Screening And Identification Of Active Substances And Lysozyme Of Marine Microorganisms

Marine organisms live in a special environment with low temperature,high salt,high pressure and oligotrophic conditions.Because of the special living environment,microbes in the ocean are different in terms of physiological traits and genetic background.So they can produce novel active substances.The development of marine microbes has great potential.In this study,samples were taken from Dalian coastal sea water,sea mud and sea cucumber farming circles and screened out the marine microbes with biologically active substances or producing lysozyme with high activity.Some microorganisms were identified and the active substances were isolated and analyzed.The main results were as follows:(1)In this study,samples were taken from Dalian coastal sea water,sea mud and 38 marine actinomycetes were isolated.Two indicator bacteria,Staphylococcus aureus,Micrococcus lysodeikticus were chosen for screening.As a result,nine strains were isolated.Further test found that two strains of marine actinomycetes showed high antibacterial activity,named HS-B31 and HS-B34.(2)Both strains HS-B31 and HS-B34 were identified by 16S rDNA sequencing.The genomic DNA of two strains was extracted,respectively.The specific gene fragments were obtained by PCR amplification.Based on 16S rDNA sequence,blast and phylogenetic analysis,the results showed that the HS-B31(Streptomyces sundarbansensis)and HS-B34(Streptomyces chumphonensis)belong to different Streptomyces species.(3)The fermentation supernatants of the two strains HS-B31 and HS-B34 were extracted by ethyl acetate.The crude extracts were concentrated to obtain the crude substances,named as B31 and B34.The analysis was done to isolate active substances by thin-layer chromatography.Five components(B31-1?B31-2?B31-3?B31-4 and B31-5)were isolated from B31,and three components(B34-1?B34-2 and B34-3)were isolated from B34.The antibacterial activity of the components were measured by the filter paper method.The results showed that the compounds B31-3 and B34-3 have stronger antibacterial activity against the gram-positive bacterium of Staphylococcus aureus and the gram-negative bacterium of Vibrio parahaemolyticus.(4)The second part of this study was focused on screening marine strains with the ability to produce lysozyme.First,1540 bacteria were isolated from samples which were taken from Dalian coastal sea water,sea mud and sea cucumber farming circles.Four indicator bacteria,Staphylococcus aureus,Micrococcus lysodeikticus,Vibrio parahaemolyticus and Pseudomonas aeruginoysa were chosen for screening.And 64 strains were screened out after subculture.Further test was done by using the filter paper method and 28 bacterial strains with high antibacterial activity were identified.Then,the ability of these 28 strains to produce lysozyme was determined.The results showed that the strain HS-C261 had a high yield.The yield of lysozyme was 418.4 U/mL.The strain HS-C261 was identified by 16S rDNA sequencing.The genomic DNA of the strain was extracted.The specific gene fragment was obtained by PCR amplification.Based on 16S rDNA sequence,blast and phylogenetic analysis,the results showed that the strain HS-C261 is Bacillus altitudinis.