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Heparosan-glucuronic acid-5-epimerase, and method for producing polysaccharide using same

A technology of glucuronic acid and heparin precursor is applied in the field of bacterial heparin precursor-glucuronic acid-5-epimerase, which can solve the problems of unclear and undiscovered products.

Active Publication Date: 2016-01-27
SEIKAGAKU KOGYO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no protein homology to the known epimerase, C5-epi, was found
In addition, for the African giant snail, the enzyme cascade involved in the biosynthesis of AS has not been elucidated, and the substrates of the enzymes that synthesize IdoA and the products obtained through enzymatic reactions are unclear

Method used

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  • Heparosan-glucuronic acid-5-epimerase, and method for producing polysaccharide using same
  • Heparosan-glucuronic acid-5-epimerase, and method for producing polysaccharide using same
  • Heparosan-glucuronic acid-5-epimerase, and method for producing polysaccharide using same

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Experimental program
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Embodiment 1

[0229] is derived from the fractionation of the mRNA of the African large snail mucus gland

[0230] The mRNA of the African giant snail was extracted from the mucus gland by the thermal phenol method (for example, VerwoerdTC, DekkerBM, HoekemaA., NucleicAcidsRes. 1989Mar25; 17(6): 2362.), and then used for the purification method using magnetic beads and mRNA Obtained by fractionation. The steps are shown below.

[0231] A large African snail was cut open with a cutter, the excised mucus gland was washed with distilled water, and then sealed in a 1.5 mL screw-cap test tube. Immerse the test tube in liquid nitrogen for 1 minute to freeze, then use a homogenizer to crush the mucus glands, add 0.5 mL of hotextraction buffer heated to 80°C in a water bath (heat to dissolve phenol and buffer in a water bath at 55°C) solution (0.1M LiCl, 100mM Tris-HCl (pH = 8.0), 10mM EDTA, 1% SDS) was added in a 1:1 and stirred solution), and stirred for 30 seconds using a test tube mixer. Th...

Embodiment 2

[0234] Preparation of the African giant snail cDNA phage library

[0235] cDNA was obtained from the mRNA obtained in the above by using AccuScript Hi-FicDNA Synthesis Kit (manufactured by Agilent Technologies) according to the protocol attached hereto. That is, using the mRNA obtained in the above as a template, a reverse transcription reaction was performed using an XhoI site-added oligo dT primer (SEQ ID NO: 6), 5-methyl dCTP, and Moloneymurineleukemiavirus reversetranscriptase (MMLV-RT) . Next, a nick translation reaction using DNApolymeraseI was performed in the presence of RNaseH to obtain double-stranded cDNA.

[0236] The PfuDNA Polymerase attached to the kit was applied to the double-stranded cDNA to blunt both ends of the cDNA, and then the EcoRI linker (described later) was prepared according to the protocol attached using DNALigationKitVer.2.1 (manufactured by TakaraBIO). ) to ligate the double-stranded cDNA. Next, restricted digestion was performed using the...

Embodiment 3

[0239] Acquisition of a DNA fragment encoding a polypeptide of HG-5epi

[0240] Escherichia coli XL-1BlueMRF' strain (manufactured by Agilent Technologies) was used as a host cell (host bacterium) for infecting the cDNA phage library obtained in the above , and the phage was amplified according to the plate lysis method described in detail below. Increment and recycling.

[0241] Plant the host bacteria in 20 mL of LB medium (containing 0.2% maltose, 10 mM MgSO 4 ) and cultured overnight at 37° C. with shaking to obtain a culture solution with an absorbance of about 1.5 at a measuring wavelength of 600 nm. The solution of the phage library obtained in the above was dispensed into three 50 mL test tubes at 30 μL portions, and 5 mL of the culture solution of the host bacteria was added to each test tube and mixed. Thereafter, the phage particles were left to stand in a water bath at 37° C. for 15 minutes to infect the host bacteria.

[0242] In order to cultivate the host b...

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Abstract

Provided are a polypeptide having heparosan-glucuronic acid-5-epimerase activity, and a means for producing a polysaccharide having an isomerized hexuronic acid residue using same. The cDNA library of Achatina fulica is screened, and the DNA that codes for the polypeptide of heparosan-glucuronic acid-5-epimerase, which is an isomerase that acts on a glucuronic acid residue of N-acetylheparosan and / or an iduronic acid residue of completely desulfated and N-acetylated heparin, is obtained. The polypeptide coded by the DNA is expressed in insect cells, and the polypeptide, which has heparosan-glucuronic acid-5-epimerase activity, is obtained. By contacting the polypeptide with N-acetylheparosan or completely desulfated and N-acetylated heparin, a polysaccharide having the isomerized hexuronic acid residue is obtained.

Description

technical field [0001] The invention relates to a bacterial heparin precursor-glucuronic acid-5-epimerase (heparosan-glucuronicacid-5-epimerase). In particular, the present invention relates to a compound having the activity of isomerizing glucuronic acid residues of N-acetyl bacterial heparin precursors into iduronic acid residues and / or completely desulfating·N-acetylating Enzyme derived from the large African snail with the activity of isomerizing the iduronic acid residue of heparin into glucuronic acid residue; the polypeptide having the activity; the nucleic acid encoding the polypeptide, the vector containing the nucleic acid, and maintaining the nucleic acid And / or the host cell of the vector, the production method of the polypeptide, the production method of the polysaccharide whose hexuronic acid residue is isomerized using the enzyme and / or the polypeptide, etc. Background technique [0002] In the description of the present application, the following abbreviatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N9/90C12P19/26C12P19/64
CPCC12N9/90C12Y501/03C08B37/0075C12P19/04C12P19/24
Inventor 望月秀雄山岸究铃木喜义金永植
Owner SEIKAGAKU KOGYO CO LTD
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