Activity expression and application of thermobifida fusca xylose isomerase in wine brewing yeast

A technology for xylose isomerase and Saccharomyces cerevisiae, which is applied in the field of extraction of xylose isomerase from Thermosporum tanus, and achieves the effects of good activity expression, good development and utilization prospects

Inactive Publication Date: 2008-09-10
GUANGXI ACAD OF SCI
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AI Technical Summary

Problems solved by technology

[0005] So far, there has been no report on the expression of xylose isomerase fro...

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0018] 1. Extraction of Genomic DNA from Brown Thermobifida fusca

[0019] Inoculate Thermophilospora tan (purchased from China Microorganism Culture Collection Management Center) in 20ml starch beef extract medium. The medium is composed of 9% soluble starch, 0.5% beef extract, 0.5% yeast extract, 0.5% NaCl, 0.2% K 2 HPO 4 and 1% trace element solution. The inoculated culture medium was cultured overnight in a constant temperature shaker at 55°C. Take 1.5ml of the culture and centrifuge for 2min, wash the obtained precipitate with sterile water for 3 times, dry the water, put the precipitate into a mortar filled with liquid nitrogen and quickly grind it into a powder, put the powder into an EP tube, and then use Beijing Tiangen The bacterial genome DNA extraction kit provided by Bioengineering Company was used to extract the total DNA.

[0020] 2. Cloning of xylose isomerase gene

[0021] Design the following primers:

[0022] 1. Forward primer:

[0023] 5'-ACTCATATGAA...

Embodiment 2

[0045] Genetically engineered yeasts use xylose as carbon source to ferment and produce ethanol:

[0046] 1. Pick out a single colony and insert it into 15ml of SC-U [0.17% (m / v) Yeastnitrogen base, (NH 4 ) 2 SO 4 0.5% (m / v), 0.01% (m / v) essential amino acids (His, Leu, Trp, without uracil U)] culture medium, shaking culture overnight at 30°C and 220rpm / min The recombinant bacteria were cultivated under the condition of xylose isomerase gene expression.

[0047] 2. Determine the OD of the overnight culture 600 , calculate the OD of adding 50ml medium 600 The amount of the overnight culture was 0.4, centrifuged at 2000rpm, 4°C for 5min, and the supernatant was discarded.

[0048] 3. The pellet was resuspended in 2ml of induction medium (SC-U medium containing 2% galactose) and inserted into 50ml of SC-U medium, and cultured with shaking at 30°C and 220rpm / min for 8-12 hours.

[0049] 4. After collecting the cells by centrifugation, wash the cells once with sterilized sali...

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Abstract

The invention discloses a method for extraction of xylose isomerases by taking brown hot crack loving spore fungus as sources and the activity expression and application of the brown hot crack loving spore fungi xylose isomerases in Saccharomyces cerevisiae. The molecule weight of the brown hot crack loving spore fungi xylose isomerases is 43 kilodaltons; the most suitable reaction temperature of the xylose isomerases is 80 to 85 DEG C; and the most suitable reaction pH of the xylose isomerases is 7.0. The xylose isomerases can convert xyloses into xyluloses under normal biochemical reaction conditions; genes of the xylose isomerases can be recombined into the Saccharomyces cerevisiae in which the activity expression is realized, and the xyloses or other carbon sources such as lignocellulose hydrolysate and so on can be utilized for production of various fermentation products such as alcohols and so on. The method utilizes the genetic engineering means to expand substrates which are converted by the alcohols and has important theoretical significance and application value on full utilization of lignocellulose resources.

Description

technical field [0001] The invention relates to the extraction and application of a xylose isomerase, in particular to a method for extracting the xylose isomerase from Thermobista cerevisiae and its active expression and application in Saccharomyces cerevisiae. Background technique [0002] Xylose isomerase (EC5.3.15) can not only catalyze the conversion of xylose into xylulose, but also catalyze the conversion of glucose into fructose. This enzyme can be applied to the production of high fructose syrup. Another important potential application of xylose isomerase lies in the development of renewable lignocellulosic resources. Lignocellulose is a renewable resource with huge annual output and has not yet been fully utilized. The content of xylose in its hydrolyzate is second only to glucose. At present, with the increasingly serious energy crisis and environmental pollution, the use of renewable resources and the search for clean energy are the general trend. Fuel ethanol ...

Claims

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Application Information

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IPC IPC(8): C12N9/90C12N15/61C12N15/81C12P1/02
Inventor 王青艳杨登峰黄日波孙靓陆雁肖代俊黄志民黎贞崇
Owner GUANGXI ACAD OF SCI
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