Pseudomonas putida strain E16 16S Ribosomal RNA is identified molecularly as bacterial symbiont spesies of macroalga Sargassum polycystum which produce L-Asparaginase (E.C.3.5.1.1), levels of optimum fermentation time and substrate concentration are 48 hours and 10g/L respectively, 60-80% Fraction is The Highest L-Asparaginase Activity (2,03 IU/ml). Gel filtration chromatography sefadeks G-100 has a purity level of 11.505 compared to the crude extract which continued to CM-Sephadex C50 ion-exchange chromatography, the pure L-Asparaginase of fraction 28 have purity level of 197.112 compared to crude extract.