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Bacillus subtilis mutant strain for highly producing NK (nattokinase) and application of Bacillus subtilis mutant strain

A Bacillus subtilis, nattokinase-producing technology, applied in the application, enzyme, peptidase and other directions, can solve the problems of lack of good strains and high production cost of nattokinase, achieve high unit activity, strong nattokinase ability, The effect of high safety

Pending Publication Date: 2018-03-23
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The object of the present invention is to provide a kind of Bacillus subtilis subspecies Bacillus subtilis subsp.UJS-18 of a kind of mutagenesis, this bacterial strain has the characteristics of high-yield nattokinase, passage stability, this bacterial strain is applied to the liquid state fermentation of nattokinase, has improved The content of nattokinase in the fermentation liquid is to solve the practical problems of the lack of good strains of large-scale industrial liquid fermentation of nattokinase and the high production cost of nattokinase

Method used

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  • Bacillus subtilis mutant strain for highly producing NK (nattokinase) and application of Bacillus subtilis mutant strain
  • Bacillus subtilis mutant strain for highly producing NK (nattokinase) and application of Bacillus subtilis mutant strain
  • Bacillus subtilis mutant strain for highly producing NK (nattokinase) and application of Bacillus subtilis mutant strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] A subspecies of Bacillus subtilis subsp.UJS-18, the screening process is as follows:

[0042]1) Take 0.2 g of nattokinase bacteria powder purchased in the market, add 1 ml of sterile saline, shake and let stand, dip the supernatant with an inoculation loop and draw a line on the LB plate to separate a single colony. Use an inoculation loop to pick a single colony, refer to the eighth edition of "Berger's Bacterial Identification Manual" to observe whether it can be drawn, select a few single colonies of Gram-positive spores that can be drawn, and put them on a new LB plate for division and marking , separated and purified again, picked a single colony and inoculated it into the seed medium, 37°C, 200rpm, cultured for 6 hours, took 0.2ml and inoculated it into 40ml of new seed medium, cultivated for 6 hours to reach the logarithmic growth phase.

[0043] 2) Take 5ml of bacterial solution in the logarithmic growth phase, centrifuge at 5000rpm for 5min, discard the superna...

Embodiment 2

[0046] The PCR amplification of 16S rRNA, its steps are as follows:

[0047] Genomic DNA of UJS-18 strain was extracted using a bacterial genomic DNA extraction kit (Tiangen Biochemical Technology Co., Ltd.), diluted to 200 ng / μl with TE buffer, and used as a template for PCR amplification. Prepare the following reaction system: 10 μl of 2×PCR mix, 2 μl of genomic DNA, 1 μl of upstream primer (5'-AGAGTTTGATCCTGGCTCA-3'), 1 μl of downstream primer (5'-GGTTACCTTGTTACGACTT-3'), and 6 μl of pure water. Amplification was performed according to the following procedure: pre-denaturation at 94°C for 3 min, 35 cycles (pre-denaturation at 94°C for 20 s, annealing at 55°C for 20 s, extension for 1 min), extension at 72°C for 5 min, and storage at 4°C. After the PCR amplification, 3 μl of the reaction solution was taken for 1.5% agarose electrophoresis detection. Ultraviolet detection showed that the size of the amplified 16S rRNA band was about 1500bp.

[0048] Physiological and bioche...

Embodiment 3

[0055] Nattokinase fibrinolytic activity assay, its steps are as follows:

[0056] 1) Preparation of agarose-fibrin plate:

[0057] Use a serum bottle to measure 25ml of 1% agarose solution prepared with PBS buffer solution, cool down to 50-55°C, draw 200ul of 0.5% fibrinogen solution, and quickly pour the fibrinogen solution three times into the bottom, middle and upper parts of the bottle Pour in and stir vigorously for 5-8 times, pour 60ul of 20U / ml thrombin solution into the fibrinogen solution and stir vigorously, and immediately pour the solution in the serum bottle into the petri dish from the wall of the petri dish After shaking for a few times, let it stand still for 1 hour, and punch a hole on the plate with a glass tube with a diameter of 2 mm.

[0058] 2) The making of urokinase standard curve:

[0059] Prepare urokinase standard products (10, 20, 40, 60, 80 IU / ml) with different activities, take 10 μl each and add them to the sample wells of the agarose-fibrin p...

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Abstract

The invention discloses a Bacillus subtilis mutant strain for highly producing NK (nattokinase) and an application of the Bacillus subtilis mutant strain and belongs to the biotechnology field. The strain is collected in CGMCC (China General Microbiological Culture Collection Center) with the collection number of CGMCC No.14433 on July 17, 2017, with the suggested classification name Bacillus subtilis. The strain and a liquid fermentation medium are used for producing an NK-containing fermentation liquid with the enzyme activity up to 27,690 u / g, high-purity NK powder prepared from the fermentation liquid has the enzyme activity up to 497,280 u / g, and a healthcare capsule which is prepared from main materials including freeze-dried natto powder and the high-purity NK powder and has functions of reducing blood pressure and preventing cardiovascular-cerebral thrombosis diseases has the characteristics of being high in NK activity, good in food therapy and free of side effects.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the preparation and application of a Bacillus subtilis mutagenic strain with high nattokinase production. Background technique [0002] As of 2016, my country's elderly population over the age of 60 was 220 million, accounting for 16.1% of the total population, and China has entered an aging society. Thrombotic disease is a high incidence of middle-aged and elderly people, and it is the second most common disease after cancer, with a high threat of death. At present, dissolving thrombus is an important means to treat this kind of diseases. The drugs used in clinical treatment today include streptokinase, urokinase, tissue-type plasminogen activator, single-chain urokinase-type plasminogen activator, Chemically modified plasmin-streptokinase activator complexes, etc., but these drugs have obvious shortcomings: systemic bleeding side effects, short drug half-life, expensive, etc. Theref...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/56A23L11/00A23P10/30A23L33/10C12R1/125A23L11/50
CPCA23L33/10A23P10/30C12N9/54C12Y304/21062A23V2002/00A23L11/50C12R2001/125C12N1/205A23V2200/326
Inventor 龚爱华朱沛煌严永敏曾建彭琬昕杜凤移崔恒林
Owner JIANGSU UNIV
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