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Oriented domestication and breeding of methylotrophic bacterium capable of producing pyrroloquinoline quinone at high yield

A technology of pyrroloquinoline quinone and methylotrophic bacteria, applied to bacteria, methods using microorganisms, methods based on microorganisms, etc., can solve problems affecting screening efficiency and narrow detection limit, so as to improve screening efficiency and shorten breeding cycle , The effect of increasing the rate of production increase

Active Publication Date: 2016-06-01
福建力多利生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has high precision, but the detection limit of this method is narrow, and the sample needs to be diluted, which affects the screening efficiency

Method used

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  • Oriented domestication and breeding of methylotrophic bacterium capable of producing pyrroloquinoline quinone at high yield
  • Oriented domestication and breeding of methylotrophic bacterium capable of producing pyrroloquinoline quinone at high yield
  • Oriented domestication and breeding of methylotrophic bacterium capable of producing pyrroloquinoline quinone at high yield

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1, establishment of pyrroloquinoline quinone PQQ detection method

[0044] 1. Full-wavelength scanning of PQQ standard and fermentation broth

[0045] Hyphomicrobium denitrificans FJNU-R8 was inoculated in a liquid culture medium at an inoculum size of 5%, fermented at 30° C. and 220 rpm for 4 days to obtain a fermented liquid, and centrifuged at 8000×g for 5 min to collect the fermented supernatant.

[0046] Take the supernatant of Hyphomicrobiumdenitrificans FJNU-R8 fermentation broth and the PQQ standard (sigma-aldrich company, product number: D7783) dissolved in the liquid medium for full-wavelength scanning (200nm-840nm) with an ultraviolet-visible spectrophotometer.

[0047] The result is as figure 1 As shown, it can be seen that PQQ has three characteristic absorption peaks at 248nm, 269nm and 330nm, and the two-dimensional spectrum of the supernatant of the fermentation broth is similar to that of the PQQ standard. Since the absorbance of PQQ at 24...

Embodiment 2

[0053] Example 2, Method for Directed Domestication and Breeding of Denitrifying Hyphobacterium FJNU-R8 Mutant Strain with High Yield of Pyrroloquinoline Quinone

[0054] 1. Stress-directed domestication of strains

[0055] 1) Seed culture solution

[0056] The starting strain Hyphomicrobiumdenitrificans (Hyphomicrobiumdenitrificans) FJNU-R8 was isolated by drawing a line on the seed preservation slope, and then the single colony was inserted into the seed medium, and cultivated at 30°C for 2 days to obtain the seed culture solution;

[0057] 2) Stress-oriented acclimatization induction culture

[0058] The seed culture solution was subjected to multiple induction cultures, as follows:

[0059] The seed culture solution was transferred to the liquid medium containing 2% methanol (volume percentage) according to the inoculum size of 5% (volume percentage content), and 30°C and 220rpm were carried out for 1 induction culture until 4 days, so that the OD 600 Increased by 1.5 c...

Embodiment 3

[0074] Example 3, Method for Directed Domestication and Breeding of Denitrifying Rhizoctonia CGMCC NO.1.12893 Mutant Strain with High Yield of Pyrroloquinoline Quinone

[0075] 1. Stress-directed domestication of strains

[0076] 1) Seed culture solution

[0077] The starting strain Hyphomicrobiumdenitrificans FJNU-6CGMCCNO.1.12893 was used to separate single colony by drawing a line on the seed preservation slope, then insert the single colony into the seed medium, and cultivate it at 30°C for 2 days to obtain the seed culture solution;

[0078] 2) Stress-oriented acclimatization induction culture

[0079] The seed culture solution was subjected to multiple induction cultures, as follows:

[0080] The seed culture solution was transferred to the liquid medium containing 0.5% (volume percentage) methanol by 5% (volume percentage) inoculum at 30 ° C and 220 rpm for 1 induction culture for 3 days, so that the OD 600 Increased by 1.5 compared with that before the first inducti...

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Abstract

The invention discloses oriented domestication and breeding of methylotrophic bacterium capable of producing pyrroloquinoline quinone at high yield. The invention provides an oriented domestication and breeding method for preparing a methylotrophic bacterium mutant strain capable of producing pyrroloquinoline quinone at high yield. The method comprises the following steps: 1) carrying out repeated induced culture on an initial methylotrophic bacterium used as a treatment object in a methanol-containing liquid culture medium to obtain the domesticated bacterium, wherein all the domesticated bacteria constitute a domesticated strain library; 2) fermenting the domesticated bacteria in the domesticated strain library, and collecting all the domesticated bacterium fermentation liquids; and 3) detecting pyrroloquinoline quinone in all the domesticated bacterium fermentation liquids, and selecting the single strain, the fermentation liquid of which has higher pyrroloquinoline quinone yield than the initial methylotrophic bacterium fermentation liquid, thereby obtaining the methylotrophic bacterium mutant strain capable of producing pyrroloquinoline quinone at high yield. The methanol concentration in the culture medium is gradually enhanced to perform stress domestication on the methylotrophic bacterium, thereby enhancing the breeding direct mutation rate and the pyrroloquinoline quinone yield increase amplitude.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to directional domestication and breeding of methylotrophic bacteria with high yield of pyrroloquinoline quinone. Background technique [0002] Pyrroloquinolinequinone (PQQ), as a bacterial dehydrogenase coenzyme, participates in the electron transfer of the respiratory chain, has the functions of promoting body growth, protecting liver damage, promoting the synthesis of nerve growth factors, regulating the level of free radicals in the body, and improving bacterial resistance to extremes such as toxicity and radiation. It is a unique physiologically active substance and has important development prospects in the fields of food, medicine and health care. The production methods of PQQ mainly include chemical synthesis and microbial fermentation. Among them, the microbial fermentation is mainly based on inorganic medium, which has low cost, high yield and is conducive to downstream purifica...

Claims

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Application Information

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IPC IPC(8): C12N1/36C12N1/32C12N1/20C12R1/01
CPCC12N1/20C12N1/32C12N1/36C12N1/205C12R2001/01
Inventor 柯崇榕黄建忠廖灿钟璐芳杨欣伟任洋
Owner 福建力多利生物科技有限公司
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