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Reductive aminase, coding gene, recombinant vector, recombinant cell and application thereof

A technology of reductive amination enzyme and amino donor, applied in application, genetic engineering, plant gene improvement, etc., can solve the problems of insufficient stereoselectivity of biological methods, high cost of noble metal ligands, general catalytic efficiency, etc. Less reaction, simple preparation method, environment-friendly effect

Active Publication Date: 2021-07-20
JIANGNAN UNIV
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  • Claims
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Problems solved by technology

[0005] Among the above synthetic methods, the cost of noble metal ligands required by the chemical method is relatively high and the reaction conditions are harsh, and the biological method has bottleneck problems such as insufficient stereoselectivity and general catalytic efficiency, and all of them need to further derivatize the hydroxyl group with an amine group to obtain end products of chiral amines

Method used

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  • Reductive aminase, coding gene, recombinant vector, recombinant cell and application thereof
  • Reductive aminase, coding gene, recombinant vector, recombinant cell and application thereof
  • Reductive aminase, coding gene, recombinant vector, recombinant cell and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Cloning of Reductive Aminase Gene

[0039] Using the strategy of gene mining, 10 hypothetical proteins from different actinomycetes were cloned. After activity measurement and comparison, it was found that the protein from Streptomyces sparsus had the highest catalytic activity. The specific operation is as follows:

[0040] (1) Firstly, the above-mentioned actinomycetes were cultivated overnight at 30°C using Gao’s No. 1 medium, and the bacterial cells were obtained by centrifugation, and then the total genomic DNA was obtained using a conventional bacterial genome extraction kit.

[0041] (2) PCR is carried out with the above-mentioned genomic DNA as a template, and the system is as follows: ddH 2 O 21 μL, 5× PrimeSTARBuffer 10 μL, upstream primer SEQ ID NO.3 (GGGAATTCCATATGAGC AACACCCCCGTGAC) 1 μL, downstream primer SEQ ID NO.4 (CGCGGATCCCTAG GCCCGGGTGCGGAGCA) 1 μL, genomic DNA 1 μL, dNTP 5 μL, PrimeSTAR DNA polymerase 1.0 μL. The PCR program was: pre-de...

Embodiment 2

[0042] Example 2: Construction and cultivation of recombinant Escherichia coli BL21(DE3) / pET28a-ssra

[0043] The gene ssra recovered in Example 1 and the plasmid pET28a were double digested with restriction enzymes NdeI and BamH I in a water bath at 37°C overnight, purified by agarose gel electrophoresis the next day, and the target was recovered using an agarose recovery kit fragment. 4°C, use T 4 DNA ligase overnight ligase cut gene ssra and plasmid pET28a to obtain recombinant expression vector pET28a-ssra ( figure 1 ). The constructed recombinant expression vector pET28a-ssra was thermally transferred into Escherichia coli BL21 (DE3) competent, coated with Kan-resistant LB solid plate, and colony PCR verification was carried out after overnight culture. The positive clone was the recombinant Escherichia coli BL21 ( DE3) / pET28a-ssra. Pick positive clones and culture them overnight in LB medium, then transfer them into fresh LB culture at 2% transfer amount the next da...

Embodiment 3

[0044] Embodiment 3: Separation and purification of reductive aminase

[0045] Suspension-cultured recombinant cells were placed in liquid A (20mmol·L -1 Sodium phosphate, 500mmol·L -1 NaCl, 20mmol L -1 imidazole, pH 7.4), the crude enzyme solution was obtained after sonication and centrifugation. The column used for purification is an affinity column HisTrapFF crude (nickel column), which is completed by using the histidine tag on the recombinant protein for affinity binding. First, use solution A to equilibrate the nickel column, load the crude enzyme solution, continue to use solution A to elute the breakthrough peak, and after equilibrium, use solution B (20mmol L -1 Sodium phosphate, 500mmol·L -1 NaCl, 1000 mmol L -1 imidazole, pH 7.4) for gradient elution to elute the recombinant protein bound to the nickel column to obtain recombinant reductive aminase. Enzyme activity assay (CPMK as substrate, NADPH as coenzyme) and SDS-PAGE analysis were performed on the purifie...

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Abstract

The invention discloses a reductive aminase, a coding gene, a recombinant vector, a recombinant cell and an application thereof, and belongs to the technical field of bioengineering. The amino acid sequence of the reductive aminase of the present invention is shown in SEQ ID NO.2, or the amino acid sequence shown in SEQ ID NO.2 is substituted, deleted or added with one or more amino acids and has reductive aminase activity amino acid sequence. The recombinantly expressed soluble SsRA enzyme of the present invention has the advantages of high expression level and high enzyme activity, and can be used to efficiently prepare (S)-(4'-chlorophenyl)-(pyridine-2'-yl)-methylamine and other current biocatalysis The difficult-to-prepare chiral bisaryl secondary amine has high economy, simple preparation method, mild reaction conditions, low energy consumption, environmental friendliness, few side reactions, and can be completed by one-step reduction.

Description

technical field [0001] The invention relates to a reductive aminase, a coding gene, a recombinant vector, a recombinant cell and an application thereof, belonging to the technical field of bioengineering. Background technique [0002] Chiral secondary amines are a very important class of compounds, and 40% of commercially available drugs contain chiral secondary amine groups. Since chiral bisaryl secondary amines have two aryl groups, more structures with medicinal value can be derived, among which chiral (4-chlorophenyl)-(pyridin-2-yl)-methylamine (CPMAm) It can be used to synthesize the antiallergic drug Bepotastine. At present, the synthesis of bepotastine is mainly through (4-chlorophenyl)-(pyridin-2-yl)-methanol, mainly including the following synthesis process: [0003] (1) Chemical method: with (4-chlorophenyl)-(pyridin-2-yl)-methanone (CPMK) as raw material, (S)-[Ru(BINAP)Cl 2 ] 2 (NEt 3 ) as a catalyst, pressurize, pass hydrogen, and reduce to obtain (S)-(4-chl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12P13/00C12P17/12
CPCC12N9/1096C12P13/001C12P17/12C12Y206/01
Inventor 刘平许国超郭曼郑香玉陈玄皂薛佳钰倪晔窦哲孙泽文
Owner JIANGNAN UNIV
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