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N-acylhomoserine lactonase QsdA-RH5 with substrate specificity and coding gene and application thereof

A technology of acyl homoserine lactone and coding gene, which is applied in the field of substrate-specific N-acyl homoserine lactonase QsdA-RH5 and its coding gene and application, which can solve the problems of normal regulation and lack of substrate specificity. Oneness and other issues

Inactive Publication Date: 2013-03-13
FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The currently reported N-acyl homoserine lactonases have a wide range of substrate specificity and lack of substrate specificity, which affects the normal regulation of non-pathogenic strains during application

Method used

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  • N-acylhomoserine lactonase QsdA-RH5 with substrate specificity and coding gene and application thereof
  • N-acylhomoserine lactonase QsdA-RH5 with substrate specificity and coding gene and application thereof
  • N-acylhomoserine lactonase QsdA-RH5 with substrate specificity and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1, acquisition of Rhodococcus erythropolis QsdA-RH5 protein and its coding gene

[0059] Inoculate Rhodococcus erythropolis (Rhodococcus erythropolis) BFXJ-1 in LB liquid medium, incubate at 30°C for 24 hours, take 1ml of the bacterial liquid, and centrifuge at 10,000 rpm for 2 minutes to collect the bacterial cells, extract genomic DNA, and use primers Qs dA-F: ​​5′ -AGTTCAGTACAAACCGTTCGTG-3′ and Qs dA-R: 5′-TCAGCTCTCGAAGTACCG-3′ were amplified by PCR; PCR reaction conditions were 95°C for 5min, 94°C for 30sec, 55°C for 30sec, 72°C for 1min, 30 cycles, 72°C for 10min Perform agarose gel electrophoresis on the obtained PCR amplification product, recover and purify a band of about 1000 bp, connect it to the vector pEASY-T3, and sequence the obtained recombinant plasmid. The results show that the recombinant plasmid is contained in the vector pEASY-T3 The DNA shown in the sequence 2 of the sequence listing from the 4th to the 969th nucleotide at the 5' end was ...

Embodiment 2

[0061] Embodiment 2, the preparation of QsdA-RH5 protein

[0062] 1. Construction of recombinant expression vector

[0063] 1. Synthesize the DNA shown in Sequence 3 of the Sequence Listing, which contains protective bases and enzyme recognition sequences at both ends.

[0064] 2. Using the DNA synthesized in step 1 as a template, perform PCR amplification with a primer pair composed of QsdA 2-F and QsdA 2-R to obtain a PCR amplification product.

[0065] QsdA 2-F: 5′-CG GAATTC ATGAGTTCAGTACAAACCGTTCGTG-3' (the underlined base is the recognition sequence of EcoR I digestion);

[0066] QsdA 2-R: 5′-AA GCGGCCGC TCAGCTCTCGAAGTACCG-3' (the underlined base is the recognition sequence of Not I restriction enzyme).

[0067] 2. Digest the PCR amplified product of step 1 with restriction endonucleases EcoR I and Not I, and recover the digested product.

[0068] 3. Digest the vector pET-28a(+) with restriction endonucleases EcoR I and Not I to recover the vector backbone (about 5...

Embodiment 3

[0105] Example 3, Characterization of QsdA-RH5 protein as N-acyl homoserine lactonase

[0106] 1. Optimal pH

[0107] Method: Prepare the QsdA-RH5 protein solution obtained in step 3 of Example 2 with 0.1mol / L PBS buffer (pH8.0) to prepare a solution with a total protein concentration of 1.0mg / mL (as the solution to be tested), according to The method in step 4 of Example 2 was used to detect N-acyl homoserine lactonase activity. The difference was that the 0.1mol / L PBS buffer solution (pH8.0) in the reaction system used the following buffer solutions with different pH values:

[0108] 0.1mol / L McIlvaine buffer (citric acid) at pH 4.0, 5.0, 6.0;

[0109] 0.1mol / L PBS buffer (PBS) with pH 5.0, 6.0, 7.0, 7.5, 8.0;

[0110] Tris-HCl buffer (Tris-HCl) at pH 8.0, 8.5, 9.0;

[0111] 0.1mol / L glycine-NaOH buffer (Gly-NaOH) with pH 9.0, 10.0, 11.0.

[0112] When 0.1mol / L PBS buffer solution (pH8.0) is used in the reaction system, the enzyme activity of the solution to be tested is...

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Abstract

The invention discloses N-acylhomoserine lactonase QsdA-RH5 with substrate specificity and a coding gene and application thereof. The protein QsdA-RH5 is (a) protein consisting of the amino acid sequences shown by sequence 1 in a sequence table, or (b) protein obtained by substituting and / or losing and / or adding one or multiple amino acid residues of the amino acid sequence of the sequence 1 in the sequence table, having the function of N-acylhomoserine lactonase and derived from the sequence 1 in the sequence table. Experiments prove that the QsdA-RH5 protein provided by the invention has substrate specificity and can degrade various N-acylhomoserine lactone without substituent groups; the specific activity of the QsdA-RH5 protein serving as N-acylhomoserine lactonase is 292.83U / mg and is stable in a pH range of 4.0-12.0, and more than 90% of enzyme activity can be maintained; and moreover, the thermal stability is better, and relative enzyme activity after keeping temperature at 70 DEG C for 10 minutes is still 100%. The QsdA-RH5 protein provided by the invention can be used for preparing a novel bio-control enzyme preparation.

Description

technical field [0001] The invention relates to a substrate-specific N-acyl homoserine lactonase QsdA-RH5, its coding gene and application. Background technique [0002] In 1994, Fuqua et al. first discovered that during the growth process of the microbial population, due to the increase of the population density, its physiological and biochemical characteristics changed, and some characteristics that a small number of bacteria or a single bacteria did not have appeared. When the population density reaches the threshold, the secreted autoinducers (AI) also reach a certain concentration. After AI binds to the receptor, it affects the expression of specific genes through signal transduction and regulates the physiological characteristics of the population. This is the so-called quorum-sensing (QS). [0003] With the increasing drug resistance of pathogens, especially multi-drug resistance, the discovery of new targets of antibacterial drugs and the development of new antibact...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/18C12N15/55C12N15/63C12N5/10C12N1/21C12N15/11C12P13/06C12R1/19C12R1/01
Inventor 周志刚张美超杨雅麟徐俐李青姚斌于会民何夙旭
Owner FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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