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Recombinant bacteria for preparing bacteriocin durancin GL, preparation method and application

A bacteriocin and recombinant bacteria technology, applied in the field of microbial biology, can solve the problems of difficult to guarantee purity, complex fermentation broth components, low concentration of target products, etc., and achieve the effect of simple components

Active Publication Date: 2018-03-30
NANJING UNIV OF FINANCE & ECONOMICS
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AI Technical Summary

Problems solved by technology

The bacteriocin durancin GL is produced by fermentation of Enterococcus durancinus, the concentration of the target product in the fermentation broth is low, and the composition of the fermentation broth is complex, so the process of isolating the active bacteriocin is time-consuming, laborious, costly, and the purity is difficult to guarantee

Method used

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  • Recombinant bacteria for preparing bacteriocin durancin GL, preparation method and application
  • Recombinant bacteria for preparing bacteriocin durancin GL, preparation method and application
  • Recombinant bacteria for preparing bacteriocin durancin GL, preparation method and application

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Embodiment 1

[0028] 1. Cloning of bacteriocin durancin GL gene

[0029] According to the sequence of the bacteriocin durancin GL gene (HQ696461.1) in the GenBank database, primers dur1 (SEQ ID NO: 5) were designed: 5′-ggg agg caa tta tat gaa gaa aaa att tgt tag tat t-3′ and dur2 ( SEQ ID NO:6): 5′-CCA gTg CCC CCA TAA ACT ATA CAC CC-3′, the gene fragment of bacteriocin durancin GL was amplified from Enterococcus durancinus 41D (a published bacterium), which was composed of the structure Gene and immune gene composition, the sequence is shown in SEQ ID NO:1.

[0030] The gene of bacteriocin durancin GL can also be prepared by chemical synthesis.

[0031] 2. Sequence verification of the bacteriocin durancin GL gene fragment

[0032] The amplified durancin GL gene fragment was recovered, and then ligated with pEASY-Blunt Simple Cloning Vector (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) to obtain the recombinant plasmid pEASY / durAB. During the ligation process, the ratio of t...

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Abstract

The invention provides a recombinant bacterium, a preparation method and an application for preparing the bacteriocin durancin GL, and relates to the field of microbial biotechnology. The recombinant bacterium carries the coding gene of the fusion protein formed by linking the bacteriocin durancin GL, the purification tag and the restriction site. The method for preparing the bacteriocin durancin GL is as follows: Induce the recombinant bacteria to express the fusion protein, crush to obtain the lysate, centrifuge to get the supernatant, use HisTrap HP column and GSTrap 4B column to separate or use GSTrap 4B column to separate, collect the elution peak, and use enterokinase Enzymolysis, removal of the purification tag, to obtain the bacteriocin durancin GL. The recombinant bacterium of the present invention is convenient to construct, can efficiently express the recombinant bacteriocin durancin GL, perform efficient separation through affinity chromatography, and obtain the active bacteriocin durancin GL after hydrolyzing the site of cleavage.

Description

technical field [0001] The invention relates to the field of microbial biotechnology, in particular to a recombinant bacterium, a preparation method and an application for preparing the bacteriocin durancin GL. Background technique [0002] Bacteriocins are a class of proteins or polypeptides produced by ribosomes with bacteriostatic activity, which widely exist in Gram-positive and Gram-negative bacteria, and their inhibition of target bacteria is characterized by low concentration and high efficiency. Today, more and more attention has been paid to the safety hazards of chemical preservatives, and the application of food-grade microbial-derived bacteriocins as biological preservatives meets consumers' requirements for safety, health and naturalness. [0003] The bacteriocin durancin GL, which is derived from Enterococcus durans and can specifically inhibit Listeria monocytogenes, has important potential application value in the food industry. The bacteriocin durancin GL w...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/31C12P21/02
Inventor 鞠兴荣陈信全都立辉袁建何荣和肖营吴学友刘凌平施荣华
Owner NANJING UNIV OF FINANCE & ECONOMICS
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