Amine dehydrogenase mutant and application thereof in synthesis of chiral amine alcohol compound

A technology for alcohol compounds and chiral amines, which can be used in applications, microorganism-based methods, and introduction of foreign genetic material using carriers, which can solve the problems of harsh reaction conditions, low safety factor, and large pollution.

Active Publication Date: 2021-05-28
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The synthetic method of above aminobutanol, chemical method needs high temperature, high pressure and metal catalyst, and reaction condition is harsh, and pollution is big, and safety factor is l

Method used

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  • Amine dehydrogenase mutant and application thereof in synthesis of chiral amine alcohol compound
  • Amine dehydrogenase mutant and application thereof in synthesis of chiral amine alcohol compound
  • Amine dehydrogenase mutant and application thereof in synthesis of chiral amine alcohol compound

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0202] Example 1. Preparation of engineering bacteria of amine dehydrogenase SpAmDH gene or its mutant

[0203] 1. Preparation of amine dehydrogenase SpAmDH genetically engineered strains

[0204] The coding gene of amine dehydrogenase (SpAmDH) derived from Sporarcina psychrophila was codon-optimized using Escherichia coli as the host cell to obtain the SpAmDH gene shown in SEQ ID No. 1 in the sequence listing. The amino acid sequence of SpAmDH is shown in SEQ ID No. 2 in the sequence listing.

[0205] The SpAmDH gene shown in SEQ ID No.1 in the full gene synthesis sequence table, the small DNA fragment between the recognition sequences of NdeI and XhoI in the pET24a(+) vector (Novagen) was replaced with the SpAmDH gene shown in SEQ ID No.1, A recombinant vector was obtained, which was designated as pET24a-SpAmDH. pET24a-SpAmDH could express SpAmDH shown in SEQ ID No. 2 in the sequence listing, and the expression of SpAmDH gene was driven by T7 promoter.

[0206] The recombi...

Embodiment 2

[0239] Example 2. Expression of amine dehydrogenase SpAmDH or its mutant and preparation of whole cells, crude enzyme powder and pure enzyme solution

[0240] The recombinant bacteria BL21(DE3) / pET24a-SpAmDH, BL21(DE3) / pET24a and each amine dehydrogenase SpAmDH gene mutant engineering strain prepared in Example 1 were induced to express, and the whole cells and crude enzyme powder of each strain were obtained. With pure enzyme solution, the operation steps for each strain are as follows:

[0241] Pick the strains into 5mL LB liquid medium containing 50μg / mL kanamycin, shake at 37°C and 220rpm overnight for 12h, and then inoculate TB containing 50μg / mL kanamycin according to the volume percentage of 1% inoculum. In liquid medium, cultivate to OD at 37°C 600 When it is 0.7, IPTG with a final concentration of 0.1 mmol / L was added. After inducing expression at 20°C and 220rpm for 12h, centrifuge at 4°C and 4,000rpm for 10min to collect the precipitated cells (that is, whole cells...

Embodiment 3

[0242] Example 3. Amine dehydrogenase SpAmDH or its mutant catalyzes the generation of (S)-2-amino-1-butanol from 1-hydroxy-2-butanone

[0243] The crude enzyme powder or whole cell of the amine dehydrogenase SpAmDH or its mutant prepared in Example 2 is used to catalyze 1-hydroxy-2-butanone to generate (S)-2-amino-1-butanol, as shown in the schematic diagram. figure 2 shown, and BL21(DE3) / pET24a was used as a control.

[0244] The reaction system of the catalytic reaction of SpAmDH or its mutant is obtained by adding the following amount of substances to 1 mol / L ammonium chloride / ammonia buffer solution (a mixture of ammonium chloride and ammonia in an equimolar ratio, pH 8.5): Substrate 1 -Hydroxy-2-butanone 20mmol / L or 40mmol / L, amine dehydrogenase SpAmDH or its mutant crude enzyme powder 20g / L or whole cell 100g / L, NAD + (Exist in the form of oxidized coenzyme I aqueous solution) 1mmol / L, GDH crude enzyme powder 2g / L, glucose 100mmol / L, lysozyme 1g / L (Beijing Soleibao Te...

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Abstract

The invention discloses an amine dehydrogenase mutant and an application thereof in synthesis of a chiral amine alcohol compound. The amine dehydrogenase mutant disclosed by the invention is a protein obtained by mutating the 40th site, the 61th site, the 68th site, the 69th site, the 111st site, the 114th site, the 116th site, the 150th site, the 187th site, the 239th site, the 288 site, the 291 site, the 292 site, the 295th site, the 297th site and/or the 294th site of the amine dehydrogenase (SpAmDH) derived from Sporosarcina psychrophila. The amine dehydrogenase mutant can catalyze hydroxy ketone substrates to synthesize chiral amine alcohol compounds represented by 1-hydroxy-2-butanone and 4-hydroxy-2-butanone, is convenient to operate, has the advantages of high optical purity of products and the like, has stereoselectivity up to 99%, and has a better industrial application prospect in preparation of the chiral amine alcohol compounds through biological catalysis.

Description

technical field [0001] The present invention relates to an amine dehydrogenase mutant and its application in the synthesis of chiral amine alcohol compounds in the field of biotechnology. Background technique [0002] Chiral amine alcohols are the structural units of many biologically active molecules, and are important pharmaceutical and fine chemical intermediates. Among them, (S)-2-amino-1-butanol is mainly used to synthesize Ethambutol hydrochloride (Ethambutol). Since then, it has been used as a first-line drug against Mycobacterium tuberculosis; (R)-3-amino-1-butanol is an important raw material for the synthesis of the anti-AIDS integrase inhibitor Dolutegravir. [0003] (S)-2-amino-1-butanol mainly has chemical method and biological enzymatic synthesis, wherein the current chemical synthesis method has: with butyraldehyde, dibenzyl azodicarboxylate and D-proline as raw materials, in NaBH 4 , H 2 Synthesize (S)-2-amino-1-butanol under the catalysis of nickel and ni...

Claims

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Application Information

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IPC IPC(8): C12P13/00C12N9/06C12N15/53C12N15/70C12N1/21C12R1/19
CPCC12N9/0014C12P13/001C12Y104/99003
Inventor 孙周通王红月曲戈蒋迎迎
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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