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《Journal of Fisheries of China》 2011-11
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Characteristics of quenching enzyme AiiO-AIO6 and its effect on Aeromonas hydrophila virulence factors expression

ZHANG Mei-chao1,CAO Ya-nan1,YAO Bin1,BAI Dong-qing2,ZHOU Zhi-gang1(1.Key Laboratory for Feed Biotechnology of the Ministry of Agriculture,Feed Research Institute, Chinese Academy of Agricultural Sciences,Beijing 100081,China; 2.Department of Fisheries Science,Tianjin Agricultural College,Tianjin 300384,China)  
The prokaryotic expression vector pET28a/aiiO-AIO6 was constructed by inserting the aiiO-AIO6 gene into expression vector pET28a and transformed into E.coli.Subsequently,the recombinants with activity of N-acyl homoserine lactonase were investigated.After purification with nickel column,enzymatic characteristic of the target protein was researched.Moreover,the effects of purified enzyme solution on the expression of Hemolysin,Cytotonic enterotoxin,Extracellular protease,Serine protease,Phospholipase A1,ea tl five virulence factors from Aeromonas hydrophila ATCC7966 were detected by real-time quantitative PCR.The purified AiiO-AIO6 showed that the N-acyl-homoserine lactonase had optimal pH and temperature at pH 8.0 and 30 ℃,respectively.The enzyme was stable between pH 6.0 and 11.0,retained over 65% enzyme activity at 80 ℃ for 30 min.It resisted various neutral proteases and chemical reagents.The fusion protein can hydrolyze many N-acyl homoserine lactones substrates.With N-(3-oxo-octanoyl)-L-homoserine lactone as substrate,the Km value of AiiO-AIO6 was 0.015 mmol/L and specific activity was 583.33 U/mg.The expression amount of five virulence factors of A.hydrophila had down-regulated trend obviously(P0.05)at 8 h and 12 h.with adding the purified enzyme liquid for the expression amount of five virulence factors In this study the optimal pH and temperature of AiiO-AIO6 were obtained by detecting enzymatic property,and AiiO-AIO6 could inhibit A.hydrophila virulence factors expression indirectly by degrading signal molecules,which established a theoretical foundation for the application in aquaculture environment and biological control of pathogenic A.hydrophila.
【Fund】: 科技部农业科技成果转化资金项目(2010GB23260591);; 中国农业科学院饲料研究所所长基金(2009);; 中国博士后科学基金(20090450472)
【CateGory Index】: S941
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