A kind of durable enterococcus mutant, its preparation method and its application
A technology of durable enterococci and mutants, which is applied in botany equipment and methods, biochemical equipment and methods, applications, etc., can solve the problem of low activity of durable enterococci, achieve high stability, simple preparation method, highly active effect
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Embodiment 1
[0021] The amino acid sequence of Durancin GL mature peptide is shown in SEQ ID NO:1. Its precursor has a signal peptide, and the coding gene is shown in SEQ ID NO:4. This example describes the specific method of substituting the lysine (K) at the 21st position in SEQ ID NO: 1 with alanine (A) to prepare a durable enterococcin mutant (named K21A). The amino acid sequence of the durable enterococcin mutant K21A is shown in SEQ ID NO: 2, and the sequence of the coding gene is shown in SEQ ID NO: 3.
[0022] 1. Amplify the bacteriocin fusion gene fragment
[0023] In order to facilitate purification and expression, primers were designed to amplify and prepare the mature peptide encoding gene consisting of His tag, enterokinase cleavage site, and durable enterococcus Durancin GL (the 85th-216th core of SEQ ID NO: 4 after removing the signal peptide). A bacteriocin fusion gene fragment consisting of a nucleotide fragment) and an immune gene (durB, GenBank accession number HQ69646...
Embodiment 2
[0037] Embodiment 2 prepares mutant
[0038] Insert the expression strain DE3-pGEX-durAB-K21A into LB liquid medium containing 50 μg / mL ampicillin with 1% inoculum, and culture it with shaking at 37°C and 200r / min until OD 600 0.6-0.8, add IPTG with a final concentration of 1mmol / L to the culture medium for induction, and continue to culture for 5-6h. The fermentation broth was centrifuged at 8000g for 5 minutes to collect the bacteria sludge, and the bacteria sludge was washed twice with physiological saline. Binding buffer (containing 140 mM NaCl, 2.7 mM KCl, 10 mM NaCl, 2 HPO 4 and 1.8mM KH 2 PO 4aqueous solution, pH 7.4) to suspend the bacterial cells, place the bacterial cells in an ice-water mixture and ultrasonically disrupt the bacterial cells until the solution is clear, and the ultrasonic parameters are: 400W ultrasonic 2s interval 9s. The cell lysate was centrifuged at 4°C and 15,000 r / min for 30 min, and the supernatant was filtered through a 0.22 μm filter me...
Embodiment 3
[0040] The property of embodiment 3 mutants
[0041] Investigate the properties of the mutants prepared in Example 2, wherein Durancin GL was prepared by the method in Example 2.
[0042] (1) activity
[0043] Listeria monocytogenes (Listeria monocytogenes) was used as the indicator bacteria to detect the antibacterial activity of the durable enterococcin mutant K21A. For specific methods, see references (Lihui Du, George A. Somkuti, John A. Renye Jr. Guicheng Huo. 2012. Properties of Durancin GL, a new antilisterialbacteriocin produced by Enterococcus Durans 41D. Journal of food safety, 32:74-83.). Among them, the concentrations of Durancin GL and the mutant were the same (both 20 μg / mL), and the dropping volume was 5 μl. from image 3 It can be seen that the antibacterial activity of mutant K21A to Listeria monocytogenes is about 2 times that of durable enterococcus Durancin GL (such as image 3 ).
[0044] (2) Stability
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