High yield expression of an AHL-lactonase from Bacillus sp B546 in Pichia pastoris and its application to reduce Aeromonas hydrophila mortality in aquaculture

文献类型： 外文期刊

第一作者： Chen, Ruidong

作者： Chen, Ruidong;Zhou, Zhigang;Cao, Yanan;Bai, Yingguo;Yao, Bin

作者机构：

期刊名称：MICROBIAL CELL FACTORIES （ 影响因子：5.328； 五年影响因子：5.588 ）

ISSN： 1475-2859

年卷期： 2010 年 9 卷

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收录情况： SCI

摘要： Background: Aeromonas hydrophila is a serious pathogen and can cause hemorrhagic septicemia in fish. To control this disease, antibiotics and chemicals are widely used which can consequently result in "superbugs" and chemical accumulation in the food chain. Though vaccine against A. hydrophila is available, its use is limited due to multiple serotypes of this pathogen and problems of safety and efficacy. Another problem with vaccination is the ability to apply it to small fish especially in high numbers. In this study, we tried a new way to attenuate the A. hydrophila infection by using a quorum quenching strategy with a recombinant AHL-lactonase expressed in Pichia pastoris. Results: The AHL-lactonase (AiiA(B546)) from Bacillus sp. B546 was produced extracellularly in P. pastoris with a yield of 3,558.4 +/- 81.3 U/mL in a 3.7-L fermenter when using 3-oxo-C8-HSL as the substrate. After purification with a HiTrap Q Sepharose column, the recombinant homogenous protein showed a band of 33.6 kDa on SDS-PAGE, higher than the calculated molecular mass (28.14 kDa). Deglycosylation of AiiA(B546) with Endo H confirmed the occurrence of N-glycosylation. The purified recombinant AiiA(B546) showed optimal activity at pH 8.0 and 20 degrees C, exhibited excellent stability at pH 8.0-12.0 and thermal stability at 70 degrees C, was firstly confirmed to be significantly protease-resistant, and had wide substrate specificity. In application test, when co-injected with A. hydrophila in common carp, recombinant AiiA(B546) decreased the mortality rate and delayed the mortality time of fish. Conclusions: Our results not only indicate the possibility of mass-production of AHL-lactonase at low cost, but also open 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.

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