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Fluoroacetic acid dehalogenase mutant and application thereof

A technology of acetate dehalogenase and mutants, applied in the biological field, can solve the problem that fluoroacetic dehalogenase cannot be used to catalyze brominated substrates

Inactive Publication Date: 2019-12-13
ABIOCHEM BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The technical problem to be solved by the present invention is that the existing wild-type fluoroacetic acid dehalogenase cannot be used to catalyze the defects of brominated substrates, especially 2-bromobutyric acid substrates, so the present invention provides a kind of fluoroacetic acid dehalogenase Mutant and its application in the preparation of (R)-2-bromobutyric acid

Method used

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  • Fluoroacetic acid dehalogenase mutant and application thereof
  • Fluoroacetic acid dehalogenase mutant and application thereof
  • Fluoroacetic acid dehalogenase mutant and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0068] Example 1 Construction of Fluoroacetic Dehalogenase Mutant Library

[0069] The primer sequences designed for the construction of the mutant library for mutations at positions 155, 156, and 219 of the fluoroacetic dehalogenase FAcD-RPA1163 (sourced as Rhodopseudomonas palustris) sequence (ie, SEQ ID NO.1 in the sequence listing) are shown in the table 3 shows:

[0070] table 3

[0071]

[0072] Wherein, N represents any nucleotide in A, G, C, T, M represents A or C, and K represents G or T; it is selected according to the coding nucleotide of the amino acid to be mutated into at the site , For example, NNK in the A166-forward primer can represent AAG (lysine), AAT (aspartic acid), AGG (arginine) or AGT (serine), etc. The nucleotides corresponding to specific amino acids can be found in Table 2.

[0073] According to the method disclosed in the literature J.Am.Chem.Soc., 2017, 139(32), 11241-11247, the plasmid template pET28a-FAcD-RPA1163 was constructed, and the t...

Embodiment 2

[0079] Embodiment 2 High-throughput screening mutant library

[0080] Screen according to the following experimental steps:

[0081] The transformant obtained in Example 1 was inoculated into a 96-well plate for culture, and induced overnight at 30° C. with IPTG. Afterwards, the bacteria were harvested, cracked with bugbuster protein extraction reagent, and centrifuged to obtain the total enzyme solution.

[0082] Weigh 250mg of racemized 2-bromobutyric acid substrate, dissolve it in 45mL 1mM Tris solution, place it on ice, adjust the pH between 8.0 and 8.5 with dilute NaOH, and adjust the volume to 50ml to prepare 2-bromobutyric acid The substrate concentration was 30 mM substrate solution. Add 100 μl of substrate solution (final concentration: 25 mM) and 20 μL of the above-mentioned total enzyme solution to each reaction. After reacting for a period of time, take 20 μl of the reaction solution to a 96-well microtiter plate, and add 30 μl of saturated Hg (SCN) in sequence. ...

Embodiment 3

[0096] Example 3 Catalytic preparation of (R)-2-bromobutyric acid by fluoroacetic acid dehalogenase mutant

[0097] Add 50mL tap water into the reaction bottle, add 10g 2-bromobutyric acid substrate, stir and dissolve, adjust the pH to 7.0 with 30% NaOH solution, add 20mL of the mutant crude enzyme solution prepared according to the method of Example 2 (that is, weigh 4g The thallus described in Example 2 was added to 20 mL of 100 mM pH7.0 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, stirred evenly, and homogeneously crushed under high pressure to obtain mutant crude enzyme liquid. Mutation with fluoroacetic acid dehalogenase Take body 3 as an example, its concentration is 10U / ml), dilute the reaction system to 100mL with tap water. During the reaction process, 2mol / L sodium carbonate solution was used to control the pH at about 7.0, and after 8 hours of reaction in a water bath at 30°C, samples were taken to detect the conversion rate of the 2-bromobutyric ...

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Abstract

The invention discloses a fluoroacetic acid dehalogenase mutant. The sequence of the fluoroacetic acid dehalogenase mutant comprises a sequence obtained by mutating the 155th amino acid residue H shown in SEQ ID NO.1 and / or the 156th amino acid residue W shown in SEQ ID NO.1. The fluoroacetic acid dehalogenase mutant has the activity of catalyzing a bromination substrate, particularly a 2-bromobutyric acid substrate. The invention also provides an application of the fluoroacetic acid dehalogenase mutant in preparation of (R)-2-bromobutyric acid and / or (R)-2-hydroxybutyric acid. When the fluoroacetic acid dehalogenase mutant is used for preparing (R)-2-bromobutyric acid, the production cost is low, the stereoselectivity is high, and industrial production is facilitated.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to a fluoroacetic dehalogenase mutant and application thereof. Background technique [0002] 2-oxo-1-pyrrolidine derivatives such as levetiracetam, buvaracetam, and seletracetam are a new type of antiepileptic drug developed by UCB Company in Belgium. EP1806339 discloses a preparation method thereof, which uses (R)-2-bromobutyric acid to perform a substitution reaction with the corresponding 2-oxo-1-pyrrolidine compound, and then reacts with triethylamine to obtain the corresponding product. Among them, (R)-2-bromobutyric acid is an important raw material, which plays a decisive role in the production of antiepileptic drug 2-oxo-1-pyrrolidine derivatives. Therefore, it is necessary to look for (R)-2 with low cost and high optical purity. - The preparation method of bromobutyric acid is very important. [0003] [0004] At present, the preparation method of (R...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/14C12N15/55C12P41/00C12P7/52C12P7/42
CPCC12N9/14C12P7/42C12P7/52C12P41/001C12Y308/01003
Inventor 瞿旭东程占冰丁少南徐艳冰黄瑶
Owner ABIOCHEM BIOTECH CO LTD
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