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Directed domestication and breeding of methylotrophic bacteria with high yield of pyrroloquinoline quinone

A technology of pyrroloquinoline quinone and methylotrophic bacteria, which is applied in the direction of bacteria, microorganism-based methods, methods using microorganisms, etc., can solve the problems of narrow detection limit and affecting the screening efficiency, so as to shorten the breeding cycle and improve the screening efficiency , the effect of improving the detection efficiency

Active Publication Date: 2019-04-16
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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  • Directed domestication and breeding of methylotrophic bacteria with high yield of pyrroloquinoline quinone
  • Directed domestication and breeding of methylotrophic bacteria with high yield of pyrroloquinoline quinone
  • Directed domestication and breeding of methylotrophic bacteria with high yield of pyrroloquinoline quinone

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 medium with 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 Hyphomicrobium denitrificans FJNU-R8 fermentation broth and the PQQ standard (sigma-aldrich company, product number: D7783) dissolved in the liquid medium to perform 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 ...

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 Hyphomicrobium denitrificans (Hyphomicrobium denitrificans) 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...

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 Hyphomicrobium denitrificans FJNU-6CGMCCNO.1.12893 was used to separate a 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 a 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 220rpm for 1 induction culture for 3 days, so that the OD 600 Increased by 1.5 compared with that before the first induction...

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Abstract

The invention discloses directional domestication and breeding of methylotrophic bacteria with high yield of pyrroloquinoline quinone. The invention provides a method for directional acclimatization and breeding of methylotrophic bacteria mutants with high yield of pyrroloquinoline quinone, comprising the following steps: 1) taking the initial methylotrophic bacteria as the treatment object in a liquid medium containing methanol for multiple Secondary induction culture to obtain the domesticated bacteria, all the domesticated bacteria form the domesticated strain library; 2) ferment the domesticated bacteria in the domesticated strain library, and collect all the domesticated bacteria fermentation liquid; 3) detect the fermentation of all the domesticated bacteria Pyrroloquinoline quinone in the liquid, select the single strain whose yield of pyrroloquinoline quinone in the fermentation broth is higher than that of pyrroloquinoline quinone in the fermentation broth of the initial methylotrophic bacteria, and is a mutant strain of methylotrophic bacteria with high yield of pyrroloquinoline quinone . In the invention, the methylotrophic bacteria are stress-acclimated by gradually increasing the concentration of methanol in the culture medium, thereby improving the positive mutation rate of breeding and the increase rate of pyrroloquinoline quinone output.

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] Pyrroloquinoline quinone (pyrroloquinoline quinone, PQQ) as a bacterial dehydrogenase coenzyme participates in respiratory chain electron transfer, 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 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 purificatio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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