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Heat-resisting N-acyl homoserine lactonase AiiA-AIO6 with high specific activity as well as coding gene and application thereof

An acyl homoserine lactone, coding technology, applied in DNA/RNA fragments, applications, genetic engineering, etc., can solve the problems of limited application value, poor heat resistance, low specific activity, etc., and achieve good degradation of various substrates. Ability, strong protease resistance, high specific activity effect

Active Publication Date: 2011-10-12
北京挑战生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The currently reported N-acyl homoserine lactonase has low specific activity and poor heat resistance, which limits its application value

Method used

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  • Heat-resisting N-acyl homoserine lactonase AiiA-AIO6 with high specific activity as well as coding gene and application thereof
  • Heat-resisting N-acyl homoserine lactonase AiiA-AIO6 with high specific activity as well as coding gene and application thereof
  • Heat-resisting N-acyl homoserine lactonase AiiA-AIO6 with high specific activity as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1, the separation and identification of Pallidum pallidus AIO6

[0059] 1. Isolation of bacteria

[0060] Soil samples were resuspended in sterile water at a ratio of 1:10 (soil:water) and diluted to 10 with sterile water. -8 Afterwards, spread on LB plates (with 5 μM C6-HSL and 3-oxo-C6-HSL), and after culturing at 25°C for 5 days, the colonies were picked into liquid LB and cultured at 25°C for 3 days, centrifuged at 10,000rpm for 1min to collect the cells, and weighed Suspended in 0.1 mol / L PBS buffer (pH 8.0), ultrasonicated and centrifuged at 12000 rpm for 5 min, the supernatant was collected to detect N-acyl homoserine lactonase activity.

[0061] A strain with enzyme activity was obtained, and the strain was named AIO6.

[0062] 2. Identification

[0063] The 16S rDNA of strain AIO6 was amplified and then sequenced, and the sequencing results were compared with the nucleotide sequence in the Genbank database, which proved that the strain AIO6 was Oc...

Embodiment 2

[0074] Embodiment 2, the preparation of recombinant bacteria

[0075] 1. Construction of recombinant expression vector

[0076] 1. Synthesize the DNA (AiiA-AIO6 gene) shown in sequence 2 of the sequence listing.

[0077] 2. Using the DNA synthesized in step 1 as a template, perform PCR amplification with a primer pair composed of A6-F and A6-R (the target sequence is shown in sequence 3 in the sequence listing) to obtain a PCR amplification product.

[0078] A6-F: 5′-CG GAATTC AAATCCCATGAAATCGAGACCAGTC-3' (the underlined part is the EcoR I restriction site);

[0079] A6-R: 5′-TTAA GCGGCCGC TCAGGCCGTGCAGTCG-3' (the underlined part is the Not I restriction site).

[0080] 2. Digest the PCR amplified product of step 1 with restriction endonucleases EcoR I and Not I, and recover the digested product.

[0081] 3. Digest the vector pET-28a(+) with restriction endonucleases EcoR I and Not I to recover the vector backbone (about 5.4kp).

[0082] 4. Ligate the digested product ...

Embodiment 3

[0121] Embodiment 3, AiiA-AIO6 protein as the enzymatic property of N-acyl homoserine lactonase

[0122] 1. Optimal pH

[0123] The AiiA-AIO6 protein solution prepared in Example 2 is prepared into a 0.02mg / mL solution (as the solution to be tested) with 0.1mol / L PBS buffer (pH8.0) to carry out the activity of N-acyl homoserine lactonase Detection (in the activity detection, except the buffer solution, it is the same as Step 6 of Example 2 and 3).

[0124] Use the following buffers:

[0125] 0.1mol / L McIlvaine buffer solution of pH 4.0, 5.0, 6.0;

[0126] 0.1mol / L PBS buffer solution with pH 5.0, 6.0, 7.0, 7.5, 8.0;

[0127] Tris-HCl buffer at pH 8.0, 8.5, 9.0;

[0128] 0.1 mol / L glycine-NaOH buffer solution (NaOH-Gly) with pH 9.0, 10.0, 11.0.

[0129] When 0.1mol / L PBS buffer solution (pH8.0) is used, the enzyme activity of the solution to be tested is recorded as 100% (enzyme activity value is 877.04U / mL). For relative enzyme activities using various buffers, see ima...

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Abstract

The invention discloses a heat-resisting N-acyl homoserine lactonase AiiA-AIO6 with high specific activity as well as a coding gene and application thereof. A protein provided by the invention is (a) or (b): the protein (a) is composed of amino acid sequences shown as a sequence 1 in a sequence table; the protein (b) is obtained through substituting and / or lacking and / or adding one or more amino acid residues of amino acid sequences shown as the sequence 1, has the activity of the N-acyl homoserine lactonase and is derived from the sequence 1. The AiiA-AIO6 protein provided by the invention is a novel N-acyl homoserine lactonase and the N-acyl homoserine lactonase is heat-resisting, has high specific activity, higher protease resistance and better capability of degrading each substrate and can be used as a novel bio-control enzyme preparation.

Description

technical field [0001] The invention relates to a heat-resistant N-acyl homoserine lactonase AiiA-AIO6 with high specific activity and its coding gene and application. Background technique [0002] Quorum sensing (QS) is a regulatory mechanism of bacteria, which means that bacteria perceive the number of themselves or other bacteria in the surrounding environment by sensing the concentration of specific signal molecules, and adjust the expression of related genes to adapt to changes in the environment. Specific signaling molecules (also known as autoinducers). [0003] The typical QS system in Gram-negative bacteria includes the luxI gene responsible for signal generation, the response regulator luxR gene and the LuxR protein binding region luxbox. The most typical class of signaling molecules in the quorum sensing system of Gram-negative bacteria are N-acyl-homoserine lactones (AHLs) compounds. Bacterial cells exchange information by producing AHLs to regulate the express...

Claims

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Application Information

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IPC IPC(8): C12N9/18C12N15/55C12N15/63C12N5/10C12N1/21C12N15/11A01N63/02A01P1/00C12R1/01C12R1/91C12R1/19
Inventor 周志刚张美超曹雅男何夙旭毛玮张宇婷霍凤敏徐俐
Owner 北京挑战生物技术有限公司
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