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Amphetamine dehydrogenase mutant and application thereof in chiral amine synthesis

An amine dehydrogenase and mutant technology, applied in the field of bioengineering, can solve the problems of narrow application range, inability to catalyze large sterically hindered carbonyl substrates, etc., and achieve high catalytic activity, high substrate concentration tolerance, and product optical high purity effect

Active Publication Date: 2021-12-28
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] Aiming at the problem that existing amine dehydrogenases cannot catalyze large sterically hindered carbonyl substrates, the present invention provides a mutant of amphetamine dehydrogenase and its application in the synthesis of chiral amines
[0009] The present invention mainly provides several amine dehydrogenase mutants with significantly improved catalytic performance on large steric hindrance substrates through the semi-rational design of the enzyme structure, solves the problem of its narrow application range, and expands its application in large steric hindrance medicines. The scope of application in the synthesis of sex amine intermediates

Method used

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  • Amphetamine dehydrogenase mutant and application thereof in chiral amine synthesis
  • Amphetamine dehydrogenase mutant and application thereof in chiral amine synthesis
  • Amphetamine dehydrogenase mutant and application thereof in chiral amine synthesis

Examples

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Embodiment 1

[0054] The construction of embodiment 1 amphetamine dehydrogenase GkAmDH mutant

[0055] Using the recombinant plasmid GkAmDH as the parent, design primers for the sites to be mutated, and the sequences of the upstream and downstream primers are shown in the table.

[0056] The gene sequence of amphetamine dehydrogenase GkAmDH is shown in SEQ ID No.1.

[0057] The primer purification method is PAGE, and the primer structure is: upstream primer: 15-20 bases before the mutation site + mutation base + 15-20 bases after the mutation site; downstream primer: reverse complementary sequence of the entire upstream primer ;Primers should be stored at -20°C for long-term storage. The PCR system contained 10 μL PrimeSTAR HS, 1 μL of upstream and downstream primers (10 ng / μL), 1 μL of recombinant plasmid (100 ng / μl), 0.5 μL DMSO and 6.5 μL ddH2O. The PCR amplification program was as follows: pre-denaturation at 98°C for 10 seconds, followed by 20 cycles of denaturation at 98°C for 10 se...

Embodiment 2

[0069] Example 2 Construction of amphetamine dehydrogenase GkAmDH mutant recombinant expression transformant

[0070] Transform the recombinant plasmid ligation solution containing the target fragment of the amphetamine dehydrogenase mutant obtained by PCR amplification in Example 1 into E.coli BL21, and pick positive clones to obtain a series of recombinant expression transformants E.coli BL21(DE3) / pET28a-GkAmDH M3~8, the correctness of the target gene was confirmed by gene sequence determination.

Embodiment 3

[0071] Embodiment 3 Preparation of amphetamine dehydrogenase GkAmDH mutant

[0072] The recombinant expression transformant obtained in Example 2 was inoculated into LB medium containing 50 μg / mL kanamycin, cultured in a shaker at 37° C. for 12 hours, and then inoculated with an inoculation amount of 1% (v / v) Put it into a shaker flask containing 100mL TB medium, put it into a shaker at 37°C and 180rpm for culture, when the OD of the culture solution 600 When it reaches about 0.6-0.8, add isopropyl-β-D-thiogalactopyranoside (IPTG) with a final concentration of 0.2mmol / L, and induce culture at 16°C for 24 hours. After the cultivation, the culture solution was centrifuged at 8000rpm to collect the cells. The collected cells were resuspended with 10 mL of Tris-HCl buffer (100 mM, pH 8.0), and subjected to the following ultrasonic treatment in an ice-water bath: 400 W power, working for 4 s, resting for 6 s, 99 cycles. The crushed solution was centrifuged at 4°C and 10,000 rpm f...

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Abstract

The invention discloses an amphetamine dehydrogenase mutant derived from geobacillus kaustophilus, a coding gene and an amino acid sequence of the amphetamine dehydrogenase mutant, and a method for preparing corresponding chiral amine by catalyzing reductive amination of a large-steric-hindrance carbonyl compound and ammonia by using the amphetamine dehydrogenase mutant. The developed amine dehydrogenase mutant can catalyze carbonyl compounds such as large steric hindrance aromatic ketones, alkyl ketones, heterocyclic ketones and functionalized ketones which are difficult to be reduced and aminated by reported enzymes at present. Compared with other preparation methods, the method for synthesizing the chiral amine by catalyzing asymmetric reductive amination of ketone and ammonia through amine dehydrogenase has the advantages that the used amino donor is cheap ammonia water, relatively expensive organic amine is replaced, a byproduct generated in the reaction is only water. Therefore, the method has the advantages of high atom economy, high optical purity of the product, mild reaction conditions, environmental friendliness, simple post-treatment of the product and the like, and has a good application prospect in synthesis of the chiral primary amine.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to an amphetamine dehydrogenase mutant and its application in the synthesis of chiral amines. Background technique [0002] Chiral amines widely exist in natural active molecules, and are also the synthetic precursors of many drugs, natural active molecules, fine chemicals, etc. Especially in drug synthesis, about 40% of chemical drugs contain one or more structural blocks of chiral amines, such chiral drugs involve anti-inflammatory drugs, anti-Alzheimer's drugs, anti-depressants, anti-AIDS Medicines, etc., occupy a very important position in the field of medicine. Due to the importance and great value of the chiral amine structure in the synthesis of drug molecules, the development of its synthesis technology has become a hot spot in the research of new drugs (Chem. Rev. 2003, 103, 2985-3012). [0003] The traditional chemical synthesis methods of chiral amines are mainly...

Claims

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Application Information

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IPC IPC(8): C12N9/06C12N15/53C12N15/70C12N1/21C12P41/00C12P13/00C12P17/04C12P17/10C12P17/12C12P17/14C12R1/19
CPCC12N9/0018C12N15/70C12P41/002C12P13/001C12P13/008C12P17/04C12P17/10C12P17/12C12P17/14C12Y104/0102
Inventor 郑高伟汪东浩潘江许建和陈飞飞陈琦丁旭伟殷赛男
Owner EAST CHINA UNIV OF SCI & TECH
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