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Gene Cloning, Expression, Characterization and Application in Aquaculture of N-acyl Homoserine Lactone Lactonase

Author: ChenRuiDong
Tutor: ZhouZhiGang
School: Chinese Academy of Agricultural Sciences
Course: Animal Nutrition and Feed Science
Keywords: N-acyl homoserine esterase Clone Pichia pastoris Highly expressed Injection challenged Aquaculture
CLC: S948
Type: Master's thesis
Year: 2010
Downloads: 86
Quote: 1
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Abstract

N-acyl homoserine esterase is a class-specific degradation of the metal of the signaling molecules of the N-acyl homoserine lactones (AHLs) proteolytic enzyme widely exists in a variety of microorganisms. N-acyl homoserine esterase in recent years become a hot research tools enzyme (quenching quorum sensing strategies) as a novel antibacterial strategy. Mainly through the construction of transgenic plants, genetically modified bacteria to study its role in quenching quorum sensing mechanism, while genetically modified plants or genetically modified bacteria in the practical application of biological safety and effectiveness issues. The purpose of this study is the use of Pichia expression system efficient expression of the N-acyl homoserine lactone and enzyme form of enzyme preparations to combat aquaculture hydrophila gas Aeromonas caused disease. The papers by enrichment culture method using 3-oxo-C6-HSL as the sole carbon filter medium from Tianjin the Wuqing pond sediment isolated a strain of producing N-acyl homoserine esterase, named B546, identified by 16s rDNA Bacillus. Through the design of the species N-acyl homoserine esterase degenerate primers, successful cloning of the N-acyl homoserine esterase gene aiiAB546. aiiAB546 fragment size of 753 bp, encoding 250 amino acids, and the predicted encoded protein molecular weight of 28.14 kDa, an isoelectric point of 4.64, one nitrogen glycosylation point (Asn-Ser-Thr) predicted in the N-terminus of the protein. The aiiAB546 the connection pPIC9 expression vector, transformed Pichia pastoris GS115 competent cells and successfully expressed. Efficient expression of 13 strains screened by Agrobacterium tumefaciens KYC 55 active plate method. 3-oxo-C8-HSL as substrate, the strains in shake flask level expression of N-acyl homoserine esterase activity of 27.1 ± 3.2 U / mL, 3.7 L fermenter high density fermentation of recombinant proteins The expression levels of up to 3,558.4 ± 81.3 U / mL, 0.24 U / mL E. coli expression of the enzyme activity only. Pichia significantly increased the expression amount of N-acyl homoserine esterase to achieve efficient expression of the target protein. Pichia yeast expression of the recombinant protein was purified by ammonium sulfate precipitation and anion column, a single band of 33.6 kDa. For the purpose of proteins identified by mass spectrometry, Endo H treatment to verify the presence of nitrogen glycosylation. The purified recombinant AiiAB546 optimal pH and temperature were 8.0 and 20 ° C, in the range of pH 6.5-8.5, the enzyme activity can be maintained at more than 80% at 0 ° C, 20 ° C to 37 ° C, AiiAB546 with 60% of the enzyme activity. AiiAB546 having good pH and temperature stability. For 60 min in the range of pH 6.5-12.0, the activity of more than 100%, indicating the AiiAB546 is very stable in neutral and alkaline conditions. AiiAB546 treated at 60 ° C for 30 min, the activity of essentially unchanged, 70 ° C for 15 min, the remaining 90% of the enzyme activity, AiiAB546 remained very stable at 60 ° C and 70 ° C conditions. AiiAB546 having a very good anti-trypsin, α-chymotrypsin, subtilisin proteases, collagenase and the capacity of the alkaline protease, AiiAB546 processed a total of 60 min with the above-mentioned protease activity were greater than 100%. In addition, most of the ion pairs AiiAB546 inhibition. The Aeromonas bacteria AiiAB546 hydrophila coinjection the koi can significantly reduce hydrophila Aeromonas the bacteria lethal koi average cumulative mortality of 25%, delay Koi median lethal time of 18 h. Alone to inject AiiAB546, koi survive in good condition after a long period of observation. According to the test results to predict the AiiAB546 harmless to fish and can reduce Aeromonas hydrophila lethal capacity, the test provides an important test data for the study of potential mechanisms. This test is not only highly expressed in Pichia pastoris success of AiiAB546, get lot of AiiAB546 become possible to make low-cost vivo injection through in the form of the enzyme preparation successfully attenuation hydrophila Aeromonas lethal in Koi . Knowledge, this is the first time high-level expression in Pichia pastoris AiiAB546 by injection AiiAB546 attenuation of the virulence of Aeromonas hydrophila. This test has the potential antibacterial Strategy aquaculture application value.

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