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Method for detecting content of threo beta-hydroxy-alpha-amino acid

An amino acid and hydroxyl technology, applied in the field of analysis and detection research, achieves the effect of simple and convenient operation and mild detection conditions

Active Publication Date: 2019-12-20
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

[0003] In the currently known reports, no quantitative and high-throughput detection method has been found for the threo-style β-hydroxy-α-amino acid content. Therefore, a threo-style β-hydroxy-α-amino acid stereo The method of isomer content is very necessary

Method used

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  • Method for detecting content of threo beta-hydroxy-alpha-amino acid
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  • Method for detecting content of threo beta-hydroxy-alpha-amino acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Example 1: Dehydratase gene obtained

[0014] 1.1 Synthesis and acquisition of dehydratase gene

[0015] The dehydratase gene is derived from Paraburkholderia xenovorans LB400 (GenBank: AIP32383). According to the codon preference of Escherichia coli, the gene is optimized and sent to the company for synthesis. The codon-optimized genes are respectively added with NdeI (CATATG) enzyme cutting site at the 5' end point, add XhoI (CTCGAG) restriction site at the 3' end, and clone into PET21a vector. Using the PET21a plasmid with the RasADH gene as a template and designing primers according to the RasADH gene sequence for rolling circle amplification, the PCR amplification reaction system is:

[0016]

[0017] The PCR amplification conditions are:

[0018]

[0019] The PCR amplification products were purified and recovered using a DNA purification kit. The PCR product purified by the DNA purification kit was digested with Dpn I restriction endonuclease, and digeste...

Embodiment 2

[0028] Example 2: Construction and induced expression of dehydratase expression bacteria

[0029] Transfer the digested and purified PCR product into Escherichia coli BL21(DE3) competent cells by chemical transformation, pick a single clone into 4 mL LB medium containing ampicillin (1 mg / mL), and take fresh bacterial liquid Sent to the sequencing company for sequencing, and the sequencing results showed the correct expression bacteria. Prepare 50 mL of seed liquid, and the medium is LB liquid medium (peptone 10g / L, yeast powder 5g / L, NaCl 10g / L), pick a single colony of genetically engineered bacteria with an inoculation loop and insert it into the medium, 37°C, 200rpm Incubate overnight. The overnight cultured seed solution was transferred to the fermentation medium at an inoculum size of 1%, and cultured at 25° C. and 200 rpm for 20 h. Take 5 mL of the fermented liquid to concentrate and ultrasonically disrupt the bacteria, and detect the dehydratase activity.

Embodiment 3

[0030] Example 3: Dehydratase Purification

[0031] After the induced expression, the fermentation broth was centrifuged at 6500rpm for ten minutes to collect the bacterial cells, and the collected bacterial cells were resuspended and washed once with buffer (0.1mol / L pH 7.0 sodium phosphate buffer and 0.5M NaCl), and centrifuged in the same way. The suspension was crushed with a high-pressure homogenizer, and the crushed liquid was centrifuged at 6000 rpm at 4°C for 20 minutes to obtain a crude enzyme liquid. The obtained crude enzyme liquid was separated and purified using a His-Trap TM / FF affinity chromatography column, The eluent (0.1M sodium phosphate, 0.5M NaCl, 0.25M imidazole, pH 7.0) was used for gradient elution, the active fraction was collected, and the purity of the enzyme protein was detected by SDS-PAGE.

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Abstract

The invention discloses a method for detecting stereisomer content of threo beta-hydroxy-alpha-amino acid. By adding dehydratase and Fe3+ to the system to be measured, and performing quantitative detection according to the absorption value of a specific ultraviolet wavelength to obtain threo-beta-hydroxy-alpha-amino acid in the system, the purpose of detecting the stereisomer content of beta-hydroxy-alpha-amino acid is achieved. The method, without using liquid chiral detection, can achieve the stereisomer detection of beta-hydroxy-alpha-amino acid high throughput.

Description

technical field [0001] The invention belongs to the field of analysis and detection research, in particular to a method for detecting content of threo-type β-hydroxyl-α-amino acid. Background technique [0002] Amino acids are the most basic substances that constitute biological proteins, and are also important components of active peptides, enzymes, and other biologically active molecules in organisms, and are closely related to life activities. Most amino acids have chiral enantiomers, L-amino acids and D-amino acids (except glycine), chiral amino acids are not only closely related to life activities, but also can be used as key intermediates in the synthesis pathway for fine chemical industry . Chiral β-hydroxy-α-amino acids and their derivatives, such as thiamphenicol, L-threo-3-(4-p-thiasulfonyl)phenylserine, L-threo-phenylserine (L -threo-phenylserine) derivatives, L-threo-3,4-dihydroxyphenylserine (L-threo-DOPS) and so on are the intermediates of some important drug...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/33
CPCG01N21/33
Inventor 陈曦陈启佳冯进辉吴洽庆朱敦明
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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