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Pseudomonas fluorescent and application thereof in biosynthesizing methionine

A technology of Pseudomonas fluorescens and methionine, which is applied in the field of nitrilase-producing strains, can solve the problems of increasing investment in equipment, increasing costs, recovering salt, and not complying with green chemistry and atomic economy, and achieves the effect of wide application prospects

Active Publication Date: 2015-03-04
ZHEJIANG UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] Although the current chemical synthesis process of methionine is relatively mature, the generation of by-products has been reduced compared with the previous ones, or recycled and reused, but it is still unavoidable to use corrosive acids, alkalis and other dangerous compounds, resulting in various sulfates. This not only needs to increase the additional cost of recycling salt, but also increases the investment in equipment, which does not meet the requirements of green chemistry and atomic economy
Some scholars are also studying the production of methionine by fermentation. Although the methionine produced by fermentation is all L-type, there is serious feedback inhibition in this way, the output is small, the yield is extremely low, the cost is high, and the industrialization is slow. Industrial value

Method used

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  • Pseudomonas fluorescent and application thereof in biosynthesizing methionine
  • Pseudomonas fluorescent and application thereof in biosynthesizing methionine
  • Pseudomonas fluorescent and application thereof in biosynthesizing methionine

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Embodiment 1: the screening of new bacterial strain ZJB-12001

[0038]Inoculate the enrichment medium with the soil samples collected from Zhejiang Province and sewage samples from pharmaceutical factories; after 72 hours of enrichment culture on a shaker at 30°C and 150rpm, take 2ml of the culture solution and inoculate it on the fresh enrichment medium. Secondary culture, so repeated 3 cycles. After the last dilution of the culture solution, spread it on the beef extract peptone plate medium, culture at 30°C for 24-72 hours to obtain a single colony, pick a single colony and inoculate it on the slant medium for preservation. Pick an inoculation loop strain from the slant culture medium and inoculate it into the fermentation medium, shake and culture at 30°C and initial pH 7.0-7.5 for 48 hours, centrifuge, discard the supernatant, and take the precipitate to obtain the enzyme-containing wet bacteria ; The enzyme-containing wet bacteria were suspended in the pH7.0 phos...

Embodiment 2

[0041] Embodiment 2: Identification of new bacterial strain ZJB-09150

[0042] In Example 1, a bacterial strain with the strongest activity was obtained from the soil through enrichment culture and then diluted coating method through high-performance liquid chromatography detection and screening. The label is ZJB-12001. figure 1 As shown, the diameter of the colony is about 1.0-2.0 cm, and the colony is milky white, spherical, bright, moist, with neat edges and slightly raised surface. Electron microscope scanning photo see figure 2 As shown, there are 3-6 polar flagella.

[0043] Using the Biolog (GENⅢ) automatic microbial identification system, 94 phenotypic tests were carried out on strain ZJB-12001, including 71 carbon source utilization tests and 23 chemical sensitivity tests (Biolog microbial automatic identification reagents and culture media were purchased from Biolog Company), the metabolic fingerprint was analyzed by Biolog reader, and the identification results o...

Embodiment 3

[0051] Example 3: Preparation of biocatalyst Pseudomonas fluorescens ZJB-12001 enzyme-containing wet thallus

[0052] (1) Incline cultivation:

[0053] The Pseudomonas fluorescens ZJB-12001 (CCTCC No: M2012449) obtained in Example 2 was inoculated into the slant medium, cultured at 30°C for 48 hours, and the slant bacteria were obtained; the final concentration of the slant medium was composed of: peptone 10.0g / L, beef extract 5.0g / L, sodium chloride 5.0g / L, agar 20.0g / L, pH7.2.

[0054] (2) Seed cultivation:

[0055] Pick a ring of bacteria from the slant of step (1), inoculate it into 50.0ml of sterile seed medium, and cultivate it at 30°C for 24 hours to obtain the seed liquid; the final concentration of the seed medium is composed of: glucose 10.0g / L, Sodium glutamate 10.0g / L, yeast powder 3.0g / L, KH 2 PO 4 0.75g / L, K 2 HPO 4 ·3H 2 O0.75g / L, MgSO 4 ·7H 2 O0.2g / L, caprolactam 1.0g / L, pH7.0, solvent is water. The medium was sterilized at 121°C for 20 minutes.

[...

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Abstract

The invention provides a rhodococcus rhodochrous strain, namely, pseudomonas fluorescens ZJB-12001, and an application thereof in 2-amino-4-methylmercapto-butyronitrile synthetic methionine. The strain is preserved in the Chinese Typical Culture Collection Center which is located in Wuhan College, Wuhan, China, the postcode is 430072, the preservation number is CCTCC NO: M 2012449, and the preservation date is November 8th, 2012. The pseudomonas fluorescens ZJB-12001 is applied to experiments for producing methionine in a biological catalysis mode, the result shows that the pseudomonas fluorescens ZJB-12001 can be used for effectively producing methionine in the biological catalysis mode, and the conversion rate of 20mM3-cyanopyridine within 60 minutes can be 60-75%, so that the pseudomonas fluorescens ZJB-12001 has a wide application prospect in the field of biological catalysis methionine production.

Description

(1) Technical field [0001] The invention relates to a nitrilase-producing strain—Pseudomonas fluorescens ZJB-12001, and its application in the biotransformation of 2-amino-4-methylthiobutyronitrile to synthesize methionine. (2) Background technology [0002] Methionine (Methionine) is also known as methionine, its chemical name is 2-amino-4-methylthiobutyric acid, and its molecular formula is C 5 h 11 NO 2 S, the appearance is white flaky crystal or powder, with special smell, slightly sweet taste, melting point 281°C (decomposition), relative density 1.34, soluble in water, dilute acid and dilute alkali, slightly soluble in alcohol, insoluble in ether, 1 The pH value of % methionine aqueous solution is 5.6-6.1, which is stable to heat and air, but unstable to strong acid, which can lead to demethylation. Methionine contains an amino group and a carboxyl group. It is the only sulfur-containing amino acid and has optical activity. It is divided into D-type and L-type. In a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P13/12C12R1/39
Inventor 郑裕国金利群李晓庆柳志强
Owner ZHEJIANG UNIV OF TECH
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