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Pseudomonas putida and method for producing nicotinic acid or isonicotinic acid through converting Pseudomonas putida

A technology of Pseudomonas putida and isonicotinic acid, applied in the direction of microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve the problem of no reports of Pseudomonas putida, etc., and achieve high yield and good reaction conditions Mild, short-cycle effects

Active Publication Date: 2014-05-21
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The currently reported strains that can be used to biocatalyze 3-cyanopyridine to prepare nicotinic acid include Bacillus subtilis, Rhodococcus rhodochrous, Nocardia, Fusarium solani, which catalyze the conversion of 4-cyanopyridine to isonicotinic acid. Strains include Fusarium solani and Aspergillus niger, but no reports of Pseudomonas putida for niacin and isonicotinic acid production

Method used

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  • Pseudomonas putida and method for producing nicotinic acid or isonicotinic acid through converting Pseudomonas putida

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: Pseudomonas putida CGMCC3830 ( Pseudomonas putida ) isolation and identification

[0036] (1) Pseudomonas putida CGMCC3830 ( Pseudomonas putida ) enrichment screening process.

[0037] Collect soil samples at 5-15 cm around the nitrile compound production plant. Take 1 g of soil sample and put it into a conical flask, add 15ml of normal saline, add glass beads, and place it on a shaker for 30 minutes. Take 0.2mL of soil suspension and spread it on the solid screening medium with 3-cyanopyridine as the only nitrogen source. The composition of the solid screening medium is: glucose 0.5%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.01%, sulfuric acid Ferrous iron 0.002%, calcium chloride 0.002%, sodium chloride 0.1%, 3-cyanopyridine 0.1%, agar 2%, pH 7.0. Cultured at 30°C for 3 days, picked colonies of different bacteria and continued to streak and separate into single colony.

[0038] Pick 20 purified strains and transfer them to liquid m...

Embodiment 2

[0041] Embodiment 2: Pseudomonas putida CGMCC3830 ( Pseudomonas putida ) shake flask fermentation culture

[0042] Shake Flask Fermentation Medium

[0043] A: Glucose 1%, soybean peptone 0.5%, yeast powder 0.3%, malt extract 0.3%, NaCl 5%, pH 7.2.

[0044] B: Glycerin 1%, tryptone 1%, yeast powder 0.5%, NaCl 0.5%, pH 7.2.

[0045] C: Glycerin 1%, Tryptone 1%, Yeast Powder 0.5%, NaCl 0.1%, KH 2 PO 4 10.1%, pH7.2.

[0046] D: Glycerin 1%, tryptone 1%, yeast powder 0.5, NaCl 0.1%, KH 2 PO 4 10.1%, urea 1%, pH6.0.

[0047] Separately pack the above-mentioned culture media, each in three parallels, and sterilize at 121°C for 20 minutes. Pseudomonas putida CGMCC3830 ( Pseudomonas putida ) were inoculated into each fermentation medium, cultured at 30° C. and 120 rpm, and the fermentation was completed in 36 hours. After the fermentation, the bacteria were collected by centrifugation at 10,000 rpm for 10 minutes, and the bacteria were washed twice with phosphate. Cells 0....

Embodiment 3

[0050] Embodiment 3: Pseudomonas putida CGMCC3830 ( Pseudomonas putida ) fermenter fermentation culture

[0051] Pseudomonas putida CGMCC3830 ( Pseudomonas putida ) was inoculated into the seed medium, cultivated at 30° C. and 120 rpm for 24 hours to obtain a seed liquid; the composition of the seed medium was as follows: 0.5% peptone, 0.5% yeast powder, 0.5% NaCl, pH 7.0.

[0052] The fermentation medium was inoculated into the fermentation medium with an inoculation amount of 1% by volume, and cultured at 30° C. for 36 hours to obtain a fermentation broth. The composition of the fermentation medium is as follows: 1% glycerol, 1% tryptone, 0.5% yeast extract, 0.1% potassium dihydrogen phosphate, 0.1% sodium chloride, and 0.1% urea. 30°C, stirring speed 150rpm-600rpm, ventilation rate 0.5-3mL / min, pH control at 7.0 with acid and alkali, after 24 hours of cultivation, collect the bacteria in the fermentation broth for reaction.

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Abstract

The invention discloses a new bacterial strain CGMCC3830 (Pseudomonas putida) and a method for catalytically producing nicotinic acid or isonicotinic acid by using the bacterial strain. In the invention, the nicotinic acid or isonicotinic acid is obtained through a hydrolysis reaction, wherein 3-cyanopyridine and 4-cyanopyridine are taken as raw materials, and the CGMCC3830 (Pseudomonas putida) with high nitrilase activity obtained through fermentation culture is taken as a catalyst. The method comprises the following steps of: adding a substrate in a reactor in a flowing mode, and reacting for 6-10 hours to obtain 147g / L-221g / L of nicotinic acid or isonicotinic acid. The CGMCC3830 (Pseudomonas putida) has the characteristics of short life cycle, strong vital force, high conversion efficiency, short conversion cycle and the like, the production cost of nicotinic acid or isonicotinic acid is reduced greatly. The production process has the characteristics of mild reaction conditions, low energy consumption and high yield, is environmentally-friendly and the like.

Description

technical field [0001] The invention belongs to the technical field of biocatalysis and relates to a strain of Pseudomonas putida CGMCC3830 ( Pseudomonas putida ) and a method for preparing nicotinic acid by converting 3-cyanopyridine and converting 4-cyanopyridine to isonicotinic acid by using the strain. Background technique [0002] Niacin, also known as 3-pyridinecarboxylic acid, is one of the 13 essential vitamins for the human body. The symptoms of niacin deficiency in humans and animals are mainly pellagra symptoms, so niacin is also called anti-pyridine vitamin. Niacin is mainly used in medicine to synthesize niacin tablets for the prevention and treatment of pellagra. Niacin is also used in food products, meat additives and feed additives to prevent pellagra. In particular, the addition of nicotinic acid in the feed can improve the disease resistance of livestock and poultry, accelerate the growth rate, improve the utilization rate of feed, save a lot of feed, an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P17/12C12R1/40
Inventor 许正宏史劲松朱小燕龚劲松陆震鸣孙文敬周强
Owner JIANGNAN UNIV
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