Isolation And Identification Of Alginate-degrading Bacteria And The Research Of The Alginate Lyase

Alginate is a kind of unbranched polysaccharide from brown algae cell wall, The ocean contains rich alginate resources. Alginate degradation bacteria can produce the alginate lyase, which is important source of alginate lyase. Alginate lyases have potential applications in the food industry, agriculture, medicine and bio-energy field, Therefore screening new alginate degradation bacteria and isolated alginate lyases have important practical significance.In this study, the16S rRNA sequence alignment,(G+C) mol%, bacterial fatty acids, physiological and biochemical characteristics, morphological characteristics and other indicators were used to identify strains. Strain SH-52is a rod-shaped, Gram-negative, anaerobic bacteria. Temperature range is20℃to45℃in the growth, the optimum growth temperature is30℃. It can be grown within the range of pH6-8.5, the optimum pH was7.5. It can be grown in the NaCl concentration range of1%-9%, the optimum concentration is3%NaCl. Sulfur and sodium thiosulfate sulfur strains can be reduced to H2S. Bacteria (G+C) mol%is44%. The major fatty acids is C15:Oiso, C17:Iw6c, C15:Iw6c, the C15:Oanteiso, C16:0and C17:0. Strain SH-52shares the highest16S rRNA gene sequence similarity with Sunxiuqinia elliptica DQHS-47. Morphology and aerobic also were big differences between the strain SH-52with Sunxiuqinia, mode strain and other bacteria of the genus, it is recommended that strain SH-52is a new genus and species, named Marinicatena alginatilytica.In order to determine the optimal bacterial fermentation conditions for enzyme production, we selected these conditions to optimize,they were temperature, pH, the concentration of carbon source, nitrogen source types, metal ions. The optimum fermentation conditions was35℃, the medium pH8. The optimization of the carbon source, the nitrogen source, and the metal ion concentration was0.5%(W/V) Alginate,0.32%(W/V) Yeast extract,2%(W/V) NaCl,0.01%(W/V) CaCl2. The enzyme production optimized medium can make the bacterial enzyme production12hours in advance, maximal activities of16U/ml,2times higher than before. Ammonium sulfate fractionation, ion exchange chromatography and gel filtration chromatography were used to purify alginate lyase from alginate degradation bacteria SH-52. And we finally got three alginate lyases:SHA-1, SHA-2and SHA-3. We got0.84mg SHA-1protein, final enzyme yield was108U, the recovery rate was6.2%, specific activity is128.5U/mg, and27.8times higher then crude. We got1.21mg SHA-2protein, final enzyme yield was119.7U, the recovery rate was6.8%, specific activity is98.43U/mg and22times higher then crude. We got0.43mg SHA-3protein, final enzyme yield wasl97U, the recovery rate was5.9%, specific activity is458U/mg and32.2times higher then crude.Protein molecular weight was determined by SDS-PAGE., the molecular weight of SHA-1was about60.2KDa, the molecular weight of SHA-2was about42.9KDa, the molecular weight of SHA-3about51.3KDaThe optimum temperature, the optimum pH value, the influence of metal ions, the substrate specificity and the shear type characteristics of alginate lyase SHA-1, SHA-2and SHA-3were studied. The study found that SHA-1was an exotype polyM specific alginate lyase, the optimum temperature was50℃, the optimum pH was8.5, remained stable below50℃. SHA-2was an endotype polyG specific alginate lyase, the optimum temperature was40℃, the optimum pH was between6-6.5, remained stable below45℃. SHA-3was an exotype polyG specific alginate lyase, the optimum temperature was40℃, the optimum pH was between6.5-7, remained stable below50℃.