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Recombinant escherichia coli immobilized cell and application to production of xylitol from xylose mother liquor

A technology for recombinant Escherichia coli and immobilized cells, applied in the direction of immobilized on or in inorganic carriers, based on microorganisms, bacteria, etc., can solve the problems of low space-time yield, complex process, high cost, etc., and achieve simple production methods , Simplify the purification process and increase the effect of added value

Active Publication Date: 2018-12-11
ZHEJIANG UNIV OF TECH
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  • Abstract
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  • Claims
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Problems solved by technology

Due to the production process, the viscosity of the xylose mother liquor is relatively high, so that the xylose mother liquor cannot use monosaccharides again through concentrated crystallization
Under normal circumstances, about 1.5 tons of xylose mother liquor is produced for every ton of xylose produced. Its price is low and its utilization rate is low, causing a lot of waste of resources and serious environmental pollution. Therefore, xylose mother liquor has strong development and application potential
At present, some scholars have used xylose mother liquor by chemical method or fermentation method, but due to the complicated process, high cost and low product price, it has not achieved large-scale industrial application
Chinese patent CN104086607B discloses a method for producing L-arabinose from xylose mother liquor, which uses yeast fermentation to remove glucose, and then uses chromatographic separation to obtain L-arabinose and xylose components, which are crystallized respectively to obtain arabinose and xylose Sugar products, the disadvantage of this method is that the operation of using yeast to remove glucose is complicated, and the utilization value of xylose is lower compared with xylitol
Chinese patent CN102603814B discloses a method for improving crystallization efficiency in xylose mother liquor, wherein yeast fermentation is used to remove glucose and galactose, and then membrane separation is used to remove impurities such as colloids and proteins to obtain a supernatant mainly containing xylose and arabinose. The method has complex fermentation steps and low added value of the product, which limits its application range
Chinese patent CN107384815A discloses an engineering strain of Saccharomyces cerevisiae, which uses xylose mother liquor to produce xylitol by fermentation method, and the conversion rate reaches 100% of the theoretical value after 60 hours of fermentation. This method has the disadvantages of long fermentation period, difficulty in repeated use of bacteria, time and space The yield is low and cannot meet the requirements of industrialized production

Method used

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  • Recombinant escherichia coli immobilized cell and application to production of xylitol from xylose mother liquor
  • Recombinant escherichia coli immobilized cell and application to production of xylitol from xylose mother liquor
  • Recombinant escherichia coli immobilized cell and application to production of xylitol from xylose mother liquor

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Experimental program
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Embodiment 1

[0027] The component analysis of embodiment 1 xylose mother liquor

[0028] Each component in the xylose mother liquor is detected and analyzed by high performance liquid chromatography. The detection method of xylitol and its by-products generated by catalyzing the xylose mother liquor is the same as the detection method of the xylose mother liquor. The detection conditions of the high performance liquid chromatography are as follows: The system is Waters 2414, RID detector. The detection conditions are: the chromatographic column is BioRad Aminex HPX-87H (300×7.8mm), the column temperature is 60°C, and the mobile phase is 5mM H 2 SO 4 solution, the flow rate is 0.6ml / min, and the injection volume is 20μL.

[0029] Sample processing method: dilute the sample to about 5g / L, in 1.2×10 4 Centrifuge at rpm for 5 min, retain the supernatant and pass through a 0.22 μm filter membrane for liquid phase analysis.

[0030]This method effectively detects the main components in the x...

