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Acetylglucosamine phosphate mutase atagm coding gene and its enzyme, preparation, application and enzyme activity detection method

A technology of glucosamine phosphate and coding gene, which is applied in the field of gene sequence and preparation of acetylglucosamine phosphate mutase AtAGM, which can solve the problems that the enzyme activity and properties cannot be guaranteed, and the activity of hexose phosphate mutase cannot be accurately measured. , achieve good separation effect, good enzymatic properties, and promote the accumulation effect

Active Publication Date: 2020-08-04
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the coupled enzyme can only be prepared by the laboratory itself, its enzyme activity and properties cannot be guaranteed, making it impossible to accurately measure the activity of hexose phosphate mutase

Method used

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  • Acetylglucosamine phosphate mutase atagm coding gene and its enzyme, preparation, application and enzyme activity detection method
  • Acetylglucosamine phosphate mutase atagm coding gene and its enzyme, preparation, application and enzyme activity detection method
  • Acetylglucosamine phosphate mutase atagm coding gene and its enzyme, preparation, application and enzyme activity detection method

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Cloning of embodiment 1 acetylglucosamine phosphate mutase AtAGM full-length gene

[0043] The mRNA of Arabidopsis leaves was extracted according to the operation steps of the RNA extraction kit (Bomad Biotechnology, Cat. No. RN0112). After analyzing the sequence of acetylglucosamine phosphate mutase in The National Center for Biotechnology Information (NCBI) database, design primers Agm-F: 5'-CACCCCCATATGATGGACGAGATCCAAATAGC-3'; Agm-R: 5'-CTCGAGGCTTGAACCAAGGAAGCTTTTGACG-3' for amplification The gene sequence encoding the mature protein of acetylglucosamine phosphate mutase AtAGM was amplified by PCR using the reversed cDNA of the extracted Arabidopsis thaliana as a template. The PCR reaction conditions were: 94°C for 2min, 1 cycle; 94°C for 30s, 55°C for 30s, 72°C for 2min, 30 cycles; 72°C for 5min, 1 cycle. After the PCR product was analyzed by agarose gel electrophoresis, the target fragment was recovered by gel cutting, and after double enzyme digestion, it was con...

Embodiment 2

[0044] Example 2 Sequence analysis of acetylglucosamine phosphate mutase AtAGM gene

[0045] The results of the sequencing were analyzed using the Basic Local Alignment Search Tool (BLAST) in the GenBank database, and the Vector NTI Suite 8.0 software was used for multiple sequence alignment and sequence information analysis.

[0046] The coding region of the obtained acetylglucosamine phosphate mutase gene (named AtAGM) is 1698 bp long, and its nucleotide sequence is shown in SEQ ID NO.3. AtAGM encodes 564 amino acids and a stop codon, its amino acid sequence is shown in SEQID NO.4, the theoretical molecular weight of the protein is 61.5kDa, and the predicted isoelectric point is 5.35. The nucleotide sequence of AtAGM is called DNA-DAMAGE-REPAIR / TOLERATION 101 (DRT101) in the Arabidopsis genome, located on the fifth chromosome of Arabidopsis (locus-tag="AT5G18070"), with acetylglucose Aminophosphomutase function, but only has sequence information, and its activity and proper...

Embodiment 3

[0047] Example 3 Recombinant expression and purification of AtAGM gene in Escherichia coli

[0048] Sequencing results showed that the AtAGM gene shown in SEQ ID NO.3 was inserted into pET23a, and the insertion direction was correct, which proved that the constructed recombinant plasmid was correct, and the recombinant plasmid was named pET23a-AtAGM.

[0049] Transform pET23a-AtAGM into Escherichia coli strain BL21(DE3) for induced expression. Add the overnight cultured seed solution to fresh LB medium with 1% inoculum size for expansion at 37°C, add IPTG when the OD600nm of the bacterial solution is 0.6-0.8, make the final concentration 0.5mM, and carry out at 16°C Induce overnight. After the cells were broken, the supernatant was collected by high-speed centrifugation for nickel column purification, and gradient imidazole elution (20-500mM imidazole, 20mM Tris-HCl, pH7.6). Polyacrylamide gel electrophoresis was used to detect the expression and purification of acetylglucos...

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Abstract

The invention discloses a gene sequence of acetylglucosamine phosphate mutase (Arabidopsis thaliana N-acetylpHospHoglucosamine mutase, AtAGM) derived from Arabidopsis thaliana (Arabidopsis thaliana) and its application. The invention provides a method for preparing the acetylglucose phosphomutase, namely cloning the gene of the enzyme into an Escherichia coli expression vector to obtain a recombinant strain of Escherichia coli capable of heterologously expressing the enzyme, and using the strain to express heterologously to prepare AtAGM. The present invention also provides the application of the acetylglucosamine phosphate mutase, that is, for the enzymatic catalytic synthesis of UDP-GlcNAc or the production of hexose phosphate isomers.

Description

technical field [0001] The invention relates to a gene sequence of acetylglucosamine phosphate mutase AtAGM and a preparation method thereof, in particular to the application of the enzyme in the production of nucleotide sugar and hexose phosphate isomers. The invention also provides a recombinant plasmid and a recombinant genetically engineered strain of the acetylglucosamine phosphate mutase, and provides a new method for detecting the activity of the hexose phosphate mutase and a method for using the enzyme to produce and separate hexose phosphate. Background technique [0002] Acetylglucosamine (GlcNAc, N-acetyl-β-D-glucosamine) is an amino sugar derivative of glucose. In organisms, it is a monosaccharide with important physiological and biochemical functions after glucose. Often in the form of UDP-GlcNAc, it participates in many biological reaction processes including the nervous system and immune system and the structural composition of various glycoconjugates in orga...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/61C12N9/90C12P19/24C12P19/02C12P19/30C12Q1/533C12Q1/48
Inventor 尹恒贾晓晨张洪艳曹海龙王文霞
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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