Immobilization and application of 1,3-dihydroxy acetone producing recombinant genetic engineering bacteria

A technology of dihydroxyacetone and genetically engineered bacteria, applied in the direction of genetic engineering, application, plant genetic improvement, etc., can solve the problems of difficulty in increasing the yield of DHA, inactivation, lysing of fermentation cells, etc., and achieve high product expression and repeatability. The effect of good usability and broad application prospects

Inactive Publication Date: 2015-08-19
XUZHOU AOGEMAN NEW MATERIAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the main problem of this method is that when the concentration of the substrate glycerol and the product DHA in the medium is too high, high osmotic pressure is generated, which makes the fermentation cells lyse and inactivate, thus making it difficult to increase the yield of DHA.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Plasma particles and strains:

[0035] Cloning vector PET28a, Escherichia coli E.Coli JM109 were purchased from commercial companies.

[0036] Restriction enzymes, Taq enzymes, and T4 DNA ligase were purchased from Dalian Bao Biological Company. MCM-41 is from Jiangnan University. Other chemical reagents are domestic analytical pure chemical reagents.

[0037] Klebsiella isolation medium: distilled water 1000mL, glycerin 5g, NaCl 5g, yeast powder 5g, glucose 20g, agar powder 15g, adjust pH to 7.0, sterilize at 0.1MPa for 20min, cool to 35°C, add Neil red ( Nile Red) 2mL / L (0.30mg Nile Red dissolved in 100mL dimethyl sulfoxide), pour it into a petri dish under sterile conditions, cool it down and set aside.

[0038] Nutrient-enriched medium: distilled water 1000mL, yeast powder 10g, agar powder 10g, glycerin 3g, (NH 4 ) 2 SO 4 5g, adjust the pH to 7.0, and sterilize at 0.1MPa for 10min.

[0039] Product fermentation medium:

[0040] Preparation of phos...

Embodiment 2

[0056] The composition and culture conditions of each medium are the same as above. Recombinant Escherichia coli was prepared as above.

[0057] Supramolecular Template Immobilization:

[0058] Add 2.7g of dealuminated ultra-stable Y zeolite and 5mL of pH7.0 phosphate buffer into a 25mL conical flask, then add 2mL of activator and 2mL of Escherichia coli engineering bacteria solution, and finally add 7.5% of Glutaraldehyde until the solution mass fraction is 0.70%. The immobilization reaction was carried out at 170 r / min and 15 degrees for 2 hours, and then filtered and washed with 50 mL of phosphoric acid solution with a pH of 7.0 to obtain immobilized Escherichia coli engineering bacteria.

[0059] Klebsiella fermentation and DHA extraction:

[0060] The pre-cultivation is in the L-test tube, 5 ml of nutrient-rich medium is added by aseptic operation, and a single colony of Klebsiella pneumoniae is inserted with a sterile toothpick. Cultivate at 35°C and 120r / min for 15h...

Embodiment 3

[0065] The composition and culture conditions of each medium are the same as above. Recombinant Escherichia coli was prepared as above.

[0066] supramolecular template immobilization

[0067] Add 3.3g of mesoporous carbon and 5mL of pH7.0 phosphate buffer into a 25mL Erlenmeyer flask, then add 2mL of activator and 2mL of Escherichia coli engineering bacteria solution, and finally add 7.5% glutaraldehyde dropwise to the solution Until the solution mass fraction is 0.85%. The immobilization reaction was carried out at 170 r / min and 15 degrees for 2 hours, and then filtered and washed with 50 mL of phosphoric acid solution with a pH of 7.0 to obtain immobilized Escherichia coli engineering bacteria.

[0068] Klebsiella fermentation and DHA extraction:

[0069] The pre-cultivation is in the L-test tube, 5 ml of nutrient-rich medium is added by aseptic operation, and a single colony of Klebsiella pneumoniae is inserted with a sterile toothpick. Cultivate at 35°C and 120r / min for...

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Abstract

The invention discloses immobilization and an application of 1,3-dihydroxy acetone producing recombinant genetic engineering bacteria; a supramolecular template is used for immobilization of the engineering bacteria of an escherichia coli expressed glycerol dehydrogenase gene (gldA). Construction of the engineering bacteria comprises that with a klebsiella pneumoniae genome DNA as a template, PCR amplification is applied to obtain the gene (gldA) encoding glycerol dehydrogenase (GDH), the gene (gldA) is cloned onto an escherichia coli expression vector PET 28a, and a cloned vector PET-gldA is constructed and is expressed successfully in E.coli JM109. A method for expressing 1,3-dihydroxy acetone comprises that the recombinant genetic engineering bacteria immobilized by the supramolecular template are fermented in a glycerol-containing culture medium to obtain the 1,3-dihydroxy acetone. With the immobilized stain, the product expression quantity is high and is up to a maximum of 120 g / L, the repeated use is good, and the product expression quantity still can reach 107 g / L after repeated use for 15 times, an active role is provided in microbial fermentation preparation of the 1,3-dihydroxy acetone, and application prospects are wide.

Description

[0001] Technical field: The invention belongs to microbial fermentation technology, and relates to the immobilization and application of recombinant genetically engineered bacteria producing 1,3-dihydroxyacetone. Background technique [0002] 1,3-Dihydroxyacetone is an important chemical and biochemical raw material, a synthetic intermediate of medicine and pesticide, and a multifunctional food additive with a wide range of uses. The synthesis methods of dihydroxyacetone mainly include precious metal catalytic oxidation method and microbial method. The synthesis method of heavy metal catalyzed oxidation of glycerol, due to the inability to overcome the fatal shortcoming of poor catalytic selectivity, causes the conversion rate of glycerol to be lower than 40%, and the productive rate of DHA is lower than 25%. The industrial production method currently utilizes microbial batch fermentation. This method is to use the dehydrogenase produced by the bacteria to carry out dehydro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/14C12N15/70C12N15/53C12P7/26C12R1/19
Inventor 不公告发明人
Owner XUZHOU AOGEMAN NEW MATERIAL TECH CO LTD
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