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Method for producing 1,3-propanediol from whole-cell mixed conversion glycerinum

A cell transformation and whole cell technology, applied in the field of bioengineering, can solve the problems of low tolerance of substrate glycerol, difficulty in separation and purification, and high production cost, so as to avoid the separation and purification process of enzymes, simple culture conditions, and improve utilization rate effect

Active Publication Date: 2018-05-22
镇江百泰生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to overcome the defects in the prior art, such as: high production cost of chemical synthesis, difficulty in separation and purification, low tolerance of production strains to substrate glycerol, conversion rate of glycerol to 1,3-PD Low-level problem, the present invention provides a kind of method that whole cell mixes conversion glycerol to produce 1,3-PD, utilizes Clostridium butyricum XYB11 and wxya Whole cells of genetically engineered bacteria are mixed and transformed into glycerol to produce 1,3-PD, reduce the accumulation of 3-HPA in the metabolic pathway, and increase the production of 1,3-PD

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Construction of Genetically Engineered Bacteria with High 1,3-Propanediol Oxidoreductase Activity

[0028] (1) According to the 1,3-propanediol oxidoreductase gene sequence and the characteristics of the multiple cloning site of the expression vector pET-28a, use bioinformatics software to design and synthesize primers: primer1:5- ACAGCAGCGGCCTGGTGCCGCGAATTTTAAATTAAAAGGAGAA-3, primer2: 5'- GGTGGTGGTGGTGCTCGAGT TTGAATTCTTTAAATATTAT-3', primer1 and primer2 underlined and passed Nhe I and Hind III The linearized pET-28a sequence is complementary.

[0029] (2) Using Clostridium butyricum genomic DNA as a template, the target gene was amplified by PCR.

[0030] (3) PCR reaction parameters: pre-denaturation, 95°C 5min; denaturation, 94°C 2min; annealing, 55°C 30sec; extension: 72°C 90sec; cycle: 30; stop extension: 72°C 10min.

[0031] (4) The obtained PCR product was detected by 1% agarose gel electrophoresis, and an electrophoretic band with a size of about 1.2Kb ...

Embodiment 2

[0039] Preparation of 1,3-propanediol by mixed transformation of whole cells

[0040] (1) Take Clostridium butyricum XYB11 stored at -80°C in 5 mL of seed medium, and culture it statically at 37°C for 12 hours in anaerobic culture to obtain Clostridium butyricum XYB11 seed liquid. Seed medium includes: yeast extract 3g / L, beef extract 10g / L, tryptone 10g / L, glucose 5g / L, soluble starch 1g / L, sodium chloride 5g / L, sodium acetate trihydrate 3g / L , cysteine ​​hydrochloride 0.15g / L.

[0041] (2) The Clostridium butyricum XYB11 seed solution obtained in step (1) was inoculated into the expansion medium at an inoculum amount of 10%, and left to stand for anaerobic culture at 37°C for 12 hours to obtain the Clostridium butyricum XYB11 bacterial solution. The expansion medium includes: yeast extract 3g / L, beef extract 10g / L, tryptone 10g / L, glucose 5g / L, soluble starch 1g / L, sodium chloride 5g / L, sodium acetate trihydrate 3g / L , cysteine ​​hydrochloride 0.15g / L.

[0042] (3) Centri...

Embodiment 3

[0047] Change the concentration of glycerol in the cell transformation fluid component in step (5) of Example 2 to 20 g / L or 80 g / L, and the conditions for other conversions to prepare 1,3-propanediol and the method for measuring the conversion rate are completely the same as in Example 2 same.

[0048] When the concentration of glycerol in the cell transformation liquid components was 20 g / L, the conversion liquid components were detected by high performance liquid chromatography, and the conversion rate of glycerol to 1,3-propanediol was measured to be 84.9%.

[0049] The concentration of glycerol in the cell transformation solution was 80 g / L. The conversion rate of glycerol to 1,3-propanediol was 77.1% when the conversion solution was detected by high performance liquid chromatography.

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Abstract

The invention belongs to the technical field of bioengineering and specifically relates to a method for producing 1,3-propanediol from whole-cell mixed conversion glycerinum. The method for producing1,3-propanediol from the whole-cell mixed conversion glycerinum disclosed by the invention comprises the following specific steps: firstly, preparing clostridium butyricum XYB11 thallus and E.coli-Cb-dhaT thallus; then mixing the two kinds of thallus according to a certain proportion to obtain mixed whole cells; finally, adding the mixed whole cells into a conversion solution containing glycerinumand oscillating and converting to obtain 1,3-propanediol under the certain condition. By means of the method for producing the 1,3-propanediol from whole-cell mixed conversion glycerinum disclosed bythe invention, 3-HPA accumulation in a metabolic pathway is reduced, a utilization rate of the glycerinum is improved, and a 1,3-PD yield is increased; enzyme activity of 1,3-propanediol oxidordeuctase in the E.coli-Cb-dhaT can reach 98U / mg; a conversion rate of the glycerinum can reach 84.9%. The whole cell preparation method disclosed by the invention has the advantage of simpleness; only washing the thallus again is needed, a complex enzyme separating and purifying process is avoided, enzyme is fixed in the cells, reaction conditions are moderate, production cost is reduced, and a wide industrial application prospect is achieved.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a method for producing 1,3-propanediol by mixing whole cells into glycerol, in particular to a method of mixing whole cells of 1,3-propanediol genetically engineered bacteria and Clostridium butyricum XYB11 to produce glycerol by mixing whole cells 1,3-propanediol method. Background technique [0002] As an important chemical raw material, 1,3-propanediol (1,3-PD) can be used to synthesize polycondensates such as polyether, polyester, and polyurethane, and has important applications in the fields of food, medicine, and chemical industry. 1,3-PD shows its great commercial value because of its broad application fields. The current production methods of 1,3-PD include chemical synthesis and microbial fermentation. Due to the high production cost of the chemical synthesis method, the need to carry out under high temperature, high pressure, and expensive catalysts,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P39/00C12P7/18C12R1/19C12R1/145
CPCC12P7/18C12P39/00
Inventor 齐向辉员君华杨苗苗苏本胡克田纳张国艳石坡
Owner 镇江百泰生物科技有限公司
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