Embodiment 2

[0033] Example 2 Construction of recombinant Escherichia coli E.coli BL21(DE3) / pCDFDuet-1-XR-GDH

[0034] Recombinant Escherichia coli construction process: the nucleotide sequence is xylose reductase gene XR shown in SEQ ID NO.1 to construct the expression plasmid pET-28b(+)-XR, and the nucleotide sequence is glucose shown in SEQ ID NO.2 The dehydrogenase gene GDH was constructed to express the plasmid pET-28b(+)-GDH. Plasmids pET-28b(+)-XR, pET-28b(+)-GDH and empty plasmid pCDFDuet-1 were respectively extracted by plasmid extraction kit. The pET-28b(+)-GDH plasmid and the empty plasmid pCDFDuet-1 were double-digested with restriction endonucleases NdeI and XhoI respectively, and the GDH gene fragment was recovered by agarose gel electrophoresis and gel recovery kit. The GDH gene fragment was ligated with the double digestion product of the empty plasmid pCDFDuet-1 to obtain the plasmid pCDFDuet-1-GDH. The pET-28b(+)-XR plasmid and the plasmid pCDFDuet-1-GDH were double-dig...

Embodiment 3E

[0036] Example 3E. coli BL21(DE3) / pCDFDuet-1-XR-GDH whole cell immobilization

[0037] 1. Preparation of immobilized cells

[0038] The present invention relates to a fixed selective cross-linking method for whole cells of recombinant genetically engineered bacteria.

[0039] Take by weighing 2g of the wet thalli cells that embodiment 2 obtains in 20mL, the pH is in the phosphate buffered saline solution of 8.5, add the activated carbon that final concentration 6g / L handles and stir at room temperature (activated carbon processing method: granular activated carbon crosses 40 mesh sieves, immerses In 1M hydrochloric acid, stir at 50°C for 1 hour. Suction filter, rinse with distilled water until the filtrate is neutral, put it in an oven to dry, and set aside), then add polyethyleneimine with a final volume concentration of 5% and stir evenly, and then add the final volume Concentration of 50% glutaraldehyde, cross-linking reaction at room temperature for 2 hours. After suctio...

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Abstract

The invention discloses a recombinant escherichia coli immobilized cell and application to production of xylitol from xylose mother liquor. The immobilized cell is prepared by the steps: jointly guiding xylose reductase gene and glucose dehydrogenase gene into host escherichia coli to obtain recombinant escherichia coli containing bi-enzyme gene, then fermenting and culturing the recombinant escherichia coli and taking wet somatic cell to be immobilized to obtain the recombinant escherichia coli immobilized cell. According to the recombinant genetically-engineered bacterium E.coli BL21(DE3) / pCDFDuet-1-XR-GDH disclosed by the invention, bi-enzyme coexpression of xylose reductase and glucose dehydrogenase is achieved, no extra coenzyme is added into a bi-enzyme coupling system, a productionmethod is simple and efficient, the space time yield is far higher than a fermentation method, the xylose conversion rate in the xylose mother liquor can reach 99% or above, and glucose is utilized asan auxiliary substrate; after 200g / L xylose is catalyzed by the recombinant cell and reacts for 30 h, the xylitol yield is 100%.

Description

technical field [0001] The invention relates to a genetically engineered bacterial strain and its application in producing xylitol from xylose mother liquor, and belongs to the technical field of biocatalysis. Background technique [0002] Xylose mother liquor is the waste sugar liquid left after xylose is produced by chemical method using biomass resources such as corncobs and bagasse. It is a dark viscous liquid left after xylose crystallization. Generally speaking, xylose mother liquor It contains about 50-60% sugar, which contains xylose, arabinose, glucose and galactose, among which xylose contains about 40-50%. Due to the production process, the xylose mother liquor has a high viscosity, so that the xylose mother liquor cannot use monosaccharides through concentrated crystallization again. Normally, about 1.5 tons of xylose mother liquor is produced for every 1 ton of xylose produced. Its price is low and its utilization rate is low, causing a lot of waste of resource...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/14C12N1/21C12P7/18C12R1/19
CPCC12N9/0006C12N11/14C12P7/18Y02P20/584
Inventor 金利群郑裕国柳志强许韦徐建妙贾东旭
Owner ZHEJIANG UNIV OF TECH
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