Yeast gene engineering bacterium and endoinulase preparation and its application method

A technology of endo-inulinase and genetically engineered bacteria, which can be applied in the directions of genetic engineering, botanical equipment and methods, biochemical equipment and methods, etc., and can solve problems such as unsatisfactory effects.

Inactive Publication Date: 2002-03-27
HUBEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The effect is not very good

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Identifying and confirming the introduction of a number of GS115 transformants (such as 8--10) of the endo-inulinase gene, cultured in shake flasks, so that the OD600 corresponding to the cell density reaches 2.0--6.0; transfer Induce culture at 28°C----30°C in the induction medium with methanol as the only carbon source.

[0021] After induction and culture for 24 hours, samples were taken every 10 hours, and the samples were analyzed by SDS-PAGE electrophoresis.

[0022] Select, induce, and cultivate the bacterial strain HY005 (GS115+ endo-inulinase gene) of high-efficiency expression endo-inulinase effect, repeat the above-mentioned process and carry out larger-scale (100----1000ml bottling quantity) induction culture, meanwhile Take a sample for SDS--PAGE analysis. When the enzyme production level reaches 250u / ml or more, stop the induction, centrifuge the bacteria, and collect the supernatant containing endo-inulinase, which is the crude enzyme solution....

Embodiment 2

[0023] Example 2: Identifying and confirming that a number of GS115 transformants (such as 8--10) have been introduced into the endo-inulinase gene, cultured in shake flasks, so that the OD600 corresponding to the cell density reaches 2.0--6.0; transfer Induce culture at 28°C----30°C in the induction medium with methanol as the only carbon source.

[0024] The culture was induced for 72 hours, during the first 48 hours, samples were taken every 10 hours, after 48 hours, samples were taken every 24 hours, and the samples were analyzed by SDS-PAGE electrophoresis.

[0025] Select, induce, and cultivate the bacterial strain HY005 (GS115+ endo-inulinase gene) of high-efficiency expression endo-inulinase effect, repeat the above-mentioned process and carry out larger-scale (100----1000ml bottling quantity) induction culture, meanwhile Sampling for SDS--PAGE analysis, when the enzyme production level reaches above 350u / ml, stop the induction, centrifuge the bacteria, collect the sup...

Embodiment 3

[0026] Example 3: Identifying and confirming the introduction of a number of GS115 transformants (such as 8--10) of the endo-inulinase gene, cultured in shake flasks, so that the OD600 corresponding to the cell density reaches 2.0--6.0; transfer Induce culture at 28°C----30°C in the induction medium with methanol as the only carbon source.

[0027] Induced culture for 168 hours, during the first 48 hours, samples were taken every 10 hours, after 48 hours, samples were taken every 24 hours, and the samples were analyzed by SDS-PAGE electrophoresis.

[0028] Select, induce, and cultivate the bacterial strain HY005 (GS115+ endo-inulinase gene) of high-efficiency expression endo-inulinase effect, repeat the above-mentioned process and carry out larger-scale (100----1000ml bottling quantity) induction culture, meanwhile Take a sample for SDS-PAGE analysis. When the enzyme production level reaches above 450u / ml, stop the induction, centrifuge and separate the bacteria, and collect t...

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PUM

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Abstract

The present invention provides a yeast gene engineering bacterium Pichia pastoris GS115/HY005 which is an endo inulase gene obtain by screening strain from Aspergillus by means of PCR method or DNA library hybridization method. It can be cloned and inserted into Pichia yeast integration expression vector, then the obtained expression vector containing endoinulase gene is introduced into Pichia yeast, then a yeast gene engineering bacterium capable of high-effectively expressing endoinulase can be screened. The enzyme activity of the endoinulase prepared with said yeast gene engineering bacterium can be up to above 300 u/ml, and the reaction time for hydrolyze inulin with it is short, and its conversion rate is high, so that it can high-effectively economically convert and prepare oligofructose.

Description

technical field [0001] The invention relates to a genetically engineered bacterium, in particular to a yeast genetically engineered bacterium highly expressing endo-inulinase, an inulinase preparation and a method for preparing fructo-oligosaccharide by hydrolyzing inulin with endo-inulinase. Background technique [0002] Oligosaccharides refer to a class of sugars in which two to ten monosaccharide units are connected by glycosidic bonds to form a straight or branched chain. It has good physiological properties such as low heat, stability, safety and non-toxicity, and it also has the ability to increase beneficial bacteria in the intestines. , The physiological function of reducing harmful bacteria. It has broad prospects in food production and pharmaceutical production. [0003] At present, there are two methods for the industrial production of fructo-oligosaccharides. One is the conversion of sucrose, and the other is obtained by acid hydrolysis or ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N9/24C12N15/56C12N15/81C12P19/00
Inventor 马立新蒋思婧
Owner HUBEI UNIV
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