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Method for rapid and efficient screening of vitamin K2 (MK-7) high-yielding strain

A technology of vitamins and high-yield strains, applied in the biological field, can solve the problems of inability to obtain high-yield strains and low efficiency in the strain selection process, and achieve the effects of simple operation, low mutagenesis cost, and high positive mutation rate

Inactive Publication Date: 2017-07-04
NANJING UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The technical problem to be solved by the present invention is that the efficiency of the strain selection process in the prior art is too low to obtain high-yield bacterial strains; in order to solve this problem, the present invention provides a method for quickly and efficiently screening vitamin K2 (MK-7) high-yield bacteria method

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  • Method for rapid and efficient screening of vitamin K2 (MK-7) high-yielding strain
  • Method for rapid and efficient screening of vitamin K2 (MK-7) high-yielding strain
  • Method for rapid and efficient screening of vitamin K2 (MK-7) high-yielding strain

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Embodiment 1

[0032] Embodiment 1: This embodiment illustrates the biological material source information of the present invention

[0033] The biological material in the present invention is Bacillus natto, which is isolated from natto products purchased in the market.

Embodiment 2

[0034] Embodiment 2: This implementation example illustrates the process of ARTP mutagenesis

[0035] Bacillus natto is activated and cultured on a solid plate, and the activated cultured cells are suspended with physiological saline. Properly dilute the cells to OD with physiological saline 600When the value is between 0.5 and 2.0, place the metal slide in the outer flame of an alcohol lamp in a clean bench and burn for 30s. After cooling, put it on a sterilized glass plate. Take 10ul of bacterial suspension and apply it evenly on the slide. Air-dried for mutagenesis.

[0036] The operating compartment of the ARTP mutagenesis system was first opened for UV sterilization for 30 minutes, and then the slide was placed into the operating compartment of the ARTP mutagenesis system with sterile tweezers, and the knob under the stage was adjusted so that the slide was at 2 mm from the airflow port. Set the power of the instrument to 100W, the gas flow parameter to 10SLM, and the m...

Embodiment 3

[0038] Embodiment 3: This embodiment illustrates the process of determining the critical value of the concentration of structural analogues

[0039] Dilute the cultured cells with normal saline to 10 -1 ~10 -7 In between, take 100ul dilution to coat the plate. Among them, the formula of the solid plate is: 30g / L glucose, 40g / L peptone, 5g / L NaCl, 5g / L beef extract, 5g / L yeast extract, 20g / L agar, 2.5~20mg / L 1,4-dihydroxy -2-Naphthoic acid, p-benzoquinone, 2-methyl-1,4-naphthoquinone or 1-naphthol.

[0040] Structural analogues can be combined with repressors and allosteric enzymes due to their structural similarity with metabolites, so that the catalytic action of enzymes is irreversibly inhibited. Different kinds of vitamin K2 structural analogues were used as pre-screening to determine the sensitivity threshold of Bacillus natto, to breed resistant mutants, and a series of analogues were added to the plate.

[0041] Table 1 Vitamin K2 structural analogue resistance conce...

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Abstract

The invention discloses a method for rapid and efficient screening of a vitamin K2 (MK-7) high-yielding strain. An original strain undergoes atmospheric and room temperature plasma (ARTP) mutagenesis, and plate breeding of the mutagenesis strain is carried out by the use of a vitamin K2 analogue and a respiratory chain inhibitor. After fermentation and re-screening through a 96-well culture plate, content of the high-yielding strain vitamin K2 is raised by 200%. By the above method, a batch of Bacillus natto having advantages of fast growth speed, high yield of vitamin K2, genetic stability and strong tolerance to the environment is bred.

Description

technical field [0001] The invention relates to a method for quickly and efficiently screening vitamin K2 (MK-7) high-yielding bacteria, which belongs to the field of biotechnology. Background technique [0002] Vitamin K2 is a fat-soluble blood coagulation vitamin. It is a series of menaquinone compounds. There are 14 forms according to the length of the isoprene side chain at the C-3 position of its molecular structure. It is represented by MK-n (n refers to The number of isoprene units on the side chain), menadione-4 (MK-4) and menadione-7 (MK-7) are the most common, and MK-7 is rapidly absorbed by the intestinal tract Advantages such as long half-life in blood have been extensively studied. Vitamin K2 has a remarkable effect in the prevention and treatment of osteoporosis, and is called "the fourth generation of anti-osteoporosis products". In addition, vitamin K2 can also quickly improve bleeding caused by vitamin K deficiency, resist arterial calcification and osteoa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N13/00C12N15/01C12R1/07
CPCC12N13/00C12N15/01
Inventor 黄和任路静罗苗苗倪擎天关荣锋
Owner NANJING UNIV OF TECH
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