Expression Of AiiA Gene From Bacillus Thuringiensis And Immobilization Of AiiA Enzyme On Functionalized Multi-walled Carbon Nanotubes

The emergence of superbugs that resist to most available antibiotics reinforces the need for the development of new antibiotics and novel strategies against microbial pathogens. The finding that many pathogens rely on quorum sensing to co-ordinate gene expression for infection and survival confers quorum quenching to a promising disease control strategy. Here we cloned and expressed aiiA (coded AHL-lactonase that hydrolyzes quorum sensing signal molecule N-acyl-homoserine lactones (AHLs) into inactive products), and immobilized the derived AHL-lactonase onto functionalized multi-walled carbon nanotubes to improve the practical application value of enzyme.Gene aiiA was amplified by PCR from Bacillus thuringiensis BF1 and then inserted into the expression vector pET-28a and finally transformed into Escherichia coli BL21.33kDa molecular weight of the recombinant enzyme AiiA containing two six-histidine tags at the N-terminus or C-terminus was overexpressed in E. coli as inclusion bodies (IBs). The IBs was solubilized by30%N-Lauroylsarcosine sodium salt and refolded by0.2%PEG6000,0.2mM GSSH, lmM GSH and5M L-Arginine. By AHL bioassay with Chromobacterium violaceum CV026, the result showed the refolded AiiA kept the AHL-degrading activity.The multi-walled carbon nanotubes possessing low diffusion resistance and high surface/volume ratio were modified with Nα,Nα-bis(carboxymethyl)-L-lysine hydrate (ANTA) as enzyme immobilization surpporters. The results confirmed that MWCNT-ANTA-Co2+-AiiA conjugates retained the native AHL-degrading activity.