A strain of Escherichia coli and its application in biocatalytic production of low by-product nicotinamide

A technology of Escherichia coli and biocatalysis, applied in Escherichia coli and its application field in the production of low-by-product nicotinamide by biocatalysis, can solve the problems of low bacterial plasmid retention rate, affecting plasmid retention rate, and cumbersome process flow, etc. To achieve the effects of increasing the scope of use, reducing irritation, and improving quality

Active Publication Date: 2022-05-03
ANHUI RUIBANG BIOLOGICAL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method for resin adsorption of nicotinic acid mentioned in US patent US3678060 has a very cumbersome technological process and is not economical and practical
[0006] Although nicotinamide is also produced by microbial transformation in the prior art, the by-products are high, and the generation of by-products can be reduced by gene knockout technology, but there is also a problem that too many passages of the strain will affect the retention rate of the plasmid, such as the patent "A Preparation and Application of a Low-by-product Pyridine Carboxamide Transformation Microorganism" (201910706179.5), the gene knockout technology involved in it makes the retention rate of the strain plasmid lower, which affects the actual use

Method used

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  • A strain of Escherichia coli and its application in biocatalytic production of low by-product nicotinamide
  • A strain of Escherichia coli and its application in biocatalytic production of low by-product nicotinamide
  • A strain of Escherichia coli and its application in biocatalytic production of low by-product nicotinamide

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] More than 100 batches of sludge samples were taken from different angles of the sewage pool of Anhui Ruibang Biotechnology Co., Ltd., and screened and identified after enrichment and cultivation in batches:

[0050] Weigh 10 grams of sludge, add 50 mL of sterile water, shake it with glass beads, inoculate 5 mL of the suspension into 45 mL of enrichment medium, place on a shaker at 30°C, and shake at 120 rpm for 3 days. Afterwards, the culture solution was taken for gradient dilution, and 10 -6 、10 -7 、10 -8 Gradient dilutions were spread on the plate containing the first screening solid medium, and cultured in a constant temperature incubator at 30°C for 3 to 6 days. Pick the grown single colony and inoculate it on the new primary screening solid medium for expanded culture, and the culture conditions are the same as above. After the bacteria grow out, scrape 3 to 5 rings and inoculate them into the liquid fermentation medium for cultivation. Shake culture at 120rpm...

Embodiment 2

[0057] Embodiment 2: bacterial strain identification

[0058] The 16S rDNA amplification and sequence analysis of the obtained strains were carried out, and the bacterial DNA was extracted by using the QIAamp kit and strictly following the kit operation steps, and the 16S rDNA of the bacteria was amplified by PCR reaction. PCR reaction system (50μL): template DNA 50ng, Primer F (5'-AGAGTTTGATCMTGGCTCAG-3') 20pmol / L, Primer R (5'-TACGGYTACCTTGTTACGACT-3') 20pmol / L, Taq DNA polymerase 2.5U, plus super Pure water to 50 μL. PCR reaction conditions: 94°C for 5 min; 94°C for 30 sec, 57°C for 45 sec, 72°C for 1 min (32 cycles); 72°C for 5 min. 2% agarose electrophoresis to separate the PCR product, after confirming the band of about 1500bp, the purified PCR product was sent to Shanghai Bioengineering Company for sequencing, and the sequencing result was processed with SerialCloner software. The 16SrDNA sequence is shown as SEQ ID NO:2 shown. It was identified as Escherichia coli (...

Embodiment 3

[0059] Embodiment 3: Escherichia coli M91001 effect verification

[0060] Transfer 1.6mL of competent cell suspension to a sterilized cold storage tube, add 0.4mL of sterilized glycerol to make the final concentration 20%, and mix well. Glycerol stock solutions were stored separately at -80°C until use. Take the screened strain M91001 and Escherichia coli E.coli BL21 (DE3) respectively into LB liquid medium, shake and culture at 120rpm on a shaker at 35°C for 3 days, take 15mL of the culture solution and centrifuge at 3000rpm for 10min, pour off the supernatant Finally, wash the precipitated cells with an equal volume of normal saline, and continue centrifuging to obtain the cells. Add the obtained cells to PBS buffer solution containing 1% (%: g / 100mL) 3-cyanopyridine and nicotinamide respectively for 2 hours, 8 hours, 24 hours, and 72 hours, and the concentration of cells in the reaction system 0.5g / L. Take 5mL of the transformation solution and centrifuge at 4000rpm for ...

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Abstract

The invention discloses an Escherichia coli strain and its application in biocatalytic production of low-by-product nicotinamide. The Escherichia coli M910001 described in the invention has a preservation number of CGMCC NO.20430. The Escherichia coli X described in the present invention after the transformation of Escherichia coli M910001 can normally carry out cell metabolism and growth and reproduction in 0-5% 3-cyanopyridine solution, 0-50% nicotinamide solution and their mixed solution, and adapt to the environment Strong ability; after 50 generations of continuous passage, the plasmid retention rate is above 91%, and after 100 generations, the plasmid retention rate is above 80%, which has better plasmid stability.

Description

technical field [0001] The invention belongs to the field of biochemical industry, and relates to a strain of Escherichia coli and its application in biocatalyzed production of low-by-product nicotinamide. Background technique [0002] Niacinamide, VB 3 , also known as Niacinamide (Nicotinamide, Niacinamide), chemical name 3-pyridine carboxamide, pyridine-3-carboxamide, etc., molecular formula C 6 h 6 N 2 O, molecular weight 122.13, white needle crystal or powder, melting point 129-131°C, specific gravity 1.400. Soluble in water, soluble in ethanol and glycerin, insoluble in ether, odorless, slightly bitter, slightly toxic. [0003] Niacinamide is a vitamin that is necessary for the human body and cannot be synthesized by itself. It belongs to the B vitamins together with niacin and is collectively referred to as vitamin PP. In practical applications, the two can be replaced in equal amounts. One of the metabolites of the acid metabolism process, both of which can parti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N1/21C12N9/88C12P17/12C12R1/19
CPCC12N9/88C12P17/12C12Y402/01084C12R2001/19C12N1/205
Inventor 董亢袁晓路杨竞成陈振钱李玉山
Owner ANHUI RUIBANG BIOLOGICAL SCI & TECH CO LTD
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