Methylotrophic bacteria and method for producing pyrroloquinoline quinine (PQQ) by fermentation thereof

A technology of methylotrophic bacteria and fermentation medium, applied in the field of methylotrophic bacteria and its fermentation to produce pyrroloquinoline quinone

Active Publication Date: 2014-07-02
WUXI NEWWAY FERMENTATION TECH RES INST
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Chinese patent CN102061278A discloses Methylovorussp.MP688 (CGMCCNo.4096), under conventional medium and culture conditions, the PQQ output reaches 125mg/L; H

Method used

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  • Methylotrophic bacteria and method for producing pyrroloquinoline quinine (PQQ) by fermentation thereof
  • Methylotrophic bacteria and method for producing pyrroloquinoline quinine (PQQ) by fermentation thereof
  • Methylotrophic bacteria and method for producing pyrroloquinoline quinine (PQQ) by fermentation thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1P

[0030] The screening of embodiment 1PQQ producing bacteria

[0031] Slant medium and plate screening medium: K medium, see "Bergey's Handbook of Systematic Bacteriology" (Second Edition, 2004, Volume Two, PartC, P421.). K medium: ammonium sulfate 2.0g / L, potassium dihydrogen phosphate 2.0g / L, sodium chloride 0.5g / L, magnesium sulfate heptahydrate 0.125g / L, ferrous sulfate heptahydrate 0.002g / L, methanol 8g / L L, pH7.

[0032] Fermentation medium used in screening: ammonium sulfate 3.0g / L, potassium dihydrogen phosphate 1.4g / L, disodium hydrogen phosphate 3.0g / L, magnesium sulfate 0.2g / L, iron citrate 30mg / L, calcium chloride 30mg / L, manganese chloride 5.0mg / L, zinc sulfate 5.0mg / L, copper sulfate 0.5mg / L, methanol 6g / L; pH7.0.

[0033] A total of more than 80 soil or water samples were collected from Wuxi and its surrounding areas, and made into appropriate concentration dilutions, which were directly spread on the K medium screening plate, cultured at 30°C for 3-5 days, and...

Embodiment 2

[0038] Identification of the strain Methylopilasp.YHT-1 of embodiment 2

[0039] The morphological characteristics, physiological and biochemical characteristics (Table 2) of YHT-1 were identified according to "Bergey's Handbook of Systematic Bacteriology" (Second Edition, 2004) and the original literature of the strains of this genus. Streak culture on the K medium plate for 2 days, the colony is milky white, round, translucent, smooth, viscous, raised in the middle, neat at the edge, and 1-2mm in diameter. Electron microscope photographs show that ( image 3 ), the bacterium is rod-shaped or (ellipsoid) spherical, with a size of 0.5-0.7×0.9-1.4μm; this strain does not produce spores, has capsules, no flagella, and does not move. Facultative anaerobic, Gram-negative, suitable growth temperature range 25-37 ℃; suitable growth pH range 6.5-8.0; can micro-grow in liquid medium without adding nitrogen source. The chromosomal DNA of YHT-1 was extracted according to the bacterial...

Embodiment 3

[0048] Purification and determination of embodiment 3 product:

[0049] First wash the column with 20mM sodium citrate buffer (pH5.5), then pass the YHT-1 fermentation supernatant filtered through a microporous membrane through a 20mLDEAE anion exchange column at a flow rate of 1mL / min, and then wash it with 2-5 times Column volume of sodium citrate buffer (pH5.5) to wash and equilibrate, and then carry out 0-100% gradient elution with sodium citrate buffer (pH5.5) containing 1M NaCl, the elution volume is 10 to 20 times the column volume , and finally the red PQQ product after preliminary purification was obtained.

[0050] Analysis of the purified product:

[0051] HPLC analysis: Dissolve the PQQ standard sample (purchased from Wuhan Nuohui Pharmaceutical Chemical Co., Ltd., purity ≧ 98%) with the same concentration of sodium citrate buffer as that of the purified product, make the solvents of the two consistent, and perform HPLC analysis on the purified product ( figure 1...

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Abstract

The invention discloses methylotrophic bacteria and a method for producing pyrroloquinoline quinine (PQQ) by fermentation thereof, and belongs to the technical field of organisms. A new bacterial strain Methylopilasp.YHT-1 is screened and separated from soil; pyrroloquinoline quinine can be produced by the bacterial strain; the bacterial strain is preserved at the China center for type culture collection on January 12, 2014; the preservation number is CCTCC NO: M2014016. The bacterial strain is subjected to aerobic culture for 3-4 days in a culture medium of taking methyl alcohol as a carbon source, so as to produce 50-113mg/L of pyrroloquinoline quinine. The condition that Methylopilasp can excessively generate PQQ and can secrete out of the cell is reported for the first time, and a new strain is provided for preparation of PQQ by a fermentation method.

Description

technical field [0001] The invention relates to a methylotrophic bacterium and a method for fermenting and producing pyrroloquinoline quinone, belonging to the field of biotechnology. Background technique [0002] Pyrroloquinolinequinone (Pyrroloquinolinequinone, PQQ), chemical name is 4,5-dihydro-4,5-dioxide-1-hydropyrrole (2,3f) quinone-2,7,9-tricarboxylic acid, alias Methaxatin , was discovered by Salisbury et al. in 1979 and was originally described in bacterial cells as a redox cofactor for membrane-bound dehydrogenases. At present, PQQ is found in many different organisms, it can prevent living cells from being damaged by oxygen in vivo or in vitro; it can also be used as a nutritional factor and vitamin to promote cell growth and improve the tolerance of bacteria under extreme conditions; mammalian cells Induces production of protein kinases during differentiation; improves crop productivity by increasing insoluble phosphate availability and as a biocontrol agent; ut...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P17/18
Inventor 郑璞姚红涛
Owner WUXI NEWWAY FERMENTATION TECH RES INST
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