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Halomonas strain and application thereof

A technology for Halomonas and strains, applied in the field of microorganisms, can solve the problems of difficulty in chemical synthesis, expensive tetrahydropyrimidine, etc., and achieves the effects of wide development and application prospects, short cycle and simple cultivation.

Active Publication Date: 2022-07-29
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The ectoine synthesis gene has been expressed in tobacco, although its expression level is relatively low, but the salt tolerance is still improved
[0006] The precursors of chemically synthesized ectoine (such as diaminobutyric acid) are expensive, and there is a chiral carbon atom in the ectoine molecule, which is difficult to synthesize by chemical methods

Method used

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  • Halomonas strain and application thereof
  • Halomonas strain and application thereof
  • Halomonas strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Isolation and identification of Halomonas strain C2-1-M8

[0032] a. Soil sample collection: Soil was collected from the bank of Longmucuo in Ngari region of Tibet, placed at 4°C and brought back to the laboratory, and separated by plate dilution coating method.

[0033] b. The medium of the isolated strain is 2216 solid medium: peptone 5.0g, yeast extract 1.0g, citric acid 0.1g, sodium chloride 19.45g, magnesium chloride 8.8g, sodium sulfite 3.24g, calcium chloride 1.3g, chlorine Potassium 0.55g, sodium bicarbonate 0.16g, potassium bromide 0.08g, strontium chloride 34.0mg, boric acid 22mg, sodium silicate 4.0mg, sodium fluoride 2.4mg, ammonium nitrate 1.6mg, disodium hydrogen phosphate 8.0mg, Agar 15g, add distilled water to volume to 1000mL, pH 7.2-7.4, sterilize at 121°C for 20min.

[0034]c. Use the plate dilution coating method for separation: fully mix the collected soil samples, weigh 10 g, put it into a triangular flask with an appropriate amount of g...

Embodiment 2

[0037] The preparation of embodiment 2 Halomonas C2-1-M8 fermentation broth

[0038] a. Bacterial activation: pick strains and streak inoculation to 2216 solid medium (5.0 g of peptone, 1.0 g of yeast extract, 0.1 g of citric acid, 19.45 g of sodium chloride, 8.8 g of magnesium chloride, 3.24 g of sodium sulfite, 3.24 g of sodium chloride Calcium 1.3g, Potassium Chloride 0.55g, Sodium Bicarbonate 0.16g, Potassium Bromide 0.08g, Strontium Chloride 34.0mg, Boric Acid 22mg, Sodium Silicate 4.0mg, Sodium Fluoride 2.4mg, Ammonium Nitrate 1.6mg, Hydrogen Phosphate Disodium 8.0mg, agar 15g, add distilled water to dilute to 1000mL, pH 7.2-7.4, sterilize at 121°C for 20min), incubate at 30°C for 14-20h;

[0039] b. Preparation of seed liquid: pick the activated strain and inoculate it in 2216 liquid medium (2216 liquid medium: formula and preparation are the same as above-mentioned 2216 solid medium, without adding agar), at 30° C., 150rpm shaking culture for 16-24 Liquid seeds are ob...

Embodiment 3

[0042] Example 3 Extraction of tetrahydropyrimidine from aseptic fermentation broth of Halomonas strain C2-1-M8

[0043] a. Centrifuge the fermentation broth in Example 2 at 12,000 rpm for 15 min, discard the supernatant, and collect the precipitated cells.

[0044] b. Wash the cells with dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (pH 7.0).

[0045] c. Centrifuge the liquid in b at 12000 rpm for 15 min, discard the supernatant, and collect the precipitate.

[0046] d. Add 2 ml of 80% ethanol to the precipitate obtained in c to suspend, and shake at room temperature overnight.

[0047] e. Centrifuge the overnight suspension in d for 15min under the condition of 12000rpm, collect the supernatant, the supernatant contains tetrahydropyrimidine, and the supernatant is determined by high performance liquid chromatography (HPLC) in the next step. sample.

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Abstract

The invention discloses a Halomonas strain and an application thereof. The strain is a new species in Halomonas, and the preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) NO.24977. The invention further discloses a preparation method of the Halomonas strain. The strain C2-1-M8 obtained by separation in the invention is a new species different from a known species of Halomonas, and can be used for synthesizing ectoine (Ectoine) in cells. The strain C2-1-M8 can utilize substrates such as D-fructose, glycerol, D-fructose-6-phosphoric acid, L-alanine, L-histamine, L-serine, D-galacturonic acid, D-glucuronic acid, glucosamide, L-lactic acid, D-malic acid, L-malic acid, Tween 40 and the like, and has a broad-spectrum property of the substrates. According to physiological and biochemical experiments, the growth temperature of the strain C2-1-M8 ranges from 4 DEG C to 30 DEG C, the NaCl concentration needed by growth ranges from 1.0% to 17.0%, and the growth pH value ranges from 5.0 to 7.5. Meanwhile, the yield of tetrahydropyrimidine synthesized by the strain C2-1-M8 under the conditions that the temperature is 30 DEG C, the NaCl concentration is 5.0%-9.0% and the pH is 5.5-7.0 is optimal. The strain has a very wide application prospect in biological preparation of ectoine.

Description

technical field [0001] The invention relates to the technical field of microorganisms, in particular to a Halomonas strain and its application in synthesizing tetrahydropyrimidine. Background technique [0002] Moderate halophiles refer to microbial groups that can grow in environments with NaCl concentrations ranging from 0.1% to 32.5%. In order to resist the damage of high osmotic pressure and maintain the osmotic pressure balance inside and outside the cell, they will synthesize organic osmotic substances in the body, such as glutamic acid, proline, glycine, betaine, trehalose, tetrahydropyrimidine and hydroxyl. tetrahydropyrimidine etc. As a compatible solute, tetrahydropyrimidine can not only serve as a stabilizer for biological macromolecules such as nucleic acids, enzymes, and cell membranes, helping cells resist such adversities as high salt, high temperature, freezing, and drying, but also as a molecular chaperone to recognize errors in proteins. folding to inhibi...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P17/12C07D239/06C12R1/01
CPCC12N1/205C12N1/20C12P17/12C07D239/06C12R2001/01Y02E50/10
Inventor 李爱华王蕊刘紫轩普布多吉周宇光
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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