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Yeast gene engineering bacteria and heat resistant alkali resistant xylanase preparation and application method

A technology of genetically engineered bacteria and xylanase, applied in the field of yeast genetically engineered bacteria, can solve the problems of low fermentation enzyme activity, insufficient heat resistance or alkali resistance, etc., and achieve high purity, high enzyme activity, and good benefits Effect

Active Publication Date: 2005-11-09
桂林精成生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] China Agricultural University Wang Hai, Li Lite, Shi Bo and others used corncob enzymatic method to prepare xylo-oligosaccharides, (see Food Science: 2002, Vol, 23, No.5, 81-83) their xylan Enzyme activity 3.6IU / ml, fermentation enzyme activity is low, heat resistance or alkali resistance is not good enough
[0007] Foreign Guptan N et al. (Cloning, Expression, and Sequence Analysis of the Gene Encoding the Alkali-Stable, Thermostable Endoxylanase from Alkalophilic, Mesophilic Bacillus sp.Strain NG-27.Appl.Environ.Microbiol, 2000, 66(6): 2631-2635) A heat-resistant and alkali-resistant xylanase gene was cloned, but they only expressed the gene in Escherichia coli, and the enzyme activity measured in a 1000ml shake flask was only 7IU / ml

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Identification and confirmation of several GS115 transformants (such as 8-10) that have introduced the heat-resistant and alkali-resistant xylanase gene, cultured in shake flasks, so that the OD600 corresponding to the cell density reaches 2.0---6.0; transfer Induce culture at 24°C-28°C in the induction medium with methanol as the only carbon source.

[0025] After 48 hours of induction culture, samples were taken every 24 hours, and the samples were analyzed by SDS-PAGE electrophoresis.

[0026] Select, induce, and cultivate the bacterial strain GS115 / HB705 (GS115+ heat-resistant and alkali-resistant xylanase gene) that highly expresses the effect of heat-resistant and alkali-resistant xylanase, repeat the above process for larger scale (100----1000ml) Bottle volume) induction culture, during which sampling is carried out SDS-PAGE analysis, when the enzyme production level reaches more than 36IU / ml, stop induction, centrifuge and separate thalline, collect t...

Embodiment 2

[0027] Example 2: Identify and confirm that several GS115 transformants (such as 8-10) that have introduced the heat-resistant and alkali-resistant xylanase gene are cultured in shake flasks, so that the OD600 corresponding to the cell density reaches 2.0---6.0; transfer Induce culture at 24°C-28°C in the induction medium with methanol as the only carbon source.

[0028] The culture was induced for 192 hours, during the first 48 hours, samples were taken every 12 hours, after 48 hours, samples were taken every 24 hours, and the samples were analyzed by SDS-PAGE electrophoresis.

[0029]Select, induce, and cultivate the bacterial strain GS115 / HB705 (GS115+ heat-resistant and alkali-resistant xylanase gene) that highly expresses the effect of heat-resistant and alkali-resistant xylanase, repeat the above process for larger scale (100----1000ml) Bottle volume) induction culture, during which sampling is carried out SDS-PAGE analysis, when the enzyme production level reaches more ...

Embodiment 3

[0030] Example 3: Identification and confirmation of several GS115 transformants (such as 8-10) that have introduced the heat-resistant and alkali-resistant xylanase gene, cultured in shake flasks, so that the OD600 corresponding to the cell density reaches 2.0---6.0; transfer Induce culture at 24°C-28°C in the induction medium with methanol as the only carbon source.

[0031] During the induction culture for 360 hours, during the first 48 hours, samples were taken every 12 hours, after 48 hours, samples were taken every 24 hours, and the samples were analyzed by SDS-PAGE electrophoresis.

[0032] Select, induce, and cultivate the bacterial strain GS115 / HB705 (GS115+ heat-resistant and alkali-resistant xylanase gene) that highly expresses the effect of heat-resistant and alkali-resistant xylanase, repeat the above process for larger scale (100----1000ml) Bottle volume) induction culture, during which sampling is carried out SDS-PAGE analysis, when the enzyme production level r...

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Abstract

A genetically engineered yeast GS115 / HB705 able to effectively express the high temp and alkali resistant xylanase is disclosed, which can be used to prepare said xylanase through culture, separation and purifying, and in turn the oligoxylose.

Description

technical field [0001] The present invention relates to yeast genetically engineered bacteria, in particular to a yeast genetically engineered bacterium that efficiently expresses heat-resistant and alkali-resistant xylanase, a heat-resistant and alkali-resistant xylanase preparation, and a heat-resistant and alkali-resistant xylanase hydrolysis preparation Xylooligosaccharide method. Background technique [0002] Plants are the main renewable organic resources in nature, and their main components are cellulose, hemicellulose and lignin. Among them, hemicellulose accounts for about 35% of the dry weight of plants, and is the most abundant polysaccharide in nature except cellulose, and hemicellulose is mainly composed of xylan and other polysaccharides. As the main enzyme for xylan degradation, β-1,4-endoxylanase degrades xylan into xylan oligosaccharides or xylose. [0003] Now, xylanase has been used in production practices such as pulp manufacturi...

Claims

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Application Information

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IPC IPC(8): C12N1/19
Inventor 屠俊陈亚兰
Owner 桂林精成生物科技有限公司
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