Methylovorus sp. MP688 and application thereof

A technology of methyl-eating bacteria and strains, applied in the direction of bacteria, fermentation, etc., can solve the problems of high cost, difficulty in realizing PQQ industrial scale production, and low output, and achieve stable output, promote industrialization, and good strain growth

Active Publication Date: 2011-05-18
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is also a method for producing PQQ by strain fermentation. The original strain PQQ output used is 0.07~7mg/L, and the fermentation time is about 2-5 days (US Pat4994382...

Method used

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  • Methylovorus sp. MP688 and application thereof
  • Methylovorus sp. MP688 and application thereof
  • Methylovorus sp. MP688 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1P

[0030] Example 1PQQ producing bacteria screening

[0031] About 3,000 soil samples will be collected from all over the country, mainly from Beijing and its suburbs, Tonghua in Jilin, Hengshui in Hebei, and Hangzhou in Zhejiang. Collect soil samples with sterilized 1.5mL centrifuge tubes, add 1mL sterile water to each tube of soil samples, add appropriate sterile water to the collected soil samples, suspend them and let them stand still, take 100μl supernatant and add them to 5ml enrichment medium (each liter contains Ammonium sulfate 3g, potassium dihydrogen phosphate 1.4g, disodium hydrogen phosphate 3g, magnesium sulfate 0.2g, ferric citrate 30mg, calcium chloride 30mg, manganese chloride 5mg, zinc sulfate 5mg, copper sulfate 0.5mg, 1mL vitamin solution, 8mL methanol, pH adjusted to 7.0. Vitamin solution: 2mg biotin, 400mg calcium pantothenate, 400mgVB1, 400mgVB6, 200mg p-aminobenzoic acid, 2mg folic acid, 2g inositol, 400mg niacin, 200mg riboflavin), anaerobic at 30°C Cult...

Embodiment 2P

[0038] Identification of embodiment 2PQQ production bacteria MP688

[0039] The MP688 strain was streaked on the MPQ plate and cultured at 30°C for 30 hours, and the colony morphology was observed. It was a small moist round colony with neat edges, and the colony protruded from the solid medium and was tightly combined with the medium. Pick a single colony for Gram staining observation, the bacterium is red, and it is a small bacillus. The MP688 strain was observed with an electron microscope at a magnification of 24000×. The thallus was short rod-shaped with flagella at the top ( Image 6 ). Primers were designed and synthesized based on the 16S rDNA conserved sequences of several known methylophagous bacteria, and the genome DNA of MP688 was used as a template to amplify part of the 16S rDNA sequence of the bacteria by PCR reaction ( Figure 7 ). The primer sequences thereof are shown in SEQ ID No.3 and 4. The T vector was connected and sent for sequencing, and its seque...

Embodiment 3

[0040] Extraction, purification and identification of embodiment 3PQQ

[0041] 1. MP688 fermentation culture

[0042] Insert the strain into the fermentation medium according to the ratio of 5% (each liter contains 5 g of yeast powder, 5 g of peptone, 3 g of ammonium sulfate, 1.4 g of potassium dihydrogen phosphate, 3 g of disodium hydrogen phosphate, 0.2 g of magnesium sulfate, and 30 mg of ferric citrate , calcium chloride 30mg, manganese chloride 5mg, zinc sulfate 5mg, copper sulfate 0.5mg, 1mL vitamin solution, 8mL methanol, pH adjusted to 7.0. Vitamin solution: 2mg biotin, 400mg calcium pantothenate, 400mg VB 1 , 400mg VB 6 , 200mg p-aminobenzoic acid, 2mg folic acid, 2g inositol, 400mg nicotinic acid, 200mg riboflavin), shake culture at 30°C for 5d. The fermenter was cultured according to 10% inoculation, and the medium concentration was 2 times normal.

[0043] 2. Preparation of PQQ

[0044] Inoculate MP688 into the enrichment medium, shake culture at 30°C for 5 day...

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Abstract

The invention discloses a new strain MP688 of Methylovorus sp., of which the preservation number is CGMCC No.4096, the preservation date is August 20th, 2010, and the preservation unit is China General Microbiological Culture Collection Center. The strain MP688 is separated from soil by using a GDH (glutamate dehydrogenase) recombinase method of PQQ (pyrroloquinoline quinone). By using the Methylovorus sp. strain disclosed by the invention, the yield of PQQ can reach 125 mg/L in a conventional culture medium under conventional culture conditions; and the Methylovorus sp. strain can be used for industrial production of PQQ, and has important meaning for promoting the industrialization of PQQ.

Description

technical field [0001] The invention relates to a microbial strain and its application, in particular to a methylophagous bacterial strain MP688 and its application. Background technique [0002] Pyrroloquinoline quinone (PQQ) is a relatively recently discovered coenzyme and biologically essential nutrient, which has the ability to stimulate the growth of certain microorganisms, animals and plants, prevent liver damage, Various physiological functions such as promoting the production of nerve growth factor, regulating the level of free radicals in the body and regulating immunity may be used in the prevention and treatment of diseases such as Alzheimer's disease, cardiovascular disease and diabetes. Breeding new PQQ high-yielding strains and determining their genes related to PQQ synthesis are of great significance for further improving PQQ production through metabolic engineering, reducing PQQ production costs, and promoting the practical application of PQQ. [0003] Th...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P17/18
Inventor 汪建华张惟材熊向华智静娟杨璐杨延新王歆李淼鑫崔艳菊董方杜宝华周满祥魏琳申云飞李月魏红玉
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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