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Yeast gene engineering fungus and beta mannosan enzyme preparation and production method of manna oligose

A technology of mannanase and genetically engineered bacteria, applied in genetic engineering, botany equipment and methods, biochemical equipment and methods, etc., can solve the problems of high production cost, high cost, complicated process, etc., and achieve low production cost , low production cost and high enzyme production level

Inactive Publication Date: 2004-03-03
HUBEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The cost of these methods is too high, or the product is difficult to purify, or the process is complicated, and most of them are not suitable for industrial large-scale production
[0004] At present, relevant domestic scientific research institutes, such as the Institute of Microbiology of the Chinese Academy of Sciences and Nankai University, have extensively conducted research on mannanase and its related content, but the reported fermentation enzyme activity is low, the purity of the enzymatic hydrolysis product is low, and the production cost is high. Alkaline bacteria are easy to cause environmental pollution in the production process, so it is difficult to carry out industrial development

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Identification and confirmation of several GS115 transformants (such as 8----10) that have introduced the β-mannanase yeast gene, cultured in shake flasks, so that the OD600 corresponding to the cell density reaches 2.0----6.0 ; Transfer to the induction medium with methanol as the only carbon source and induce culture at 28°C----30°C. After induction and culture for 24 hours, samples were taken every 8 hours, and the samples were analyzed by SDS-PAGE electrophoresis.

[0023] Select, induce, and cultivate the bacterial strain HBM047 (GS115+β-mannanase) that highly expresses β-mannanase, repeat the above-mentioned process to carry out induction culture on a larger scale (100----1000ml bottling volume), During this period, samples were taken for SDS-PAGE analysis. When the enzyme production level reached above 300u / ml, the induction was stopped, the bacteria were separated by centrifugation, and the supernatant containing β-mannanase was collected, which was t...

Embodiment 2

[0024] Example 2: Identifying and confirming that several GS115 transformants (such as 8----10) have been introduced into the β-mannanase gene, and cultured in shake flasks, so that the OD600 corresponding to the cell density reaches 2.0----6.0; Received into the induction medium with methanol as the only carbon source and induced culture at 28°C--30°C. The culture was induced for 72 hours, during the first 48 hours, samples were taken every 8 hours, and after 48 hours, samples were taken every 12 hours, and the samples were analyzed by SDS-PAGE electrophoresis.

[0025] Select, induce, and cultivate the bacterial strain HBM047 (GS115+β-mannanase) that highly expresses β-mannanase, repeat the above-mentioned process to carry out induction culture on a larger scale (100----1000ml bottling volume), During this period, samples were taken for SDS-PAGE analysis. When the enzyme production level reached above 960u / ml, the induction was stopped, the bacteria were separated by centrif...

Embodiment 3

[0026] Example 3: Identification and confirmation of a number of GS115 transformants (such as 8--10) that have been introduced with the β-mannanase gene, cultured in shake flasks, so that the OD600 corresponding to the cell density reaches 2.0--6.0; Transfer to the induction medium with methanol as the only carbon source and induce culture at 28°C--30°C. After induction and culture for 168 hours, during the first 48 hours, samples were taken every 8 hours, and after 48 hours, samples were taken every 24 hours, and the samples were analyzed by SDS-PAGE electrophoresis.

[0027] Select, induce, and cultivate the strain HBM047 (GS115+β-mannanase gene) that highly expresses the effect of β-mannanase, and repeat the above process to induce large-scale (100----1000ml bottling volume) During cultivation, samples were taken for SDS-PAGE analysis. When the enzyme production level reached above 1080u / ml, the induction was stopped, the bacteria were centrifuged, and the supernatant conta...

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Abstract

A genetically engineered yeast Pichia pastoris (GS115 / HBMO47) able to effectively express beta-mannase is prepared through screening beta-mannase gene from the environmental microbes, cloning it to the expression carrier of Pichia yeast, introducing the carrier to Pichia yeast, and screening. The process for preparing the high-activity beta-mannase and the high-purity oligomannosan from said Pichia pastoris is also disclosed.

Description

technical field [0001] The present invention relates to a genetically engineered bacterium, in particular to a yeast genetically engineered bacterium highly expressing β-mannanase, a β-mannanase preparation and a production method for preparing mannan oligosaccharides by hydrolyzing β-mannanase . Background technique [0002] Plants are the main renewable organic resources in nature, and their main components are cellulose, hemicellulose and lignin. Among them, cellulose accounts for 35% of the dry weight of plants, and isomannan, as the second component of hemicellulose, is widely distributed in plants. The endosperm of leguminous plant seeds, some plant gums (such as carob gum, guar gum, safflower horn gum, etc.), coconut meat powder, coffee beans, lettuce, leucaena, phoenix wood, chickweed, etc. are rich in semi-milk Mannan, konjac bulbs, Australian palmetto, and onetail root are rich in glucomannan (Marga F., Ghki C.et aI.AppI.Environ.Microb...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N9/24C12N15/31C12N15/56C12N15/70C12N15/81C12P19/00C12Q1/68
Inventor 王华伟李静
Owner HUBEI UNIV
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