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High-yield nattokinase gene engineering strain constructing method

A technology of genetically engineered strains and nattokinase, which is applied in the field of bioengineering, can solve the problems of low enzyme activity, long fermentation time, complicated separation and purification process, etc., and achieves the effects of good safety, less impurities in fermentation products and low cost.

Inactive Publication Date: 2019-02-01
WUHAN INSTITUTE OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Wild-type Bacillus natto produces nattokinase, which not only has a long fermentation time and low enzyme activity, but also has a variety of proteins in the fermentation products, and the later separation and purification process is cumbersome

Method used

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Experimental program
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Effect test

Embodiment 1

[0024] Construct nattokinase genetically engineered bacterial strain, comprises the following steps:

[0025] 1) Obtain the sequence of nattokinase gene fragment from GenBank, use Clone Manager software to design primers, and synthesize them by Shanghai Sangon Bioengineering Company. Using PCR technology to amplify the nattokinase gene fragment;

[0026] 2) Perform BamH I and Sma I double-enzyme digestion treatment on the PCR amplification product and the carrier plasmid, and use T4DNALigase to connect to construct a recombinant plasmid;

[0027] 3) Transform the recombinant plasmids into Bacillus subtilis using an electric transformation apparatus, culture overnight at 37°C on a plate containing antibiotics, and select positive transformants for sequencing verification.

[0028] 4) Enzyme activity detection: Inoculate the seed bacteria liquid into 25mL fresh liquid medium with 2% inoculum amount, and culture on a shaking table at 30°C until OD 600nm Between 0.6 and 0.8, 0.1...

Embodiment 2

[0031] Optimizing the expression conditions of the recombinant strains, including the following steps:

[0032] 1) Determination of optimal IPTG concentration

[0033] Inoculate the seed bacterium solution of the recombinant strain in 25mL of fresh liquid medium with 2% inoculum amount, and culture it on a shaking table at 30°C to OD 600nm Between 0.6-0.8, different concentrations (0.2mM, 0.4mM, 0.6mM, 0.8mM, 1.0mM, 1.2mM, 1.4mM and 1.6mM) were added to induce expression of IPTG for 24 hours, and then the enzyme activity of nattokinase was detected . image 3 The optimal IPTG concentration was shown to be 1.0 mM.

[0034] 2) Determination of the optimal expression temperature

[0035] Inoculate the seed bacterium solution of the recombinant strain in 25mL fresh liquid medium with 2% inoculum amount, and culture it on a shaking table until OD 600nm At 0.6-0.8, 1.0 mM IPTG was added to induce expression at 18°C, 25°C, 30°C, 37°C and 40°C, respectively. Figure 4 The optimum...

Embodiment 3

[0039] Optimization of recombinant strain fermentation medium, comprising the following steps:

[0040] 1) Determination of the best carbon and nitrogen sources

[0041] In order to determine the impact of optimum carbon source and nitrogen source type and mass concentration on nattokinase, select carbon source to have: glucose, sucrose, maltose, xylose, glycerol, lactose, cellulose, soluble starch, mass concentration is 20g / L; nitrogen source: soybean peptone, peptone, tryptone, yeast powder, NH 4 NO 3 , (NH 4 ) 2 SO 4 , NH 4 Cl, NaNO 3 , the mass concentration is 20g / L. The mass concentration gradients of nitrogen source and carbon source were 0, 10, 20, 30, 40, 50, 60, 70 g / L. The results showed that the optimum nitrogen source was peptone 20g / L, and the optimum carbon source was glucose 20g / L.

[0042] 2) Effects of sodium glutamate and sodium citrate on the activity of nattokinase

[0043] The concentration gradients of sodium glutamate and sodium citrate were ...

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Abstract

The invention discloses a high-yield nattokinase gene engineering strain constructing method, which comprises: obtaining a nattokinase gene fragment by using Bacillus natto genome as a PCR amplification template through a PCR technology; carrying out double restriction enzyme digestion on the gene fragment of nattokinase and plasmid pHT43 by using BamH I and Sma I; carrying out enzyme linking on the nattokinase DNA fragment and the obtained plasmid vector to obtain a recombinant vector pHT-NK; and transforming the recombinant vector pHT-NK into Bacillus subtilis, and screening positive recon so as to obtain the nattokinase transgenic engineering strain. According to the present invention, the nattokinase gene engineering strain is constructed by using the Bacillus subtilis-deficient strainas the host cell, such that the yield of nattokinase is high, the impurity protein in the fermentation product is less, the late-stage separation and purification process is simplified, and the production cost is saved.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for constructing a high-yield nattokinase genetic engineering strain. Background technique [0002] Nattokinase (Nattokinase, NK), also known as subtilisin NAT (subtilisinNAT), was first isolated from natto by Japanese scholar Sumi et al. in 1987. Natto is traditionally used as a medicine for heart and vascular diseases. At the same time, natto is also used as a medicine for relieving fatigue and anti-beriberi. NK is a protease produced by the food-grade microorganism Bacillus subtilis in the natto fermentation process, and belongs to the serine protease of the subtilisin class. NK has strong fibrinolytic activity both in vivo and in vitro, and its fibrinolytic activity is 4 times that of plasmin. NK can not only directly degrade fibrin, but also increase the amount of tissue plasminogen activator (t-PA) in plasma, which will lead to the conversion o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N9/56C12R1/125
CPCC12N15/75C12N9/54C12Y304/21062
Inventor 张佑红周文科田莉钱泽栋
Owner WUHAN INSTITUTE OF TECHNOLOGY
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