Purification, characterization and sequence analysis of Alginate Lyase Gene Bacterium

Bacterial isolate MAK4 were isolated from brown algae collected from coastal area of Red sea, Hurghada, Egypt. And confirmed for alginate lyase production by halo formation around the colonies after flooding with cetylpyridenium chloride or Gram's iodine. The isolate MAK4 were identified according to morphological, biochemical and phylogenetic identification through 16s rRNA. The 16s rRNA gene was sequenced and deposited to Gene Bank. The bacterial isolate was identified as Martelella sp. Strain was named MAK4. It is aerobic, mesophilic, Gram -ve, none spore forming none motile, rod-shaped organism and produces catalase and oxidase. PCR was performed for alginate lyase gene using two pairs of degenerative primers. The alginate lyase gene was sequenced and also deposited to the Gene Bank. The alginate lyase enzyme were isolated and purified from the culture medium by ammonium sulfate precipitation, sephadex G-100 and DEAE-Cellulose chromatography. The isolated enzyme has specific activity 62.9 u/mg, 6.15 purification folds and has 35 kDa molecular weight. The alginate lyase enzyme showed highest activity at 37 degrees C and pH 7. The enzyme was stable over pH range 6-9 and retained 80% of activity after incubation at 40 degrees C for 90 minutes. The enzyme was active in absence of metal ions but the activity was enhanced by addition of NaCl, KCl and Ca+2. And the activity was lost with addition of EDTA. The enzyme activity was strongly decreased with Cu+2, Zn+2, Co+2, Hg+2 and Mn+2 but the enzyme activity is slightly affected with Mg+2, Fe+2 and Ba+2. Novel bifunctional alginate lyase could be used in eradication of resistant bacterial biofilm in clinical samples and production of alginate oligosaccharides in industry.