recombinant expression vector, genetic engineering bacteria and their preparation of ursodeoxycholic acid and tauroursodeoxycholic acid in the application. Background technique [0002] Ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid (T-UDCA) are the main active ingredients of the famous traditional Chinese medicine - "bear bile powder", which have been used in the treatment of various gallstone diseases, various acute and chronic Liver disease, has obvious curative effect. The traditional preparation method of UDCA and T-UDCA is to extract from bear bile, but the extraction process of this method is difficult, the product yield is low, the method is too cruel, and it violates the animal protection law. [0003] At present, in addition to the "bear bile extraction method...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P33/00C12R1/19
CPCC12N9/0006C12N15/70C12P33/00C12Y101/01201
Inventor 黄斌周晶辉赵强许岗
Owner HUNAN FLAG BIOTECHNOLOGY CO LTD
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