Gene Cloning, Expression, Characterization And Application In Aquaculture Of N-acyl Homoserine Lactone Lactonase

N-Acyl homoserine lactonase, widely identified in a range of bacterial species, is a group of metal-hydrolases that can specially hydrolyze N-Acyl homoserine lactone. In recent years, N-Acyl homoserine lactone lactonase has been attracted much attention as a new enzyme in antimicrobial therapeutics (quorum quenching strategy). Transgenic plants and bacteria are used to study the mechanism of quorum quenching; however, application of transgenic plants and bacteria is limited due to problems of safety and efficacy. The objective of this study was to overexpress AHL-lactonase in Pichia pastoris and attenuate the Aeromonas hydrophila virulence with AHL-lactonase to control A. hydrophila-related diseases.A strain, denoted as B546, producing AHL-lactonase was isolated from the mud of a fish pond at Wuqing, Tianjin, China using minimal medium containing 3-oxo-C6-HSL as the sole carbon source and was identified as Bacillus sp. by its 16S rDNA sequence. Based on the conserved amino acid sequences of AHL-lactonases from Bacillus sp., a degenerate primer set was designed. Using the degenerate primers, the full-length 753-bp AHL-lactonase gene, aiiAB546, was cloned from Bacillus sp. B546. aiiAB546 encoded a 250-amino acid polypeptide with a calculated molecular mass of 28.14 kDa and a pI of 4.64. One potential N-glycosylation site (Asn-Ser-Thr) was identified at the N terminus.The recombinant plasmid pPIC9-aiiAB546 was constructed and transformed into P. pastoris GS115 competent cells. Positive transformants were screened using well-diffusion assays with reporter strain Agrobacterium tumefaciens KYC 55, and clone 13 with highest enzyme activity was selected for further assay. In the shake-flask level, AHL-lactonase activity was up to 27.1±3.2 U/mL. In the 3.7 L fermenter, the expression level of recombinant AiiAB546 reached 3,558.4±81.3 U/mL after induction, which was significantly higher than that expressed in Escherichia coli (0.24 U/mL). Expression of aiiAB546 in P. pastoris enhanced the production of AiiAB546 significantly.Recombinant AiiAB546 in P. pastoris was purified to electrophoretic homogeneity by ammonium sulfate precipitation and anion exchange chromatography. The molecular weight of the purified recombinant AiiAB546 was 33.6 kDa. Further LC-ESI-MS/MS analysis and deglycosylation of AiiA with Endo H confirmed the identity of the objective band and the occurrence of N-glycosylation. Purified recombinant AiiAB546 showed optimal activity at pH 8.0 and 20°C, retained more than 80% of the maximum activity at pH 6.5–8.5. At 0 and 20–37°C, the enzyme maintained more than 60% of the highest activity. AiiAB546 exhibited excellent pH stability, retaining more than 100% activity after pre-incubation at 37°C, pH 6.5–12.0 for 1 h. AiiAB546 was thermostable at 60°C and 70°C, retaining more than 100% and 90% of the initial activity after pre-incubation at 60°C for 30 min and 70°C for 15 min, respectively. The purified recombinant AiiAB546 was protease-resistant. After incubation with trypsin, subtilisin A, collagenase and proleather at 37°C for 60 min, the enzyme retained almost all of the enzymatic activity. In addition, AiiAB546 was also resistant to many metal ions.Co-injection of AiiAB546 and A. hydrophila decreased the mortality rate of common carp by nearly 25%. Compared with the fish injected with A. hydrophila, the LT50 of the fish with co-injection of AiiAB546 and A. hydrophila was delayed 18 h. After injection with AiiAB546 alone, the fish displayed no signs of stress or disease and no mortalities were observed. The results indicate that AiiAB546 is safe for carp and can attenuate A. hydrophila virulence, and provide more novel data for further study.This study not only achieves overexpression of AiiAB546 in P. pastoris and makes it possible to mass-produce AHL-lactonase at low cost, but also opens up a promising foreground of application of AHL-lactonase in fish to control A. hydrophila disease by regulating its virulence. To our knowledge, this is the first report on heterologous expression of AHL-lactonase in P. pastoris and attenuating A. hydrophila virulence by co-injection with AHL-lactonase. This study has potential values for antimicrobial strategies and aquaculture applications.