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Amine Dehydrogenase Mutants and Their Applications in the Synthesis of Chiral Amino Alcohols

A technology of alcohol compounds and chiral amines, which can be used in applications, methods based on microorganisms, and the use of vectors to introduce foreign genetic materials, etc., can solve problems such as low conversion rate, low safety factor, and large pollution

Active Publication Date: 2021-11-09
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 low
The reaction conditions of the biological method are mild, but the conversion rate is low, so there is an urgent need for a green and efficient method for the synthesis of chiral aminoalcohol compounds

Method used

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  • Amine Dehydrogenase Mutants and Their Applications in the Synthesis of Chiral Amino Alcohols
  • Amine Dehydrogenase Mutants and Their Applications in the Synthesis of Chiral Amino Alcohols
  • Amine Dehydrogenase Mutants and Their Applications in the Synthesis of Chiral Amino Alcohols

Examples

Experimental program
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Effect test

Embodiment 1

[0202] Embodiment 1, the preparation of the engineering bacterium of amine dehydrogenase SpAmDH gene or its mutant

[0203] 1. Preparation of amine dehydrogenase SpAmDH genetic engineering strain

[0204] Codon-optimized the coding gene of amine dehydrogenase (SpAmDH) derived from Sporosarcina psychrophila using Escherichia coli as a 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 as SEQ ID No.2 in the sequence listing.

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

[0206] The recombinant ...

Embodiment 2

[0239] Example 2, Expression of amine dehydrogenase SpAmDH or its mutants 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 of each strain are as follows:

[0241] Pick the strain into 5mL LB liquid medium containing 50μg / mL kanamycin, shake overnight at 37°C and 220rpm for 12h, and then inoculate the TB containing 50μg / mL kanamycin according to the volume percentage content of 1% inoculum Cultured in liquid medium at 37°C to OD 600 When the concentration is 0.7, add IPTG with a final concentration of 0.1mmol / L, induce expression at 20°C and 220rpm for 12h, centrifuge at 4°C and 4,000rpm for 10min, collect the precipitated cells (i.e. whole cell...

Embodiment 3

[0242] Example 3. Amine dehydrogenase SpAmDH or its mutants catalyze 1-hydroxy-2-butanone to generate (S)-2-amino-1-butanol

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

[0244] The reaction system of the catalyzed reaction of SpAmDH or its mutants is obtained by adding the following amount of substances to 1 mol / L ammonium chloride / ammonia aqueous buffer (ammonium chloride and ammonia aqueous equimolar ratio are mixed, 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 + (in the form of oxidized coenzyme I aqueous solution) 1mmol / L, GDH crude enzyme powder 2g / L, glucose 100mmol / L, lysozyme 1g / L (Beijing Solaibao Technology Co., Ltd., CAS...

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Abstract

The invention discloses an amine dehydrogenase mutant and its application in the synthesis of chiral aminoalcohol compounds. The amine dehydrogenase mutant disclosed in the present invention is the 40th, 61st, 68th, 69th, 111th, 113th, 114th, 116th to the amine dehydrogenase (SpAmDH) derived from psychrophilic bacillus sarcina (Sporosarcina psychrophila) , 134, 150, 187, 239, 288, 291, 292, 295, 297 and / or 294 positions are mutated. The amine dehydrogenase mutant of the present invention can catalyze the synthesis of hydroxyketone substrates into chiral aminoalcohol compounds represented by 1-hydroxy-2-butanone and 4-hydroxy-2-butanone, and is easy to operate and has optical purity of the product High advantages, the stereoselectivity can be as high as 99%, and the biocatalytic preparation of chiral aminoalcohol compounds has good industrial application prospects.

Description

technical field [0001] The invention relates to an amine dehydrogenase mutant and its application in the synthesis of chiral aminoalcohol compounds in the field of biotechnology. Background technique [0002] Chiral amino alcohols are the structural units of many bioactive molecules, and are important pharmaceutical and fine chemical intermediates, among which (S)-2-amino-1-butanol is mainly used for the synthesis of ethambutol hydrochloride (Ethambutol), long-term (R)-3-amino-1-butanol is an important raw material for the synthesis of anti-AIDS integrase inhibitor Dolutegravir. [0003] (S)-2-Amino-1-butanol is mainly synthesized by chemical method and biological enzymatic method, wherein the current chemical synthesis methods include: using 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 nickel; use lithium aluminum hydride to reduce 2-aminobutyric acid to obtain t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/06C12P13/00C12N15/53C12N15/70C12N1/21C12R1/19
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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