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7[beta]-hydroxysteroid dehydrogenase mutant and application thereof in preparation of UDCA

A technology of hydroxysteroids and dehydrogenases, applied in the direction of enzymes, oxidoreductases, biochemical equipment and methods, etc., can solve problems such as complex processes, difficult industrial applications, and impossible complete conversion of substrates, achieving high conversion rates, The effect of improving enzyme activity and simplifying the extraction and refining process

Pending Publication Date: 2020-12-04
JIANGXI BONTAC GREEN BIOCATALYSIS ECOIND PARK CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The whole reaction requires three enzymes, as well as the coenzyme regeneration system (lactate dehydrogenase and glucose dehydrogenase), the process is complicated
Due to the balance of the catalytic reaction, it is impossible to completely convert the substrate, making it difficult for industrial application

Method used

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  • 7[beta]-hydroxysteroid dehydrogenase mutant and application thereof in preparation of UDCA
  • 7[beta]-hydroxysteroid dehydrogenase mutant and application thereof in preparation of UDCA
  • 7[beta]-hydroxysteroid dehydrogenase mutant and application thereof in preparation of UDCA

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Mutant library construction and high-throughput screening methods:

[0028] Construction of mutant library:

[0029] In order to improve the transformation rate of wild-type 7β-HSDH enzyme, using the recombinant expression vector PET28a(+)-RT-7β-HSDH as DNA template, a random mutant library was constructed by error-prone PCR method, and by adjusting the error-prone PCR reaction Mg in the system 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 . Error-prone PCR reaction system and conditions:

[0030] Error-prone PCR reaction system:

[0031]

[0032] The error-prone PCR reaction conditions are: 95°C pre-denaturation for 5 minutes; then 94°C denaturation for 30 seconds, 55°C annealing for 1 minute, 72°C for 1.5 minutes, a total of 30 cy...

Embodiment 2

[0039] Mutation subcrystal structure simulation

[0040] Using the reported 7β-HSDH enzyme (PDB code: ID: 5FYD) from C. aerofaciens as a template (Structural and biochemical insights into 7β-hydroxysteroid dehydrogenase stereoselectivity. Published in 2016) (the amino acid similarity between the two is 76%), Using the SWISS-MODEL online server ( http: / / www.swissmodel.expasy.org / ) to construct the three-dimensional structure diagram of Clostridia 7β-HSDH mutant (SEQ ID NO.2). Using AutoDock software, the substrate 7-KLCA and NADPH were docked into the mutant structure ( figure 1 ). The three-dimensional structure of 7-KLCA and NADPH is downloaded from the Pubchem database ( https: / / pubchem.ncbi.nlm.nih.gov / ).

[0041] Virtual saturation mutation of amino acid 189

[0042] A virtual saturation mutation was performed on amino acid 207 in SEQ ID NO.2, and the binding affinity of the enzyme protein-ligand complex was analyzed with the help of Discovery Studio.

[0043...

Embodiment 3

[0050] Weigh 99% 7-KLCA (final concentration 200mmol / L), suspend in 100mL 50mmol / L potassium phosphate buffer (pH8.0), add equimolar NADPH, then add 100mL wild-type 7β-HSDH and mutant Sub-enzyme solution (2000U). The total reaction volume was 500 mL, and the reaction was carried out at 35°C and 200 rpm for 8 hours. The conversion rates of wild-type 7β-HSDH and mutant type were 29.8% and 56.8%, respectively.

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Abstract

The invention relates to a 7[beta]-hydroxysteroid dehydrogenase mutant and application thereof in preparation of UDCA, and belongs to the technical field of enzyme engineering. An amino acid sequenceshown in SEQ ID NO: 2 is obtained through mutant library construction and a high-throughput screening method. Key amino acids influencing the activity of 7[beta]-HSDH are determined through error-prone PCR, site-specific saturation mutagenesis is carried out, 200 mmol / L 7-KLCA99% can be converted into UDCA through the screened 7[beta] hydroxysteroid dehydrogenase mutant SEQ ID NO: 2, and the industrial production requirement is basically met.

Description

technical field [0001] The present invention relates to mutants of a 7β-hydroxysteroid dehydrogenase from the genus Clostridia, sequences encoding these enzymes, methods of producing such enzymes, and ursodeoxycholic acid (UCDA) enzymes Use in synthetic methods. Background technique [0002] Ursodeoxycholic acid (UDCA) is the main active ingredient contained in the precious traditional Chinese medicine bear bile, and it is clinically used to treat various gallstone diseases and various acute and chronic liver diseases. The synthesis methods of UCDA mainly include total chemical synthesis method and chemical-enzymatic combination method, and the starting material is generally animal-derived cholic acid (CA) or deoxycholic acid (CDCA). [0003] The classic chemical synthesis method of UDCA is as follows. Since the chemical oxidation is non-selective, the 3[alpha]- and 7[alpha]-hydroxyl groups must be protected by means of esterification. In addition, the reduction of 7-keto...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12P33/02
CPCC12N9/0006C12P33/02C12Y101/01201
Inventor 宋鹏
Owner JIANGXI BONTAC GREEN BIOCATALYSIS ECOIND PARK CO LTD
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