Amine dehydrogenase as well as coding nucleic acid and application thereof

A technology of amine dehydrogenase and amino acid dehydrogenase, applied in amine dehydrogenase, the application field of amine dehydrogenase, can solve the problems of narrow substrate spectrum, few kinds of AmDH, limited industrial application, etc.

Pending Publication Date: 2022-05-06
HEBEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Different dehydrogenases are almost all named according to the names of their substrates. Amine dehydrogenase is one of the dehydrogenases. According to reports, amine dehydrogenase

Method used

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  • Amine dehydrogenase as well as coding nucleic acid and application thereof
  • Amine dehydrogenase as well as coding nucleic acid and application thereof
  • Amine dehydrogenase as well as coding nucleic acid and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] Example 1, Screening and Determination of Mutation Sites

[0107] The natural amino acid dehydrogenase protein derived from Jeotgalicoccus aerolatus (as shown in SEQ ID NO: 2, its coding gene (AmDH gene) as shown in SEQ ID NO: 1) was subjected to sequence analysis, mutation and functional verification, specifically selected 12 amino acid sites, and 4 important amino acid sites were screened out from the 12 amino acid sites through further research and analysis. The four amino acid sites are mutated in different forms to obtain mutant proteins.

[0108] The four amino acid sites and their mutant forms are shown in Table 1.

[0109] Table 1: 15 mutant forms

[0110] mutant number mutant form a1 K67S a2 K67Q a3 K67M a4 N260L a5 N260C a6 N260F a7 N260I a8 K67S\N260L a9 K67S\N260C a10 K67S\N260F a11 K67S\N260I a12 K67S\N260L\E113V a13 K67S\N260L\E113V\V291A a14 K67S\N260L\E113V\...

Embodiment 2

[0119] Embodiment 2, the preparation of recombinant bacteria

[0120] 1. Construction of wild-type recombinant expression vector

[0121] The nucleotide sequence shown in SEQ ID NO: 1 was inserted between the EcoRI and XhoI restriction sites of the pET28a vector to obtain the recombinant expression vector pET28a-AmDH, which was verified to be correct by sequencing.

[0122] 2. Construction of mutant recombinant expression vector

[0123] The nucleotide sequences encoding the mutants a1-a15 (see Table 2) were respectively inserted between the EcoRI and XhoI restriction sites of the pET28a vector to obtain the recombinant expression vector pET28a-1-15, which was verified to be correct by sequencing.

[0124] 3. Preparation of recombinant bacteria

[0125] 1. Transform Escherichia coli BL21(DE3) with the recombinant expression vector pET28a-AmDH obtained in step 1 to obtain wild-type recombinant bacteria.

[0126] 2. Transform the recombinant expression vector pET28a-1-15 prep...

Embodiment 3

[0127] Embodiment 3, the enzyme activity assay of amino acid dehydrogenase mutant protein

[0128] The wild-type recombinant bacteria and mutant recombinant bacteria 1-15 obtained in Example 2 were cultivated, the protein was extracted, and the enzyme activity was detected.

[0129] 1. Inoculate the recombinant bacteria in 10 mL of LB liquid medium containing kanamycin at a final concentration of 50 μg / mL, shake and culture at 37° C. and 180 rpm for 16 hours to obtain seed liquid. Then the seed solution was inoculated into 50 mL of TB medium (inoculum size 1%), containing a final concentration of 50 μg / mL kanamycin in the medium, and cultured with shaking at 37° C. and 180 rpm until the OD of the bacterial solution 600nm 0.6-0.8, add IPTG with a final concentration of 0.5 μM, continue shaking culture at 20°C and 180rpm for 20-24h, and centrifuge at 5000rcf for 15min to collect the bacteria.

[0130] The recombinant bacterial cells were washed and resuspended twice with 50mM 5...

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Abstract

The invention provides amine dehydrogenase as well as coding nucleic acid and application thereof, the amine dehydrogenase is obtained by mutating amino acid dehydrogenase as shown in SEQ ID NO.2, and the mutation comprises at least one mutation in 67th, 113th, 260th and 291th amino acids as shown in SEQ ID NO.2. The amine dehydrogenase disclosed by the invention has relatively high reductive amination activity and high yield, meets the requirement of 99% + stereoselectivity, and has a very wide application prospect.

Description

technical field [0001] The invention relates to the technical field of protein modification, in particular to an amine dehydrogenase. Meanwhile, the present invention also relates to the coding nucleic acid of the amine dehydrogenase and the application of the amine dehydrogenase. Background technique [0002] Among many amines, chiral primary amines are very important motifs or building blocks in many bioactive molecules, and are widely used in the field of medicine. The main synthesis methods are chemical and biological methods. The former relies on heavy metals while the conversion rate of the latter needs to be improved. The latter can use cheap ammonia as an amino donor through amine dehydrogenase (AmDH), asymmetric reductive amination of potential chiral hydroxyketones to generate chiral primary amines, and the theoretical conversion rate is high. , is an ideal green synthetic route. [0003] Optically pure (R)-α-phenylethylamine is a kind of chiral amine, which is t...

Claims

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Application Information

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IPC IPC(8): C12N9/06C12N15/53C12N15/70C12N1/21C12P13/00C12R1/19
CPCC12N9/0014C12N15/70C12Y104/99003C12P13/001
Inventor 刘运亭孔维茜贺莹姜艳军高静
Owner HEBEI UNIV OF TECH
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