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Microbacterium, broad spectrum glycosaminoglycan lyase expressed by microbacterium, and coding gene and applications of broad spectrum glycosaminoglycan lyase

A technology of microbacteria and bacterial strains, applied in the field of microbacteria, can solve problems such as no glycosaminoglycan lyase

Active Publication Date: 2017-06-30
济南唐泰生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, from Flavobacterium sp. [15] , Bacillus sp. [16] , Bacteroides sp. [17] , Streptomyces sp. [18] , Vibrio sp. [19] Glycosaminoglycan lyases have been isolated from various microorganisms, but no glycosaminoglycan lyases from Microbacterium sp.

Method used

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  • Microbacterium, broad spectrum glycosaminoglycan lyase expressed by microbacterium, and coding gene and applications of broad spectrum glycosaminoglycan lyase
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  • Microbacterium, broad spectrum glycosaminoglycan lyase expressed by microbacterium, and coding gene and applications of broad spectrum glycosaminoglycan lyase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1. Obtaining of Microbacterium sp. WS15 strain

[0057] Weigh 10g of sea mud sample (the sea mud sample was collected from Qingdao trestle in May 2015), add 10mL sterile water to obtain the extract, respectively take 1mL supernatant and add it to 9mL sterile water, dilute to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 Wait for 5 concentrations, spread them on the sole carbon source separation medium, incubate at 28°C for 72 hours, count the colonies, then select the colonies with obvious differences in colony morphology, and repeat the streaking on the TSB solid plate until the purified single The colonies are then transferred to the corresponding agar slope for later use.

[0058] The cultured strains were respectively inoculated into the only carbon source liquid medium, cultured at 200 rpm and 28°C for 72 hours, observing the turbidity of the bacterial liquid, and taking the culture supernatant for carbazole reaction to detect the consumption of the carbon source. Selec...

Embodiment 2

[0064] Example 2. The cultivation method of Microbacterium sp. WS15 strain, the steps are as follows:

[0065] (1) Inoculate the microbacterium WS15 strain into a liquid medium, and culture it on a shaker for 1 day under the conditions of a temperature of 28°C and a rotation speed of 200 rpm to prepare a seed liquid;

[0066] (2) Take the seed liquid prepared in step (1), inoculate it in liquid culture medium at a volume percentage of 1%, and expand the culture for 1 day under the conditions of a temperature of 28°C and a rotation speed of 200 rotations per minute. Obtain Microbacterium sp. WS15 bacterial liquid;

[0067] The components per liter of the liquid medium are as follows:

[0068] Chondroitin sulfate 5g, tryptone 10g, yeast extract 5g, NaCl 30g, KH 2 PO 4 3g, K 2 HPO 4 7g, (NH4) 2 SO 4 2g, MgSO 4 ·7H 2 O 5mg, FeSO 4 ·7H 2 O 2mg, constant volume of water to 1 liter, pH value 7.2;

[0069] The liquid medium can also be TSB medium, which is composed of: tryptone 17g / L, plant...

Embodiment 3

[0070] Example 3. Extraction of genomic DNA of Microbacterium sp. WS15

[0071] According to the cultivation method described in Example 2, cultivate the WS15 strain to 600nm absorbance (OD 600 ) Is 1.2; take 20mL of the culture broth, centrifuge for 20min under the condition of 12,000×g (g, the earth's gravitational constant), collect the bacterial pellet; suspend the bacteria with 20mL of lysozyme buffer (10mM Tris-HCl, pH8.0) Centrifuge at 12,000×g for 20 minutes to collect the bacterial pellet;

[0072] Add 12.0 mL of lysozyme buffer to the above bacterial pellet to obtain 14.0 mL of bacterial solution. Add 560 μL of lysozyme with a concentration of 20 mg / mL to the final concentration of 800 μg / mL; place in an ice-water bath for 1.0 h , Then transfer to 37℃ water bath, warm bath for 2h, until the reaction system is viscous; add 0.82mL 100mg / mL sodium cetyl sulfonate solution, 100mg / mL proteinase K solution 60μL, warm bath 1.0 at 52℃ h; Add 15 mL of Tris-equilibrated phenol / chl...

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Abstract

The present invention relates to microbacterium, broad spectrum glycosaminoglycan lyase expressed by the microbacterium, and a coding gene and applications of the broad spectrum glycosaminoglycan lyase. According to the present invention, the Microbacterium sp. WS15 strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) on December 05, 2016, and has the preservation number of CGMCC No.13421, wherein the address is Institute of Microbiology, Chinese Academy of Sciences, 3# Court No.1, Beichen West Road, Chaoyang District, Beijing; and the glycosaminoglycan lyase TT16 is firstly obtained from the genome of Microbacterium WS15, can degrade the hyaluronic acid with no sulfation modification, can further degrade chondroitin sulfate A (CS-A), chondroitin sulfate C (CS-C) and chondroitin sulfate E (CS-E) being subjected to different sulfation modifications, and is the broad spectrum glycosaminoglycan lyase.

Description

Technical field [0001] The invention relates to a microbacterium, a broad-spectrum glycosaminoglycan lyase expressed by the bacterium and its coding gene and application, and belongs to the field of biotechnology. Background technique [0002] Glycosaminoglycan (GAGs), also known as amino polysaccharides, acid mucopolysaccharides, etc., are linear polysaccharides formed by the polymerization of repeating disaccharide structural units [1] . They are widely present in animal connective tissues, and most of them exist in the body by covalently bonding with core proteins to form proteoglycans. [0003] GAGs can be divided into hyaluronic acid (HA), heparin (HP), and heparin sulfate (HS) according to their monosaccharide composition, glycosidic bond connection form, and the degree and position of sulfation. , Chondroitin sulfate (CS), dermatan sulfate (DS) and kermatan sulfate (KS) [2] . The structure of HA is relatively simple. The disaccharide unit composed of D-glucuronic acid and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/88C12N15/60C12P19/26C12P19/12C12R1/01
CPCC12N1/20C12N9/88C12P19/12C12P19/26C12N1/205C12R2001/01
Inventor 李博刘会会蔡晓娟颜颢张传伟韩文君王申
Owner 济南唐泰生物科技有限公司
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