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Microbial xyloglucan Endotransglycosylase (XET)

A technology of xyloglucan and glycosylase, applied in the directions of microorganisms, microorganism-based methods, transferases, etc., can solve problems such as XET that have not been reported

Inactive Publication Date: 2000-03-22
NOVO NORDISK AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To our knowledge, no XET derived from microorganisms has been reported

Method used

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  • Microbial xyloglucan Endotransglycosylase (XET)
  • Microbial xyloglucan Endotransglycosylase (XET)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0177] Embodiment 1: screening XET positive bacterial strain

[0178] culture medium

[0179] PD agar: 39g potato dextrose agar, DIFCO 0013; add deionized water to 1000ml, autoclave at 121°C for 15-20 minutes.

[0180] YPG agar: 4g yeast extract (DIFCO 0127), 1g KH 2 PO 4 (Merck4873), 0.5g MgSO 4 ·7H 2 O (Merck5886), 15g glucose (Roquette 101-0441), 20g agar (Merck1614), make up to 1000ml with deionized water, and sterilize by high pressure steam at 121°C for 15-20 minutes.

[0181] MEA: 20g malt extract powder (DIFC00186), 1g peptone (DIFC00118), 20g glucose (Roquette France 1010441), 20g agar (Merck1614), make up to 1000ml with deionized water, and autoclave at 121°C for 15 minutes.

[0182] Medium A (per bottle): 30g wheat bran, 45ml of the following solution: 10g rofec (Roquette 101-0441), 10g NH 4 NO 3 (Merck1187), 10g KH 2 PO 4 (Merck 4873), 40g Solcafloc (Dicacel, purchased from Dicalite-Europe-Nord, 9000 Gent, Belgium), 0.75g MgSO 4 .7H 2 O (Merck 5886), 15g...

Embodiment 2

[0208] Example 2: Purification and identification of Dichotomocladium hesseltinei XET

[0209] Dichotomocladium hesseltinei (CBS164.61) was inoculated on 15 PDA agar slants, and cultured at 26° C. for 7 days. The bacterial cells were washed with about 250 ml of sterile distilled water containing 0.1% Tween80, and used to inoculate 80 shake flasks containing medium B (2-3 ml / bottle). The shake flask was cultured at 26° C. with shaking at 200 rpm for 5 days, and after that time, the culture solution was centrifuged at 4000 rpm for 15 minutes.

[0210] Purify the supernatant containing xyloglucan endotransglycosylase (XET) as follows:

[0211]A filter aid was added to the culture solution filtered through the filter cloth, and the solution was further filtered with a Chua depth filter disc to obtain a clear solution. The pH of the filtrate was adjusted to pH 8.0, and the filtrate was diluted with deionized water to achieve the same conductivity as 20 mM Tris / HCl (pH 8.0).

[0...

Embodiment 3

[0217] Example 3: Purification and identification of Tiarosporella phaseolina XET

[0218] Tiarosporella phaseolina (CBS446.97) was inoculated on 15 PDA agar slants and cultured at 26°C for 7 days. The slant was washed with approximately 250 ml sterile distilled water containing 0.1% Tween80 and used to inoculate 80 shake flasks containing medium B (2-3 ml / flask). The shake flask was cultured at 26° C. with shaking at 200 rpm for 7 days, and after the time was up, the culture solution was centrifuged at 4000 rpm for 15 minutes.

[0219] Purify the supernatant containing xyloglucan endotransglycosylase (XET) as follows:

[0220] A filter aid was added to the culture solution filtered through the filter cloth, and the solution was further filtered with a Chua depth filter disc to obtain a clear solution. The filtrate was concentrated by ultrafiltration on a polyethersulfone membrane with a molecular weight cut-off of 3 Kda, followed by dialysis against distilled water to reduc...

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Abstract

It has been found by a screening assay that XET activity is produced by an overwhelming array of phylogenetically dispersed microorganisms. Accordingly, the present invention relates to a xyloglucan endotransglycosylase preparation which is producible by cultivation of a microorganism expressing an XET.

Description

field of invention [0001] The present invention relates to microbial xyloglucan endotransglycosylase (xyloglucanendotransglycosylase, XET), its production method and application. Background of the invention [0002] Xyloglucan endotransglycosylase (XET) is an enzyme known from plants. To our knowledge, no XETs derived from microorganisms have been reported. [0003] Stephen C. Fry et al. in the Journal of Biochemistry (1992, 282, 821-828) imply that XET is responsible for cutting and reconnecting intermicrofibrillar xyloglucan chains, so XET can cause plant cell expansion. cell wall relaxation. [0004] XETs have been proposed for regulating plant morphology (see pages 21, 27-28 in EP562836). [0005] It is believed that XET is present in all plants, especially all terrestrial plants. XET has been extracted from dicotyledonous plants, monocotyledonous plants, specifically gramineous monocotyledonous plants and liliaceae monocotyledonous plants, liverworts and mosses (doc...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N1/19C12N9/10C12N9/24C12N9/42C12R1/645C12R1/69C12S3/04
CPCC12N9/1051
Inventor R·I·尼尔森
Owner NOVO NORDISK AS
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