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Lyase with high temperature tolerance as well as preparation method and application of lyase

A lyase, tolerance technology, applied in the field of new antibiotic substitutes, which can solve the problems of protein loss, weak tolerance, and uncommon phage lyase

Pending Publication Date: 2022-07-29
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Through the above analysis, the existing problems and defects of the prior art are: most of the lytic enzymes found at present are not very resistant to temperature, and most of them are intracellular proteins. The heat generation will lead to the loss of protein; in addition, phage lyases that can lyse bacteria across species are not common

Method used

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  • Lyase with high temperature tolerance as well as preparation method and application of lyase
  • Lyase with high temperature tolerance as well as preparation method and application of lyase
  • Lyase with high temperature tolerance as well as preparation method and application of lyase

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preparation example Construction

[0054] like figure 1 As shown, the preparation method of the lyase with strong temperature tolerance provided by the embodiment of the present invention comprises:

[0055] S101, designing, editing and predicting the sequence of the tagged cleavage enzyme, using artificial synthesis to obtain the coding sequence of the amino acid, digesting and recovering it with double restriction endonucleases, and inserting it into the prokaryotic expression vector pET-28a(+) plasmid;

[0056] S102, transforming the plasmid into the engineered bacteria Escherichia coli BL21 expressing the protein by heat shock method, screening positive clones, and performing PCR amplification and sequencing on the positive clones to verify the correctness of the sequence;

[0057] S103, inoculate the positive cloned strain in the LB liquid of kanamycin and cultivate it to log phase, add IPTG, induce, and centrifuge the obtained culture medium to obtain a supernatant; also contains lyase;

[0058] S104, t...

Embodiment 1

[0096] Example 1 Effect of Lysase Lys22 on Enterococcus faecalis Biofilm Formation and Mature Biofilm

[0097] 1. Effect of Lysase Lys22 on Biofilm Formation by Enterococcus

Embodiment approach

[0099] (1) Enterococcus faecalis 18 (MH236318) culture (OD600=0.5-0.6) was mixed with lyase Lys22 so that the concentration of lyase Lys22 was 50 μg / mL.

[0100] (2) The mixed solution was added to the 96-well plate in an amount of 100 μl / well, and cultured for 6h, 12h, and 24h respectively.

[0101] (3) Take out the bacterial liquid respectively, and measure the absorbance of the bacterial liquid at 600 nm with a microplate reader, which is used to calibrate the concentration of planktonic bacteria in different time periods.

[0102] (4) Wash the culture wells three times with 100 μl of 0.1% PBS and dry at room temperature.

[0103] (5) Add 0.1% crystal violet staining solution in an amount of 100 μl / well, stain for 10 min, and aspirate the staining solution.

[0104] (6) Wash three times with 0.1% PBS buffer to remove excess dye, air dry at room temperature, and add an equal volume of 95% ethanol to decolorize for 10 min.

[0105] (7) Use a microplate reader to detect the ...

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Abstract

The invention belongs to the technical field of new antibiotic substitutes, and discloses a lyase Lys22 with strong temperature tolerance, a preparation method and application, the lyase with strong temperature tolerance has six histidine tags, and the coding sequence of the lyase is SEQ ID NO: 1; the preparation method of the lyase Lys22 with strong temperature tolerance comprises the following steps: artificially synthesizing an amino acid coding sequence, digesting and recycling double restriction endonuclease, and inserting into a prokaryotic expression vector pET-28a (+) plasmid; transforming the plasmid into engineering bacteria escherichia coli BL21 for expressing protein, screening positive clones, and verifying the correctness of the sequence; inoculating the positive clone strain into LB liquid of kanamycin, culturing to a logarithmic phase, adding IPTG, inducing, centrifuging, filtering supernate, centrifuging, collecting filtrate, concentrating, purifying and verifying the filtrate. According to the method, two-step molecular sieve ultrafiltration is adopted, and the effect of reducing interference of other proteins possibly containing multiple histidines in the background can be achieved.

Description

technical field [0001] The invention belongs to the technical field of new antibiotic substitutes, and in particular relates to a lyase with strong temperature tolerance, a preparation method and an application. Background technique [0002] At present, antibiotic resistance is a common problem faced by the world, and it is imminent to find new alternatives to antibiotics. Many bacteria can form biofilms. Biofilms are a group structure formed by bacteria and their metabolites, which are helpful for bacterial adhesion and colonization. Biofilms increase bacteria’s tolerance to the external environment and antibiotics by hundreds of times. . [0003] Enterococcus is a gram-positive coccus that widely exists in nature, human and animal intestines. Enterococcus is a common nosocomial infection that can cause multi-organ system infections in the body. Enterococci have serious drug resistance and are difficult to treat. Vancomycin-resistant Enterococcus ranks third in the list o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70A61K38/51A61P31/04A23C9/12A23L3/3571
CPCC12N9/88C12N15/70A61K38/51A61P31/04A23L3/3571A23C9/1216A23V2002/00A23V2200/10Y02A50/30
Inventor 史红艳胡南南贾玉玺张宇思张娜
Owner JILIN UNIV
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