Novel alkali-resistant glycoside hydrolase family 10 xylanase produced from micobacterium sp. HY-17 strain

A technology of xylanase and bacterial bacteria, which is applied in the field of xylanase, can solve environmental problems such as harmful

Inactive Publication Date: 2016-04-27
KOREA RES INST OF BIOSCI & BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the bleaching process of wood pulp, organic solvents have traditionally been used to effectively remove lignin, but this process is harmful to the environment

Method used

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  • Novel alkali-resistant glycoside hydrolase family 10 xylanase produced from micobacterium sp. HY-17 strain
  • Novel alkali-resistant glycoside hydrolase family 10 xylanase produced from micobacterium sp. HY-17 strain
  • Novel alkali-resistant glycoside hydrolase family 10 xylanase produced from micobacterium sp. HY-17 strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Production of xylanase (Xylanase) Bacillus (Micobacterium sp.) isolation of bacterial strains

[0086] The present inventors isolated microorganisms having enzymatic activity for hydrolyzing xylan from commensal microorganisms in the intestines of mole crickets.

[0087]Specifically, in order to selectively isolate microorganisms that hydrolyze xylan, M9mineralsaltsagarmedium (Difco) was used as a minimal medium, and a selective medium containing 5g / Lyeastextract (Difco) and 2g / Lazo-xylan (Megazyme) was used . In the selection medium, after diluting the microbial suspension in physiological saline by serial dilution method, inject 100 μl into each selection medium, and incubate at 25-35° C. for 18-36 hours. In each medium, a colony of microorganisms forming a transparent ring due to the decomposition of azo-xylan was selectively isolated. In order to identify the isolated microorganism, PCR on the 16S rDNA sequence was performed with a pair of 27F primer (SEQ ID NO: ...

Embodiment 2

[0090] Cloning of Xylanase (Xylanase)

[0091] In the genomic DNA of the Bacillus (Micobacteriumsp.) HY-17 bacterial strain (preservation number: KCTC12338BP) that decomposes xylan, utilize the gene sequence based on the xylanase gene sequence of GH10 (glycosidehydrolasefamily10) series, amplify The polynucleotide sequence encoding the xylanase protein was amplified and cloned.

[0092] Specifically, after the genomic DNA was isolated from the strain, using the genomic DNA as a template, using 10× buffer (MgCl2), 2.5mMdNTPs, 5× buffer, FastStartTaq DNA polymerase (Roche) and MF-10 forward The primer (SEQ ID NO: 3) was paired with the MR-10 reverse primer (SEQ ID NO: 4), and PCR for the xylanase gene was performed. At this time, the PCR conditions were denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 minutes, annealing at 50°C for 30 seconds, and stretching at 72°C for 40 seconds. A final stretch was performed for 7 minutes at 72°C. The PCR product of the 375...

Embodiment 3

[0098] Refining of xylanase (Xylanase)

[0099] Recombinant XylH (rXylH) obtained by overexpressing the pET-XylH expression vector produced in in Escherichia coli was isolated.

[0100] Specifically, Escherichia coli transformed with each expression vector was inoculated into a liquid LB medium, and cultured with shaking at 37°C. When the OD600 value of each Escherichia coli culture solution reached 0.4 to 0.5, 1.0 mM IPTG was added, followed by shaking and culturing at 30° C. for 5 hours. The culture solution was centrifuged, and the result observed after the cells were pulverized by a sonic pulverizer, rXylH activated inclusion bodies with 20 mM sodium phosphate buffer solution (sodium phosphate buffer pH 7.4) containing 20 mM imidazole (imidazole) and 0.5 M sodium chloride. (inclusion bodies) soluble. The solubilized rXylH cell crush was refolded and purified using a HisTrapHP (GE Healthcare, Sweden) (5-ml) column, and then a liquid chromatography (LC) system (Amphamasi...

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Abstract

The present invention relates to novel xylanase having alkali resistance, which corresponds to glycoside hydrolase family 10 (GH10) isolated from Micobacterium sp. HY-17 strain. More specifically, the xylanase is a novel enzyme which is stable in an alkali environment, effectively hydrolyzes various xylans, hydrolyzes beta-glucan, and degrades cellobiose. Thus, the zylanase of the present invention can be used as an enzyme in the food industry, an enzyme for feed, a biomass pretreatment enzyme, and a useful enzyme for various industry groups including a pulp process.

Description

technical field [0001] The present invention relates to a new xylanase with alkali-resistance properties, which is isolated from the strain of Micobacterium sp. HY-17 and belongs to glycoside hydrolase family 10 (glycosidehydrolase family 10, GH10). Background technique [0002] A great deal of research has been conducted to use various carbon sources as raw materials. Starch polysaccharides contained in this raw material are easily hydrolyzed and are mainly used in the food industry. Therefore, in order to use carbon sources as raw materials in various industrial groups, the necessity of nonstarch polysaccharides (Nonstarch polysaccharides) is increasing. [0003] Xylan (Xylan) is a non-starch polysaccharide that exists in woody, plant and grain by-products. Non-starch polysaccharides can be divided into cellulose and hemicellulose, and the important component of hemicellulose is xylan. Xylanase is an enzyme that hydrolyzes this xylan (xylan) into xylose, xylobiose, and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/42A23L33/17A23K20/189C12R1/01
CPCA23K10/14A23K20/189A23K50/30A23K50/75C12N9/24C12N1/205C12R2001/01C12N1/20
Inventor 朴镐用孙光熙金到永申东夏赵汉永
Owner KOREA RES INST OF BIOSCI & BIOTECH
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