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AHLs are signaling molecules produced by Gram-negative bacterium during their proliferation. These molecules are closely related with bacteria pathogenicity. AiiA protein, functioning as a intracellular lactonase, hydrolyses AHLs at lactone loop in the molecule to reduce its biological activity and also dramatically reduces pathogenicity of bacterium at the same time. In this study, DNA fragment encoding aiiA gene was amplified from the plasmid derived from Bacillus thuringiensis by PCR. Expression vector, pET29a-aiiA, was constructed and transformed into bacteria strain of E. coli BL21 (DE3) for expression of AiiA protein. After 25 h induction with 0.8 mmol/L IPTG at 20 degrees C, the protein AiiA expressed in soluble form by recombinant strain reached about 54.4 microg/mL. The recombinant protein was purified with Ni-affinity chromatography. Biological activity analysis showed that AiiA protein had a capability to hydrolyse AHLs, and strong antimicrobial activity for Eriwinia carotovora.

Objective: Amorphophallus soft rot disease, caused by Erwinia carotovora, affects Amorphophallus industry development. We identified and characterized protein APn5 against Erwinia carotovora, isolated from Bacillus subtilis strain BSn5., Methods: Protein APn5 was purified from BSn5 culture by ammonium sulfate precipitation with 30% relative saturation and ultrafiltration. Inhibition activity was tested by agar well diffusion assay. Protein APn5 was treated at different temperatures, pH conditions and proteinase. The growth curve of BSn5 was examined; meanwhile, the inhibition activity of supernatant of BSn5 culture in different growth phase was tested. Amino acid sequence of protein APn5 was analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) and quadrupole-time-of-flight (Q-TOF)., Results: Protein APn5 showed a narrow inhibition spectrum, mainly strongly inhibiting the growth of a few plant pathogenic bacteria. Protein APn5 was sensitive to high temperature and alkaline pH, and partial sensitive to trypsin, proteinase K and pronase E. During strain BSn5 growth phase, the inhibition activity was unstable, which would gradually lose on stationary phase and disappeared finally., Conclusion: On the basis of the difference on inhibition spectrum and characteristics, protein APn5 was suggested as a novel antimicrobial protein.

An endophytic antagonistic bacterium was isolated from Amorphophallus konjac calli. In order to identify this bacterium, 16S rDNA was amplified and partially sequenced. Sequence comparison showed that this sequence has the highest similarity to that in Bacillus subtilis, with 99.0% identities. That demonstrated this bacterium belongs to Bacillus subtili , named BSn5. The extracted extracellular protein from strain BSn5 had antibacterial activity against Erwinia carotovora subp. carotovora, which was unstable after heated, sensitive to proteinase K and resistant to trypsin. There was only a 31.6kDa protein component as by SDS-PAGE detection. Nondenaturing polyacrylaminde gel was used to purify this protein. The purified 31.6kDa protein exhibited inhibitory activity against Erwinia carotovora subp. carotovora. This protein is different from all known metabolites from Bacillus subtilis, suggesting that it may be a novel antibacterial protein.