Quorum-quenching enzyme OLB-26, and coding gene and application thereof

A quorum sensing and genetic technology, applied in the direction of transferase, hydrolase, microbial-based methods, etc., to achieve good thermal stability

Inactive Publication Date: 2013-09-04
FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no protein that has the proper

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  • Quorum-quenching enzyme OLB-26, and coding gene and application thereof
  • Quorum-quenching enzyme OLB-26, and coding gene and application thereof
  • Quorum-quenching enzyme OLB-26, and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1, the preparation of fusion protein OLB-26

[0061] 1. Preparation of fusion gene olb-26

[0062] 1. Preparation of AiiO-AIO6 quencher gene fragment

[0063] ① artificially synthesized quencher enzyme gene aiiO-AIO6 shown in the 7th to 810th nucleotides from the 5' end of the sequence 2 of the sequence listing.

[0064] ②Using the gene synthesized in step ① as a template, perform PCR amplification with a primer pair consisting of A6-F (underlined EcoRI digestion sequence) and L2-Raiia-AIO6 (character shading marked Link sequence) to obtain a PCR product.

[0065] A6-F: 5′-CG GAATTC AAATCCCATGAAATCGAGACCAGTC-3′;

[0066] L2-Raiia-AIO6: 5'-GCTACCGCCGCCACCGGCCGTGCAGTCGCGCATGAAA-3'.

[0067] The enzyme used for PCR amplification was LA Taq (Takara, Japan). PCR amplification conditions: 95°C for 5min; 30 cycles of 94°C for 30s, 60°C for 30s, 72°C for 1min; 72°C for 10min.

[0068] ④Recover the PCR product.

[0069] 2. Preparation of AiiA-AI96 quencher gen...

Embodiment 2

[0114] Example 2. Characterization of the fusion protein OLB-26 as a quorum sensing quenching enzyme

[0115] 1. Optimal pH

[0116] The purified OLB-26 protein solution (protein concentration: 0.49 mg / mL) prepared in Step 4 of Example 1 was used as the solution to be tested, and the quenching enzyme activity was detected at 35°C under different pH conditions to determine its maximum Appropriate pH.

[0117] The signal molecule in enzyme activity determination is 3-oxo-C8-HSL, and the following buffers are used respectively:

[0118] pH5.0, 0.1mol / L McIlvaine buffer (McIlvaine);

[0119] pH6.0, 0.1mol / L McIlvaine buffer;

[0120] pH6.0, 0.1mol / L PBS buffer (PBS);

[0121] pH6.5, 0.1mol / L PBS buffer;

[0122] pH7.0, 0.1mol / L PBS buffer;

[0123] pH7.5, 0.1mol / L PBS buffer;

[0124] pH8.0, 0.1mol / L PBS buffer;

[0125] pH8.0, 0.1mol / L Tris-HCl buffer (Tris-HCl);

[0126] pH9.0, 0.1mol / L Tris-HCl buffer;

[0127] pH9.0, 0.1mol / L glycine-NaOH buffer solution (NaOH-Gly); ...

Embodiment 3

[0180] Embodiment 3, fusion protein OLB-26 as the kinetic constant determination of quenching enzyme

[0181] 1. Use the purified OLB-26 protein solution (protein concentration: 0.49 mg / mL) prepared in Step 4 of Example 1 as the solution to be tested, and detect the enzyme activity of the quenching enzyme. The buffer solution is pH 8.0, 0.1 mol / mL L of PBS buffer, the reaction temperature is 35 ° C, the signal molecule is 3-oxo-C8-HSL, the final concentration in the reaction system is 18 μmol / L, in the reaction 1, 2, 3, 5, 10, 15, 20 , Stop the enzymatic reaction at 30min.

[0182] By calculating the ratio of enzyme activity to reaction time, if the ratio of the enzyme does not change within a certain period of time, the enzymatic reaction within this period of time is a first-order reaction to determine the K m and V max optimal response time.

[0183] Based on the determined first order reaction time, the K of the fusion protein OLB-26 was determined m value and V max T...

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Abstract

The invention discloses a quorum-quenching enzyme OLB-26, and a coding gene and application thereof. The protein OLB-26 is a fusion protein disclosed as (a) or (b): (a) protein composed of amino acid sequence disclosed as Sequence 1 in the sequence table; or (b) protein derived from Sequence 1 in the sequence table with quorum-quenching enzyme functions, which is subjected to substitution and/or deletion and/or addition of one or more amino acid residues of the amino acid sequence disclosed as Sequence 1 in the sequence table. The experiment proves that the fusion protein OLB-26 disclosed by the invention has substrate specificity, can degrade long-chain and short-chain N-acylhomoserine lactones, has the specific activity of 7.59 U/mg as a quenching enzyme, is very stable within the pH range of 6.0-8.0, and can maintain more than 80% of enzyme activity; and the quorum-quenching enzyme OLB-26 has favorable heat stability, and the relative enzyme activity at 50 DEG C for 20 minutes is still 100%. The fusion protein OLB-26 disclosed by the invention can be used for preparing a novel biocontrol enzyme preparation.

Description

technical field [0001] The invention relates to a quorum sensing quenching enzyme OLB-26 and its coding gene and application. Background technique [0002] N-acetyl homoserine lactones (N-acyl homoserine lactones, AHLs) are a class of signaling molecules that widely exist in Gram-negative bacteria to regulate the quorum sensing system. These signal molecules are produced by the pathogenic bacteria themselves, can freely penetrate the cell wall and cell membrane of bacteria, and participate in the expression regulation of many pathogenic bacteria gene expression. When the signal molecule concentration reaches a certain threshold, the expression of pathogenic gene can be activated. Quorum quenching (QQ) is a class of non-secretory proteins that degrade signal molecules. This type of protein hydrolyzes the lactone bonds of AHLs molecules entering the cell, reducing its concentration, so that the pathogenic genes cannot expression, thereby preventing the infection of pathogenic...

Claims

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Application Information

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IPC IPC(8): C12N9/18C12N9/10C12N15/62C12N15/63C12N5/10C12N1/21C12N15/11C12R1/19
Inventor 周志刚张美超杨雅麟徐俐何夙旭李青余强
Owner FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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