Study on metabolism and biosynthesis of paralytic shellfish toxins using toxim-producing bacteria

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PSP toxins are toxins produced by toxic dinoflagellates such as Alexandrium tamarense. On the contrary, we suggested that toxins originate from endocellular bacteria of dinoflagellates, based on the facts that endocellular bacteria of A. tamarense produce saxitoxins, though the productivity is small. We propose here the hypothesis that bacteria produce precursor of toxins which is changed to toxins in dinoflagellates, Based on the hypothesis, we tried to find the precursor substance in bacterial. First of all, the specific antibody against saxitoxins which detects substance(s) possessing ligand with saxitoxin structure, by development of specific antibody against saxitoxin with the antigen utilizing the reaction between toxins and thiols. Western blot analysis of bacterial proteins by the antibody showed the protein bands stained by the antibody, indicating that these proteins possess ligands with saxitoxin structure. Then, the bacterial protein fraction was digested with nonspecific protease to obtain the digested protein fragments. ELISA using the antibody showed that low molecular fragment in the hydrolyzate reacts with the antibody. Trial to isolate the ELISA-positive substance showed the difficulty to isolate the substance, because of contamination of many impurities contaminated in the hydrolyzed. On the other hand, the substance was found to be effectively isolated, just in one step, when immunoprecipitation using the antibody was applied. Isolation of the substance for structure identification is on the way.