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Threonine aldolase, mutants and their application in the preparation of substituted phenylserine derivatives

A technology of threonine aldolase and phenylserine, applied in the application field of phenylserine derivatives, can solve the problem of insufficient chiral selectivity, limited application, and poor stability of threonine aldolase And other issues

Active Publication Date: 2021-09-17
王喆明 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the poor stability of threonine aldolase, the chiral selectivity, especially the insufficient chiral selectivity of β-hydroxyl, limits the application of this enzyme in chiral synthesis.

Method used

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  • Threonine aldolase, mutants and their application in the preparation of substituted phenylserine derivatives
  • Threonine aldolase, mutants and their application in the preparation of substituted phenylserine derivatives
  • Threonine aldolase, mutants and their application in the preparation of substituted phenylserine derivatives

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Amplification of threonine aldolase gene PSALD

[0026] According to the threonine aldolase gene information from Pseudomonas putida included in Genebank (the gene sequence is shown in SEQ ID NO: 1), the total genomic DNA of the thalline was extracted with a rapid nucleic acid extraction instrument, Using the genomic DNA as a template, PCR amplification was performed under the action of primer 1 (ATGAATGGTGAAA CCAGCCG) and primer 2 (TTAACGTTCCTGGGTGCGAT). PCR reaction system (total volume 50 μL): 5 μL of 10×Pfu DNA Polymerase Buffer, 1 μL of 10 mM dNTP mixture (2.5 mM each of dATP, dCTP, dGTP, and dTTP), 1 μL of cloning primer 1 and primer 2 each at a concentration of 50 μM, 1 μL of genomic DNA, Pfu DNA Polymerase 1μL, nucleic acid-free water 40μL.

[0027] Using BioRad PCR instrument, PCR reaction conditions: pre-denaturation at 95°C for 5min, denaturation at 95°C for 30s, tempering at 65°C for 45s, extension at 72°C for 1min, a total of 30 cycles, and fina...

Embodiment 2

[0028] Example 2: Amplification of Threonine Aldolase Gene CCALD

[0029] According to the threonine aldolase gene information from Caulobacter crescentus included in Genebank (the gene sequence is shown in SEQ ID NO: 88), the total genomic DNA of the thalline was extracted with a rapid nucleic acid extraction instrument, Using the genomic DNA as a template, PCR amplification was performed under the action of primer 3 (ATGACCCAFACCGCGCCCCGCTA) and primer 4 (CTAAGCCACTCGCTTCAGCGCC). PCR reaction system (total volume 50 μL): 5 μL of 10×PfuDNA Polymerase Buffer, 1 μL of 10 mM dNTPmixture (2.5 mM each of dATP, dCTP, dGTP, and dTTP), 1 μL of cloning primer 1 and primer 2 each at a concentration of 50 μM, 1 μL of genomic DNA, Pfu DNA Polymerase 1μL, nucleic acid-free water 40μL.

[0030] Using BioRad PCR instrument, PCR reaction conditions: pre-denaturation at 95°C for 5min, denaturation at 95°C for 30s, tempering at 65°C for 45s, extension at 72°C for 1min, a total of 30 cycles, a...

Embodiment 3

[0031] Embodiment 3: the construction of the recombinant escherichia coli of threonine aldolase gene PSALD

[0032] 1. Obtaining the recombinant plasmid pET28a-PSALD

[0033] Obtain the PSALD gene sequence, use Nde I and Xho I restriction endonuclease (TaKaRa) to process the amplified fragment after sequencing, and use T 4 DNA ligase (TaKaRa) ligated the fragment with the commercialized vector pET28a treated with the same restriction endonuclease to construct the expression vector pET28a-PSALD.

[0034] 2. Transformation of Escherichia coli E.coli BL21(DE3) with recombinant plasmid

[0035] Add 10 μL of PCR product to 100 μL E.coli BL21 (DE3) competent cell suspension in each tube, mix gently, and let stand in ice bath for 30 min. Transfer to a 42°C water bath and heat shock for 90s. Quickly transfer to an ice bath to cool for 3 min. Add 700 μL LB liquid medium to each tube, and incubate at 37° C. for 1 hour on a shaker at 100 rpm. After culturing, the bacterial solution ...

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Abstract

The invention discloses threonine aldolase, mutants, threonine aldolase and mutant coding genes, recombinant vectors constructed by coding genes, recombinant genetically engineered bacteria obtained by transformation of recombinant vectors, and threonine aldolase and the application of mutants in the preparation of 2- / 3- / 4-position substituted phenylserine derivatives. The present invention uses threonine aldolase and mutants as biocatalysts, 2- / 3- / 4-position substituted benzaldehyde as substrate, pyridoxal 5-phosphate as coenzyme, glycine / glycine ester as Auxiliary substrate, carry out enzyme-catalyzed reaction in appropriate conditions and media, separate and prepare a series of phenylserine derivatives with different substituents, the total yield of this method is in the range of 76-99%, the ee value of the product is greater than 99%, and the de value is greater than 99%. The range is 70‑99%.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to threonine aldolase, a mutant and its application in the preparation of phenylserine derivatives substituted at 2- / 3- / 4-positions. Background technique [0002] 2- / 3- / 4-substituted phenylserine derivatives are important pharmaceutical intermediates and chemical intermediates, especially 4-thiamphenicol phenylserine, which are widely used in the preparation of human antibiotics, poultry and livestock antibiotics, etc. , which represent drugs such as chloramphenicol, thiamphenicol, florfenicol and so on. [0003] The synthesis methods of 2- / 3- / 4-position substituted phenylserine derivatives mainly include chemical synthesis and enzyme catalysis. Among them, the chemical synthesis method is mainly to obtain chiral 2- / 3- / 4-position substituted phenylserine derivatives through addition reaction, esterification reaction and chemical resolution method. Although the chemical method is simple...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N15/75C12N15/81C12N1/21C12N1/19C12P13/00C12R1/865C12R1/19C12R1/125
CPCC12N9/88C12N15/70C12N15/75C12N15/81C12P13/00C12Y401/02005C12Y401/02042
Inventor 王喆明张荣珍谭昊李利宏
Owner 王喆明
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