Study On Tetrodotoxin And Insecticidal Metabolites From Two Marine Microbes

Living in marine environment of special conditions,marine microbes are able to create unique metabolites exhibiting special biological activities compared with 'terrestrial' microbes.Using microbial fermentation techniques to produce bioactive substances with high value,is more friendly to environment,of shorter cycle of production,more convenient to get raw material and easier for massive production,therefore considered to be an exciting and effective way to develop marine medicines and other industrious products.Tetrodotoxin(TTX)is a marine biotoxin with high value in medical research and clinical treatments.In this study,a strain of TTX-producing bacteria N141 was isolated from Fugu rubripes in Bohai Sea,China,identified as Bacillus fusiforms.TTX fermentation by N141 was carried out in 10 L fermenter,through which the curves of growth of N141,pH,DO and the yield of TTX were studied.And then the fermentation techniques for TTX production were applied in 500 L-scale fermenter.Our Results showed that marine bacteria N141 grew rapidly and produced TTX steadily in 10 L and 500 L fermenter by using the fermentation techniques in this study.We also found that TTX production by Bacillus fusiforms occurred during the stationary phase of growth,which suggests that TTX is a secondary metabolite of microbe.During the fermentation process,pH was stable after the stationary phase probably due to high content phosphate.And DO kept at a high value,at about 95%,indiacting that strain N141 costed little O₂ during the process of TTX biosynthesis.Crude toxin was isolated from fermentation broth by filtration,ultra-filtration,active carbon absorption,Sephdex G10 gel chromatography and freezing dryness,and then was identified by thin layer chromatogram(TLC),mouse bioassay,ELISA and LC-MS.Quantitative detection of microbial-origin TTX was performed by ELISA kit which is based on anti-tetrodotoxin monoclonal antibodies,showing that this method was quick,sensitive,and specific to determine TTX in fermentation broth of bacteria,offering an easy and reliable detection tool to research TTX-bearing microbes.Pursuing marine microbes of insecticidal activity and their bioactive metabolites and developing novel biopesticides were regarded as a promising research field.In this study,we screened 59 strains of marine actinomyces isolated from marine sponge,Hymeniacidon perleve and deep-sea sediment from South China Sea using Artemia Salina(brine shrimp)bioassay performed in 96-well plates as a preliminary screen and second screen.Fifteen strains killing above 70% population of A.salina were obtained,among which sponge-associated actinomycete S19 showed the highest insecticidal activity with 100%lethal rate against A.salina within 24 hours. We also found that strain S19 was able to kill larvae of Spodoptera exigua and nematodes. Through morphological,physiological,biochemical analysis and 16S rRNA gene sequence analysis,marine sponge actinomycete S19 was identified as Streptomyces griseus.However, strain S19,originated from marine sponge,Hymeniacidon perleve,is different from other reported strains of Streptomyces griseus in characteritics and bioacivities.Leavening of S19 was extracted by EtOAc after pretreatments.And then the bioactive extracts was isolated by flash dry-column chromatography of silica and preparative HPLC while using brine shrimp bioassay to test fractions.Finally,six pure compounds were obtained,all of which possessing potent bioactivties against A.salina.Preliminary toxicology studies on the fermentation broth and cells of S19 to rats and rabbits indicated that sponge-associated actinomycete S19 might be safe to mammal animals and human beings.Our results suggest that marine actinomycetes might be an exciting resource for novel and potent insecticidal compounds with environmental compatibility and that strain S19 has potential as a new marine-microbial pesticide.