Durable enterococcin mutant and its application
A technology of durable enterococcus and mutants, which can be applied in the application, introduction of foreign genetic material using a carrier, food preservation, etc. The effect of high activity and high stability
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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 I (isoleucine) at position 30 of SEQ ID NO: 1 with A (alanine) to prepare a durable enterococcin mutant (named I30A). The amino acid sequence of the durable enterococcin mutant I30A is shown in SEQ ID NO:2, and the coding gene sequence 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 coding gene consisting of His tag, enterokinase cleavage site, and durable enterococcus Durancin GL (SEQ ID NO: 85-216 after removing the signal peptide coding sequence A bacteriocin fusion gene fragment consisting of a nucleotide fragment of 2-bit nucleotide) and an immune gene (durB, GenBank accession number HQ696461...
Embodiment 2
[0041] Example 2 Preparation of mutants
[0042] Insert the expression strain DE3-pGEX-durAB-I30A into the 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 4 aqueous solution, pH 7.4) to suspend the cells, place the cells in an ice-water mixture and sonicate the cells until the solution is clarified. 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 membrane to obtain a crude extract of the bacteriocin fusion protein. The crude extract of the bact...
Embodiment 3
[0045] Example 3 Properties of Mutants
[0046] The properties of each mutant prepared in Example 2 were investigated, wherein Durancin GL was prepared by the method in Example 2.
[0047] (1) Antibacterial spectrum
[0048] The antibacterial activity of durable enterococcin mutants I30A and V31A was detected by the double dilution method, and the indicator bacteria are shown in Table 1. For specific methods, see references (Lihui Du, George A. Somkuti, John A. Renye Jr. Guicheng Huo. 2012. Properties of Durancin GL, a new antilisterial bacteriocin produced by Enterococcus Durans 41D. Journal of food safety, 32:74-83.) . The results of the antibacterial spectrum test are shown in Table 1. From Table 1, it can be seen that the durable enterococcin mutants I30A and V31A have a significantly expanded antibacterial spectrum compared with the durable enterococcin Durancin GL. Coccus 1363 had significant inhibitory effect.
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