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7[beta] hydroxy cholesterol dehydrogenase mutant and application thereof

A technology of hydroxysteroids and dehydrogenases, applied in the field of enzyme engineering, to achieve the effects of increasing tolerance concentration, improving enzyme production capacity and reducing production costs

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

[0010] Although domestic and foreign scientific researchers have used genetic engineering and protein engineering techniques to mutate and transform 7β-hydroxysteroid dehydrogenase, making its activity doubled, but there is still a lot of research on enhancing its substrate tolerance. rarely reported

Method used

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  • 7[beta] hydroxy cholesterol dehydrogenase mutant and application thereof
  • 7[beta] hydroxy cholesterol dehydrogenase mutant and application thereof
  • 7[beta] hydroxy cholesterol dehydrogenase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Mutant library construction and high-throughput screening methods:

[0025] Construction of mutant library:

[0026] In order to improve the activity of wild-type 7β-HSDH enzyme, using the recombinant expression vector PET28a(+)-RT-7 β-HSDH as a DNA template, a random mutant library was constructed by error-prone PCR, 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:

[0027] Error-prone PCR reaction system:

[0028]

[0029] 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 cycles; finally 7...

Embodiment 2

[0035] Mutant daughter crystal structure simulation:

[0036] 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%), The three-dimensional structure diagram of Clostridia 7β-HSDH mutant (SEQ ID NO: 2) was constructed by using the SWISS-MODEL online server (http: / / www.swissmodel.expasy.org / ). Using AutoDock software, the substrate 7-KLCA and NADPH were docked into the mutant structure ( figure 1). The three-dimensional bodies of 7-KLCA and NADPH are downloaded from the Pubchem database (https: / / pubchem.ncbi.nlm.nih.gov / ).

[0037] Virtual saturation mutation of amino acid 207:

[0038] 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 by Discovery Studio.

[0039] step:

[0040]...

Embodiment 3

[0046] Determination of 7β-HSDH enzyme activity:

[0047] 2.7mL 50mM phosphate buffer (pH 8.0), 0.2mL 7-ketolithocholic acid (7-KLCA) (final concentration 200mmol / L) (dissolved in buffer), 50mM phosphate buffer (pH 8.0) Dilute 0.05 mL of 7β-HSDH crude enzyme solution sample by a certain factor, mix it in a cuvette, place it in an ultraviolet spectrophotometer, and return the absorbance to zero.

[0048] Take 0.05ml NAD(P)H (50mg / mL) into the cuvette, mix well and start timing for 2min, read the absorbance change value of 340nm wavelength within 2min, and calculate △OD / min;

[0049] Blank control: The operation procedure is the same as above, but the enzyme in the reaction system is replaced by an equal amount of Tris / HCl buffer solution, and the measured result is a negative control.

[0050] Enzyme activity unit definition:

[0051] Enzyme activity (U / mL) = △OD / min*Vt*df / (6.22*1.0*Vs)

[0052] Vt: total reaction volume 3.05mL

[0053] Df:: dilution factor

[0054] 6.22: ...

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Abstract

The invention relates to a 7[beta] hydroxy cholesterol dehydrogenase mutant and application thereof, and belongs to the technical field of enzyme engineering. Through mutant library construction and ahigh throughput screening method, an amino acid sequence disclosed by SEQ ID NO: 2 is obtained. Through error-prone PCR (Polymerase Chain Reaction), a key amino acid which affects the activity of 7[beta]-HSDH (hydroxysteroid dehydrogenase) is determined, and the tolerated concentration of a 7[beta]-HSDH substrate is improved. The endurance capacity of the mutated 7[beta]-HSDH substrate is obviously enhanced than original 7[beta]-HSDH, and the mutated 7[beta]-HSDH substrate can endure 200 mmol / L of 7-ketone-lithocholic acid (7-KLCA (Ketolithocholic acid)) substrate concentration. The recombinant escherichia coli, which is constructed by the invention, of which the 7[beta]-HSDH secretion capacity is enhanced can improve 7[beta]-HSDH enzyme activity by 2.47 times than an original strain. The enzyme production ability of an improved genetically engineered bacterium is obviously improved, the genetically engineered bacterium is more suitable for industrial application, production cost isimproved, and production efficiency is improved.

Description

technical field [0001] The invention relates to one or more 7β-hydroxysteroid dehydrogenase (7β-HSDH) enzyme mutants transformed by error-prone PCR / point-fixed saturation mutation and applications thereof, belonging to the technical field of enzyme engineering. Background technique [0002] Ursodeoxycholic acid (UDCA) is an effective drug for the treatment of various gallstone diseases and various acute and chronic liver diseases. The traditional preparation method of UDCA is to extract from bear bile, but the extraction process of this method is difficult, the product yield is low, and the means are too cruel, which violates the animal protection law. [0003] At present, in addition to the "bear bile extraction method", the industrial production methods of UDCA mainly include chemical synthesis and enzymatic methods. Among them, the chemical synthesis method has been gradually developed due to the disadvantages of high cost, many process steps, serious "three wastes" and h...

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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