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Bacillus subtilis strain for excreting nattokinase at high efficiency and preparation technology of high-purity nattokinase

A technology of Bacillus subtilis and nattokinase, applied in the direction of enzymes, peptidases, bacteria, etc., can solve the problem of not being able to express NK

Inactive Publication Date: 2017-08-18
上海诺金科生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the early stage of this experiment, it was verified that B. subtilis 168 contained NK gene sequence, but could not express active NK

Method used

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  • Bacillus subtilis strain for excreting nattokinase at high efficiency and preparation technology of high-purity nattokinase
  • Bacillus subtilis strain for excreting nattokinase at high efficiency and preparation technology of high-purity nattokinase
  • Bacillus subtilis strain for excreting nattokinase at high efficiency and preparation technology of high-purity nattokinase

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Experimental program
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Effect test

Embodiment 1

[0044] Step 1: Amplification and optimization of NK-aprN gene fragment

[0045] LB liquid medium: 10g / L peptone, 10g / L sodium chloride, 5g / L yeast extract.

[0046] LB solid medium: LB liquid medium, 15g / L agarose.

[0047] Amp solution: 100 mg / mL.

[0048] According to the nattokinase gene sequence included in GenBank (GI: 608796180), according to the codon preference of host bacteria B.subtitle (http: / / www.kazusa.or.jp / codon / cgi-bin / showcodon.cgi?species=1423) , using the biological software oligo to design primers (Table 1), amplify the aprN gene and optimize its first 30 amino acid codons. Primers were synthesized by Sangon Bioengineering Co., Ltd.

[0049] After the preserved B. subtilis 168 strain was activated, it was inoculated in LB medium according to the inoculum size of 2%, and cultured at 37° C. and 200 rpm for 12 hours. The cloning of the target gene adopts the method of over-lapping PCR, that is, the bacteria solution is used as the template for the first PC...

Embodiment 2

[0063] Inoculation: activate bacterial classification, prepare primary seed liquid and secondary seed liquid under 37 ℃, 200rpm culture condition, the secondary seed liquid that seed age is 12h is inoculated to fermentation medium according to 10% inoculum (chloramphenicol final concentration 5 μg / mL, antifoaming agent 0.01%). Fermentation medium (1L): glucose 28.5g, glycerol 57g, yeast extract 60g, dipicolinic acid 0.167g, NH 4 Cl 3g, K 2 HPO 4 ·3H 2 O 1g, MnSO 4 ·H 2 O 0.032g, FeSO 4 ·7H 2 O 0.05g, CoCl 2 ·6H 2 O 0.01g, ZnCl 2 0.01g, MgSO 4 ·7H 2 O 2g, CaCl 2 2H 2 O 5g, add water to 1000ml.

[0064] Fermentation: The stirring rate is initially set to 800rpm, and the ventilation rate is 12L / min. Set the parameters, control the temperature in the tank to 37°C, the dissolved oxygen to 30%, and the pH of the fermentation broth to 7.0, and adjust the stirring rate and ventilation according to the dissolved oxygen.

[0065] Protein induced expression: detection of...

Embodiment 3

[0070] The fermentation broth was refrigerated and centrifuged at 4°C and 9000r / m for 10min, and the supernatant was taken; the finely ground (NH 4 ) 2 SO 4 Add to the fermentation supernatant according to 20% saturation, store overnight at 4°C, centrifuge at 10,000r / m for 30min, discard the precipitate; continue to add ammonium sulfate to the fermentation supernatant to 60% saturation, store overnight at 4°C , centrifuged at 10000r / m for 30min, discarded the supernatant, and the recovery rate reached 72%.

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Abstract

The invention relates to a bacillus subtilis strain for excreting nattokinase at high efficiency. An aprN gene of bacillus subtilis NK is amplified by a PCR (polymerase chain reaction) technique; codons of initial thirty amino acids are optimized according to the codon preference of the bacillus subtilis, a recombination expression plasmid pHT01-aprN is established, and the correctness is verified through the restriction enzymes digestion, PCR amplification and sequencing. The recombination expression plasmid pHT01-aprN is imported into the bacillus subtilis 168 by an electric shocking method, and is performed with resistance screening by adopting chloramphenicol, so as to obtain engineering bacteria. After the fermenting condition is optimized, and the expression is induced by IPTG, the highest enzyme activity in a shake culture fermenting liquid is 289U / ml; the highest enzyme activity in a high-density fermenting liquid is 7778U / ml; the highest enzyme activity of a nattokinase preparation prepared by affinity column chromatography is 981731U / g.

Description

technical field [0001] The invention relates to a microbial strain and a process for fermenting and preparing a high-purity enzyme preparation, in particular to a bacillus subtilis strain that efficiently secretes nattokinase and a process for preparing a high-purity nattokinase enzyme preparation. Background technique [0002] Natto is a fermented soybean food made from soybeans fermented by Bacillus subtilis natto. Nattokinase (NK) is a serine protease produced by Bacillus subtilis natto and present in natto. In addition to its strong fibrinolytic activity in vivo and in vitro, NK also has the functions of promoting blood flow, preventing platelet aggregation and lowering blood pressure. NK was first discovered by Dr. Sumi Yoko in 1980. Its relative molecular mass (Mr) is 27000 and its isoelectric point is 8.6±0.3. Cardiovascular disease caused by thrombus seriously endangers human health. At present, thrombolytic agents include injectable degradative drugs (urokinase, s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/75C12N9/54C12R1/125
CPCC12N15/75C12N9/6424C12Y304/21062
Inventor 张建华崔青龚文秀钱丙俊李希强姚晓敏王杰刘华奇
Owner 上海诺金科生物科技有限公司
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