Bacillus licheniformis engineering bacteria for nattokinase production and method for producing nattokinase by using bacillus licheniformis engineering bacteria

A technology for Bacillus licheniformis and nattokinase production, applied in the field of microbial genetic engineering and enzyme engineering

Active Publication Date: 2015-05-20
HUAZHONG AGRI UNIV
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Problems solved by technology

Nattokinase has been expressed to varying degrees in Escherichia coli and Bacil...

Method used

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  • Bacillus licheniformis engineering bacteria for nattokinase production and method for producing nattokinase by using bacillus licheniformis engineering bacteria
  • Bacillus licheniformis engineering bacteria for nattokinase production and method for producing nattokinase by using bacillus licheniformis engineering bacteria
  • Bacillus licheniformis engineering bacteria for nattokinase production and method for producing nattokinase by using bacillus licheniformis engineering bacteria

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Experimental program
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Embodiment 1

[0029] Embodiment 1: Construction of engineering bacteria 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 were designed,

[0031] PspF: 5'-GAGAGGAATGTACACATGAATTGAGAAAAAAGTATCGTGCG-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 genome of B. subtillis168 published by NCBI,

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

[0035] P43R3: 5'-CGCACGATACTTTTTTCTCAATTCATGTGTACATTCCTCTC-3'

[0036] The promoter P43 fragment was amplified by PCR. A pair of primers PaprNF and PaprNR are designed according to the sequence of the nattokinase (NK) gene aprN (accession number is FJ374767) of B....

Embodiment 2

[0046] Example 2: Expression of nattokinase and determination of plasmin activity

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

Embodiment 3

[0048] Embodiment 3: nattokinase liquid fermentation

[0049] Pick the colony B. licheniformis BL10 (pP43SNT) and inoculate it in 5 mL of LB medium with tetracycline (20 μg / mL), and cultivate overnight at 37° C. with shaking at 180 r / min. Transfer to 50 mL of fresh LB medium with 4% inoculum until OD 600 When it is 1.0, inoculate into pre-prepared nattokinase liquid fermentation medium (10g / L soybean peptone; 10g / L yeast extract powder; 10g / L maltose; pH7.0-7.2) with 5% inoculum size (1mL) ), 37℃, 200r / min, fermentation time 32h, the enzyme activity of BL10(pP43SNT) reached 11.37FU / mL.

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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 in particular relates to a nattokinase-producing bacillus licheniformis engineering bacterium and a method for producing nattokinase with the bacterium Background technique [0002] Nattokinase (NK) is a fibrinolytic enzyme synthesized by Bacillus subtilis, which has strong fibrinolytic activity and has been widely used in food and health care fields. Its active center is a catalytic triad composed of Asp32, His64, and Ser221. Nattokinase (E.C.3.4.21.62) is a single-chain polypeptide enzyme consisting of 275 amino acid residues with a molecular weight of 27,728Da. At room temperature, the enzyme activity is most stable in the range of pH 7-9, and the enzyme is unstable when the pH is lower than 5. Metal ions can affect the activity of nattokinase (NK), Cu 2+ , Zn 2+ 、Al 3+ , Ca 2+ Plasma has different degrees of inhibition of the enzyme, Fe ...

Claims

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Application Information

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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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