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A nattokinase-producing bacillus licheniformis engineered bacterium and method for producing nattokinase with the bacterium

A technology for producing Bacillus licheniformis and nattokinase, which is applied in the fields of microbial genetic engineering and enzyme engineering

Active Publication Date: 2018-01-23
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Nattokinase has been expressed to varying degrees in Escherichia coli and Bacillus subtilis, but there is no report of secreted expression in Bacillus licheniformis

Method used

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  • A nattokinase-producing bacillus licheniformis engineered bacterium and method for producing nattokinase with the bacterium
  • A nattokinase-producing bacillus licheniformis engineered bacterium and method for producing nattokinase with the bacterium
  • A nattokinase-producing bacillus licheniformis engineered bacterium and method for producing nattokinase with the bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Construction of engineering strain B.licheniformis BL10 (pP43SNT)

[0030] According to the vpr gene sequence in the whole genome sequence of B.licheniformis WX-02 measured in this experiment, a pair of primers PspF and PspR are designed,

[0031] PspF: 5’-GAGAGGAATGTACACATGAATTGAGAAAAAGTATCGTGCG-3’

[0032] PspR: 5’-CTGTACTGCTTTTTCCGGCTGCCTGCACTCCGGTGAG-3’

[0033] Using the genomic DNA of B. licheniformis WX-02 as a template, the signal peptide Svpr fragment of vpr was amplified by PCR; a pair of primers P43F and P43R3 were designed according to the sequence of the P43 promoter on the B.subtillis168 genome published by NCBI,

[0034] P43F: 5’-GGAATTCTGATAGGTGGTATGTTTTCG-3’(EcoRI)

[0035] P43R3: 5’-CGCACGATACTTTTTCTCAATTCATGTGTACATTCCTCTC-3’

[0036] The promoter P43 fragment was obtained by PCR amplification. Design a pair of primers PaprNF and PaprNR according to the sequence of the nattokinase (NK) gene aprN (accession number FJ374767) of B.subtilis MBS04-6 publish...

Embodiment 2

[0046] Example 2: Expression of Nattokinase and Determination of Fibrinolytic Enzyme Activity

[0047] Pick the bacteria B.licheniformis BL10 (pP43SNT), B.licheniformis BL10 (pHY300PLK) and B.licheniformis BL9 (pP43SNT) respectively and inoculate them in 5mL LB medium with tetracycline (20μg / mL), 37℃, 180r / min Incubate with shaking overnight. Then transfer 4% of the inoculum to 50 mL of fresh LB medium, until the OD600 is 1.0, inoculate 50 mL of fresh LB medium with 1% of the inoculum, and culture at 37°C, 200r / min with shaking for 28 hours. After culturing for 12 hours, samples were taken every 4 hours to determine the fibrinolytic activity of nattokinase, and the fibrinolytic enzyme activity was determined according to the fibrinolysis method (Liu Huizhou et al. 2012). Then take 0.9ml of Bacillus fermentation supernatant or cell crushing liquid in a centrifuge tube, add 1 / 9 volume of 100% TCA, invert 10 times to mix, place overnight at 4℃, centrifuge at 13000r / min for 10-20min...

Embodiment 3

[0048] Example 3: Liquid fermentation of nattokinase

[0049] The colony B. licheniformis BL10 (pP43SNT) was picked and inoculated in 5 mL of LB medium with tetracycline (20 μg / mL), and cultured overnight at 37° C. with shaking at 180 r / min. Then transfer 4% of the inoculum to 50mL fresh LB medium until OD 600 When it is 1.0, inoculate a pre-prepared nattokinase liquid fermentation medium (10g / L soy peptone; 10g / L yeast extract powder; 10g / L maltose; pH7.0-7.2 ), at 37°C, 200r / min, and fermentation time of 32h, the enzyme activity of BL10 (pP43SNT) reached 11.37FU / mL.

[0050]

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Abstract

Disclosed are bacillus licheniformis engineering bacteria for nattokinase production and a method for producing nattokinase by using the bacillus licheniformis engineering bacteria, which belong to the technical fields of microorganism genetic engineering and enzyme engineering. According to the invention, a nattokinase gene of bacillus subtilis MBS04-6 is obtained through PCR technology augmentation. Bacillus licheniformis BL10 is utilized as an expression host; promoter P43 of bacillus subtilis 168 is taken as a promoter; a signal peptide of extracellular serine protease Vpr of bacillus licheniformis WX-02 is taken a signal peptide; and a terminator sequence of alpha-amylase of bacillus licheniformis WX-02 is taken as a terminator. The expression elements are connected to shuttle vector pHY300PLK to form an expression vector, and the expression vector is transformed into the bacillus licheniformis BL10 to obtain the bacillus licheniformis engineering bacteria BL10 (pP43SNT) for nattokinase production. The bacterial strain has been deposited with the China Center for Type Culture Collection (CCTCC) at September 10th, 2013, and the accession number is CCTCC NO:M2013401. The invention also provides a method of the bacterial strain for producing nattokinase, and the maximum enzyme activity can reach 11.37 FU / mL in a liquid fermentation medium.

Description

Technical field [0001] The invention belongs to the technical field of microbial genetic engineering and enzyme engineering, and specifically relates to a Bacillus licheniformis engineering strain producing nattokinase and a method for producing nattokinase using the strain Background technique [0002] Nattokinase (NK) is a fibrinolytic enzyme synthesized by Bacillus subtilis. It has strong fibrinolytic activity and has been widely used in food and health care. Its active center is a catalytic triad composed of Asp32, His64, and Ser221. Nattokinase (E.C.3.4.21.62) is a single-chain peptidase consisting of 275 amino acid residues and a molecular weight of 27,728 Da. At room temperature, the enzyme activity is most stable in the pH range of 7-9, and the enzyme is unstable when the pH is lower than 5. Metal ions will affect the activity of Nattokinase (NK), Cu 2+ , Zn 2+ , Al 3+ , Ca 2+ Plasma can inhibit the enzyme to varying degrees, Fe 2+ It has a strong inhibitory effect on i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/75C12N9/56C12R1/10
CPCC12N9/54C12Y304/21062
Inventor 陈守文周银华魏雪团陈敬帮祁高富冀志霞
Owner HUAZHONG AGRI UNIV
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