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P450 enzyme mutant and application thereof

A mutant, the technology of P450BM3F87A, is applied in the field of P450 enzyme mutants that catalyze the synthesis of 25-hydroxyvitamin D3 from vitamin D3, and achieves the effects of high protein expression, broad application prospects and mature technology

Pending Publication Date: 2022-05-17
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, according to literature and patent searches, there is no P450 BM3 wild type or mutant used to catalyze vitamin D at home and abroad. 3 Synthesis of 25-hydroxyvitamin D 3 report

Method used

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  • P450 enzyme mutant and application thereof
  • P450 enzyme mutant and application thereof
  • P450 enzyme mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Construction of P450 BM3 mutant library

[0045] According to the crystal structure of P450 BM3 complexed with natural fatty acid substrate N-palmitoylglycine (PDB: 1JPZ), 11 key amino acid residues (L75, V78, F81, A82, A180) within 5 angstroms of the active pocket of the substrate were selected , L181, A184, L188, A328, A330, I263) were divided into 6 groups based on the principle of proximity, followed by A (L75, V78), B (F81, A82), C (A180, L181), D (A184, L188) , E (A328, A330), F (I263). Using P450BM3 F87A as the starting template, the mature degenerate NDT codon technology was used to carry out combined mutations of 12 different amino acids, and a mutant library with a coverage rate of >95% and a library capacity of >2000 was constructed.

[0046] The specific steps are: use the gene whose nucleotide sequence is SEQ ID NO.6 as a template, and use A-F / A-R or B-F / B-R or C-F / C-R or D-F / D-R or E-F / E-R or F-F / F-R as primers to carry out PCR amplification res...

Embodiment 2

[0050] Example 2 Screening of Mutant Library and Confirmation of Highly Active Transformation Vitamin D 3 Produces 25-hydroxyvitamin D 3 mutant

[0051] Inoculate the P450 BM3 mutant bacterial liquid of the A library or B library or C library or D library or E library or F library stored in the 96 deep-well plate in Example 1 into 300 μL LB containing 50 μg / mL kanamycin respectively. Liquid medium in sterile 96 deep well plates. The culture was cultured overnight at 37°C and 220 rpm, and 40 μL of the bacterial solution from each well was transferred as a seed solution to a new sterile 96 deep-well plate containing 400 μL of TB liquid medium containing 50 μg / mL kanamycin.

[0052] After the medium was cultured at 37°C and 220rpm for a period of time (OD 600 =1.0 or so), add 0.2mM isopropyl-β-dithiogalactopyranoside (IPTG) and 0.5mM 5-aminolevulinic acid (5-ALA) to each individual well and cultured at 18° C. and 230 rpm for 24 hours to obtain mutant protein cells of library ...

Embodiment 3

[0056] Example 3 Construction of L75 position saturation mutant library

[0057] In the screening process of the mutant library in Example 2, it was found that the mutant P450 BM3 F87A / L75H at the L75 position had higher catalytic activity. Considering that the NDT degenerate codon can only derive 12 amino acid residues, this position was carried out. Saturation mutation of the point (L75) to realize the catalytic activity comparison of 20 kinds of amino acids at this point.

[0058] The mature degenerate NNK codon technology was used for saturation mutation, and the specific steps were: PCR amplification was performed with the gene whose nucleotide sequence was SEQID NO.6 as a template and L75-F / L75-R as primers (Table 2). PCR program: 5×PrimeSTARGXL Buffer 10μL, 200μM dNTPs, 0.3μM upstream and downstream primers, appropriate amount of DNA template (10-100ng), high-fidelity polymerase (PrimeSTAR GXL DNA polymerase) 2.5U, ddH 2 O was added to 50 μL; the reaction conditions we...

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Abstract

The invention discloses a P450 enzyme mutant for catalyzing vitamin D3 to synthesize 25-hydroxyvitamin D3, which is formed by respectively mutating the 75th amino acid of P450 enzyme with an amino acid sequence shown as SEQ ID NO.1 into alanine, glycine, threonine or histidine from leucine, and the amino acid sequence of the P450 enzyme is shown as SEQ ID NO.2-NO.5. The invention further discloses a preparation method of the P450 enzyme mutant. The invention also discloses a preparation method of the P450 enzyme mutant and an application of the P450 enzyme mutant in production of 25-hydroxyvitamin D3. Experiments prove that the efficiency of the P450 enzyme mutant for catalyzing the synthesis of the 25-hydroxyvitamin D3 by the vitamin D3 can be up to 3.1 times of that of P450BM3F87A at most. Moreover, the whole operation process for producing the 25-hydroxyvitamin D3 is simple, the process is mature and environment-friendly, and the method has a wide application prospect.

Description

technical field [0001] The invention belongs to the technical fields of enzyme engineering, biocatalysis, biochemistry and molecular biology, and relates to a class of catalytic vitamin D 3 Synthesis of 25-hydroxyvitamin D 3 P450 enzyme mutants and their applications. Background technique [0002] Vitamin D 3 (vitamin D 3 , cholecalciferol) is a sterol derivative and is a fat-soluble vitamin that plays an important regulatory role in the growth and development, calcium and phosphorus metabolism, immunity, cell proliferation and differentiation of humans and mammals. In humans, vitamin D 3 It is converted from 7-dehydrocholesterol in the skin by exposure to ultraviolet light. Vitamin D 3 It does not have biological activity, and needs to be converted into the physiologically active form 25-hydroxyvitamin D by the oxidation of cytochrome P450 enzymes in the human body 3 (calcifediol) and 1α,25-dihydroxyvitamin D 3 (calcitriol), and then play a role in regulating the ab...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12P7/18C12N15/70C12N1/21C12R1/19
CPCC12N9/0042C12N15/70C12Y106/02004C12P7/18
Inventor 李盛英蒋媛媛李众马莉
Owner SHANDONG UNIV
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