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Bacillus licheniformis engineering bacterium capable of efficiently secreting nattokinase

A technology of Bacillus licheniformis and nattokinase, which is applied in the fields of enzyme engineering and microorganisms, and can solve the problems such as the lack of universality of signal peptides

Active Publication Date: 2016-03-02
武汉骏安生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At the same time, they also proposed that signal peptides are not universal

Method used

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  • Bacillus licheniformis engineering bacterium capable of efficiently secreting nattokinase
  • Bacillus licheniformis engineering bacterium capable of efficiently secreting nattokinase
  • Bacillus licheniformis engineering bacterium capable of efficiently secreting nattokinase

Examples

Experimental program
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Effect test

example 1

[0031] Example 1 Construction of nattokinase-producing engineered strain Bl10 (pP43SacCNK) from Bacillus licheniformis

[0032] According to the genome sequence of Bacillus subtilis 168 published by NCBI, the sequence of promoter P43 and the signal peptide sequence of fructanase SacC were amplified by PCR. The primers are:

[0033] P43-F: CGGAATTCTGATAGGTGGTATGTTTTCG

[0034] P43-R: CTTGAATCAGTCTCTTTTTTCATTTCATGTGTACATTCCTCTC

[0035] Sp-SacC-F:GAGAGGAATGTACACATGAAATGAAAAAGAGACTGATTCAAG

[0036] Sp-SacC-R: CTGTACTGCTTTTTCCGGCTGCATCTGCCGAAAATGCCATAG

[0037] According to the genome sequence of Bacillus subtilis natto published on NCBI, the nattokinase gene was amplified by PCR wxya sequence. Primers are as follows:

[0038] AprN-F: CTATGGCATTTTCGGCAGATGCAGCCGGAAAAAAGCAGTACAG

[0039] AprN-R: ATCCGTCCTCTCTGCTCTTTTGTGCAGCTGCTTGTACGTTGAT

[0040] According to the genome sequence of Bacillus licheniformis WX-02 published on NCBI, the amylase gene was amplified by PCR am...

example 2

[0043] Example 2 Fermentation experiment and enzyme activity determination of Bacillus licheniformis Bl10 (pP43SacCNK)

[0044] Seed solution: 10g / L peptone, 5g / L yeast extract powder, 10g / L sodium chloride pH7.2-7.4 250mL triangular bottle liquid volume is 50mL

[0045] Seed cultivation time: 10h

[0046] Fermentation medium: 20g / L glucose, 10g / L peptone, 15g / L yeast extract powder, 10g / L sodium chloride, 6g / L ammonium sulfate; 10g / L soybean peptone, 5g / L corn steep liquor pH7.0-7.2 250mL triangular bottle The volume is 30mL

[0047] Inoculum size: 3%

[0048] Training time: 48h

[0049] Fermentation broth pretreatment: Take 2mL of fermentation broth in a 2mLEP tube, centrifuge at 10000rpm for 5min, transfer the supernatant to another 2mLEP tube, and store at 4°C for later use.

[0050] Determination of enzyme activity by fibrinogen plate method: At 50°C, 10 mL of fibrinogen solution (2.0 mg / mL) and 10 mL of agarose solution (1.0%, w / v) were quickly mixed,...

example 3

[0051] Example 3 Determination of nattokinase secretion at the end point of fermentation of Bacillus licheniformis Bl10 (pP43SacCNK)

[0052] Preparation of BSA standard curve: BSA solutions of different concentrations (0.2mg / mL, 0.4mg / mL, 0.6mg / mL, 0.8mg / mL, 1mg / mL) were mixed with the same amount of 2*SDS-PAGEbuffer, and then 10ul samples were taken. After electrophoresis, Obtain the standard curve of BSA by detecting the depth and area of ​​its different concentrations of BSA bands, which is: y=1.3522x+0.7429 (R 2 =0.9955, y: depth area product of protein band x: total amount of protein)

[0053] Fermentation broth sample pretreatment: According to the method, pretreat the supernatant of the fermentation broth, mix it with 2*SDS-PAGEbuffer, take 10ul spotting, and compare the color depth and area of ​​the protein band with the standard curve of BSA to obtain The protein content of nattokinase in the fermentation broth was 163.52mg / L.

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Abstract

The invention relates to a Bacillus licheniformis engineering bacterium capable of efficiently secreting nattokinase, and belongs to the technical fields of enzyme engineering and microbes. The method adopts a molecular biology technology to screen and replace signal peptides with important effects in an exogenous protein transhipment process, and the signal peptides are replaced by signal peptides from levanase SacC in Bacillus subtillis 168 from signal peptides from extracellular serine proteinase Vpr in Bacillus licheniformis WX-02 on the basis of nattokinase producing Bacillus licheniformis engineering bacterium BL10 (pP43SNT) preserved in a laboratory in advance in order to afresh construct the nattokinase producing Bacillus licheniformis engineering bacterium BL10 (pP43SacCNK). The bacterium disclosed in the invention can substantially improve the nattokinase secreting level under liquid fermentation conditions, and the maximum enzyme activity can reach 33.83 FU / mL. The bacterium is preserved in China Center for Type Culture Collection on June 16, 2014 with the preservation number of CCTCC NO: M2014253.

Description

technical field [0001] The invention discloses a bacillus licheniformis engineering bacterium capable of efficiently secreting nattokinase, which belongs to the field of enzyme engineering and microorganism technology. Background technique [0002] Nattokinase is a single-chain serine protease secreted by Bacillus subtilis natto. The enzyme has good thrombolytic activity. The catalytic triplet structure composed of Asp32, His64 and Ser221 constitutes its active center. Compared with urokinase, Fibrinolytic enzymes such as t-PA and streptokinase, and nattokinase have good thrombolytic effects and biological safety, making them specific drugs that can be used to treat cardiovascular diseases in the future. At the same time, as a functional health food, nattokinase food is more and more favored by consumers. [0003] Currently reported bacillus for the production of nattokinase mainly includes Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus licheniformis. Because o...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N9/56C12R1/10C12R1/125
Inventor 陈守文蔡冬波魏雪团周银华陈敬帮祁高富冀志霞
Owner 武汉骏安生物科技有限公司
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