Screening And Identification Of Alginolytic Bacteria And The Research Of The Alginate Lyases

Aginolytic bacteria can synthesize alginate lyase in order to decompose and assimilate alginate. Alginate lyases are able to catalyze the degradation of alginate. Alginate lyases have important application value in biological, chemical and other fields. Aginolytic bacteria are important sources of alginate lyase, which produces many kinds of alginate lyase and has potential economic value. In the past few decades, alginolytic bacteria were studied as the producer of alginate lyase. In recent years, along with the biological activity of alginate oligosaccharides have been found and the research of converting seaweed biomass to energy is increasingly further, the research on alginolytic bacteria and the alginate lyase will become a hot field.In this research, we used sodium alginate as the sole carbon source and have isolated two alginolytic bacteria that we named ZHO and ZH1from the compost of brown seeweed. Furthermore, we identified the characteristic of them. The results of identification showed that the strain ZHO is a slightly yellowish, rod, Gram-negative, mesophilic, neutrophils, aerobic bacterium, its16S rRNA gene sequence similarity with Sphingomonas sp. Al was98%, the optimum temperature and pH value of the growth are37℃and7.0respectively. According to the result of the identification experiment, we denominated ZHO as Sphingomonas sp. ZHO. The strain ZH1was a slightly yellowish, long rod, Gram-negative, mesophilic, acid-fast, aerobic bacterium. The strain ZHO had capable of degrading alginate fast. The strain ZH1was able to decompose alginate. Its16S rRNA gene sequence similarity with the Flavihumibacter petaseus was93%, the optimum temperature and pH value of the growth were42℃and5.5respectively. The content of G+C mol%was60%. On the basis of the significant differences demonstrated in the phenotypic and chemotaxonomic characteristics, it was suggested that the strain ZH1is classified as a novel species and the name Sphingobacteriales sp. ZH1is proposed.In this study, the properties of the alginate lyases that were produce by the strain ZHO and ZHlwere also studied. The results showed that the strain ZHO was able to produce intracellular endotype alginate lyase and the alginate lyase specifically acted on polyG and polyMG the optimum temperature is42℃, the optimum pH value was 7.5. the relative activity was0.615U/mg under the optimum conditions. The strain ZH1can also generate intracellular endotype alginate lyase and the alginate lyase acted on polyG and polyMG specifically. the optimum temperature was57℃and the optimum pH value was5.5, the relative activity was0.027U/mg under the optimum conditions.Through the experiment of optimizing the conditions of producing alginate lyase, we determined the components of optimal culture medium of the strain ZH1:1%(w/v)sodium alginate,1%(w/v) yeast extract,0.3%(w/v)MgSO4,4%(w/v)(NH4)2SO4,0.05%(w/v)Na2HPO4and0.05%(w/v)KH2PO4. The Optimum fermentation conditions were initial pH value was7.0, the cultural temperature was42℃, the speed of the cradle was180rpm, the cultural time was120h. The enzyme activity was106U/mL in the culture medium. The enzyme activity was increased by4.4times than before.In this research, we also made the research on cloning alginate lyase gene of the the strain ZHO, and adopted bioinformation methods to analysis the phylogeny, the conserved domains and three-dimensional structure of the alginate lyase gene. The results showed that the alginate lyase gene ZHO-aly sequence similarity with alginate metabolic gene of Sphingomonas sp. Al (GenBank:AB011415.1) was89%. the alginate lyase gene ZHO-aly could encoded two different alginate lyase.