Recombinant saccharomyces cerevisiae for producing ethanol by xylose fermentation and construction method thereof

A technology for recombining Saccharomyces cerevisiae and xylose isomerase, applied in the biological field, can solve the problems of no enzyme activity of xylose isomerase, unfavorable yeast growth, slow xylose metabolism, etc., and achieve important economic and social benefits. Effect

Active Publication Date: 2011-01-12
ANHUI BBCA FERMENTATION TECH ENG RES
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AI Technical Summary

Problems solved by technology

The disadvantages of this recombinant S. cerevisiae are: 1. In the fermentation process, the xylose metabolism is slow, and the residual xylose is as high as 1%; 2. Xylitol is easy to cause accumulation during the fermentation process, which increases the toxicity to cells, which is not conducive to yeast growth
Under th

Method used

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  • Recombinant saccharomyces cerevisiae for producing ethanol by xylose fermentation and construction method thereof
  • Recombinant saccharomyces cerevisiae for producing ethanol by xylose fermentation and construction method thereof
  • Recombinant saccharomyces cerevisiae for producing ethanol by xylose fermentation and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Identification of xylose metabolism characteristics of Candida boidinii

[0033]Candida boidinii (CGMCC 2.1645, purchased from China General Microorganism Culture Collection and Management Center) was inoculated on YPD medium at 30°C for activation, and then inoculated on YP (without Glucose) plate medium, cultivated for 2 to 3 days, and as a result, all strains on the plate can use xylose to grow. Pick the cells with the largest colonies on the plate and inoculate them in YP liquid medium containing only xylose as the sole carbon source, and they can grow at 25-32°C and pH4.0-6.0. Explain that Candida boidinii can metabolize xylose.

Embodiment 2

[0034] The mensuration of xylitol content during the xylose fermentation of embodiment 2

[0035] Ferment xylose according to the conditions of Example 1, homogenate and break the cells, centrifuge at 5000 rpm for 5 minutes to obtain supernatant, take 10 mL of supernatant and boil at 100 ° C for 5 minutes, centrifuge to remove protein, and the supernatant after centrifugation is used for chromatographic detection. Agilent HPLC 2D chromatographic workstation: the chromatographic column is ZORBAX carbohydrate, the column temperature is 30°C, the mobile phase is acetonitrile:water (80:20 (V / V)), the flow rate is 1.5mL / min, and the differential refractive index detector detects. Xylitol was detected well within the range of 0.3-50.0 mg / mL. The results showed that xylitol was not detected.

Embodiment 3

[0036] Example 3 Separation and purification of xylose isomerase and enzyme characteristics

[0037] The activated Candida boidinii is inoculated in YPD liquid medium containing 1% xylose, fermented at 25-32 DEG C and pH 4.0-6.0, and the fermented liquid is obtained for purification.

[0038] 1. Purification: ①. Adjust the pH of the fermentation broth to 7.0, add 2% protamine sulfate dropwise, let it stand for 30 minutes, and centrifuge to remove the precipitate. ②. Add 30% ammonium sulfate under the condition of pH 7.0, let it stand for 1 hour after dissolving, centrifuge to remove impurity protein, then add ammonium sulfate to make the saturation to 80%, stand overnight at 4°C, collect the precipitate by centrifugation, and re-dissolve in a small amount of buffer. ③. The above-mentioned concentrated enzyme solution is loaded onto DEAE-Sepharose column chromatography, and the active part is eluted by 0.1M NaCl and further concentrated. ④. The sample in step ③ was purified b...

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Abstract

The invention provides recombinant saccharomyces cerevisiae for producing ethanol by xylose fermentation and a construction method thereof. In the invention, candida boidinii is led to the saccharomyces cerevisiae to metabolize a DNA fragment of a coding gene of related enzymes of the xylose, so that the saccharomyces cerevisiae can secrete and express xylose isomerase the enzymatic reaction conditions of which are close to growth conditions of the saccharomyces cerevisiae, so the recombinant saccharomyces cerevisiae can effectively utilize the xylose to produce the ethanol, thus solving the technical problem that the saccharomyces cerevisiae can not utilize the xylose to produce the ethanol. The invention lays a foundation for industrial application of the saccharomyces cerevisiae for producing the ethanol by using xylose fermentation, thus having great economic benefit and social benefit.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant Saccharomyces cerevisiae capable of fermenting xylose to produce ethanol, as well as a construction method and application of the recombinant bacterium. Background technique [0002] The rising price of fossil fuels such as oil and the decreasing storage, the emission of waste gas, the harmonious development of environmental protection and sustainability, etc., force people to look for an environmentally friendly renewable energy source. As a renewable clean energy, bioethanol can undoubtedly solve this problem. However, the traditional method is to use food crops such as starch to produce ethanol, the price of raw materials is relatively high, and there is also a contradiction of "competing with others for food". Lignocellulose is the most abundant biomass resource in the world, mainly composed of cellulose, hemicellulose and lignin. If the hydrolysis pro...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/81C12N1/19C12P7/06C12R1/865
CPCY02E50/17Y02E50/10
Inventor 李荣杰薛培俭尚海涛徐斌段绪果薛亮胡长浩
Owner ANHUI BBCA FERMENTATION TECH ENG RES
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