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Recombinant bacterium capable of high efficiently secreting and expressing natto kinase

A nattokinase, secretory expression technology, applied in the field of recombinant bacteria, can solve the problem that the nattokinase expression level cannot meet the industrialization and other problems

Active Publication Date: 2014-07-23
BEIJING YANJING BREWERY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the expression level of nattokinase in the above article still cannot meet the needs of industrialization, and further optimization and improvement of the production strains are still needed

Method used

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  • Recombinant bacterium capable of high efficiently secreting and expressing natto kinase
  • Recombinant bacterium capable of high efficiently secreting and expressing natto kinase
  • Recombinant bacterium capable of high efficiently secreting and expressing natto kinase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1, the construction of the recombinant expression vector containing nattokinase Pro-NK gene

[0049] (1) Extraction of chromosomal DNA from Bacillus subtilis subsp natto

[0050] Inoculate the spore suspension or slant mycelium of Bacillus subtilis natto (preservation number CGMCC1.1086, purchased from China General Microorganism Culture Collection Management Center) stored at -70°C into 5mL LB medium, shake at 37°C Incubate for 48 hours. Take 3 mL of the bacterial liquid and centrifuge to collect the mycelium, and wash the mycelium twice with 500 μL of STE buffer. Then use 500 μL of STE to fully suspend the mycelium, add lysozyme to a final concentration of 2 mg / mL, at this time, the bacterial suspension becomes translucent gel, and then add 250 μL of 2% SDS (Sodium dodecyl sulfate, 16 Sodium Alkyl Sulfate), vortexed to mix. Then add 250 μL of neutral phenol / chloroform mixed solution and mix well, wherein the volume ratio of phenol and chloroform is 25:24...

Embodiment 2

[0068] Embodiment 2, the preparation of the yeast strain expressing nattokinase and its functional detection

[0069] 1. Preparation of Strains Expressing Nattokinase

[0070] (1) Preparation of electrotransformed Pichia pastoris cells

[0071] Inoculate Pichia pastoris strain GS115 (purchased from Invitrogen, CA.18100) in 500mL YPD medium, cultivate to OD at 30°C and 200r / min 600 =1.3-1.5. Centrifuge at 4°C and 4500r / min for 5 minutes to collect the bacterial cells, and wash once with 500 mL, 250 mL of pre-cooled sterilized water and 20 mL of pre-cooled 1mol / L sorbitol, respectively. After each wash, the cells were collected by centrifugation at 4°C and 4500 r / min for 5 minutes, and finally suspended with 1 mL of pre-cooled 1 mol / L sorbitol to obtain electroshock competent cells.

[0072] (2) Preparation of recombinant Pichia pastoris strain containing recombinant expression vector pAOα-PNK

[0073] Take about 10 μg of the recombinant expression vector pAOα-PNK obtained i...

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Abstract

The invention discloses a recombinant bacterium capable of high efficiently secreting and expressing natto kinase, and the recombinant bacterium can prominently increases the output of natto kinase. The recombinant bacterium is a recombinant pichia pastoris containing at least two copies of a natto kinase Pro-NK gene and a Haclp regulatory factor gene. After the recombinant bacteria carry out fermentation for 96 hours in a shake flask, the enzyme activity of the natto kinase can reach 470 IU / mL.

Description

technical field [0001] The invention relates to a recombinant bacterium, in particular to a recombinant bacterium capable of efficiently secreting and expressing nattokinase. Background technique [0002] Nattokinase (Nattokinase, NK) is a serine protease produced by Bacillus natto or Bacillus subtilis natto during the fermentation of natto. In 1980, Professor Sumi Yoko of Japan first discovered that the enzyme could dissolve artificial thrombus, then studied the mechanism of action of the enzyme, and named it nattokinase. [0003] Since the first report on natto and nattokinase in China in 1995, many articles have been published so far, and research and development have gone hand in hand. At present, the properties of nattokinase (nattokinase gene sequence, protein amino acid sequence, active center and catalytic center, pI, temperature and pH stability, optimum reaction temperature and pH, inhibitor and activator, and the solubility of nattokinase Thrombosis mechanism in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N1/19C12R1/84
Inventor 朱泰承孙红兵李鹏飞李寅邓启华沈建国陆英林智平贾凤超
Owner BEIJING YANJING BREWERY
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