Isolation Of Alginate-Degrading Bacteria, Fermation And Some Properties Of Alginate Lyase

Based on many enrichments and domestications in liquid medium containing alginate as unique carbon source,the strain X8,an effectively producing alginate lyase producer,was isolated and selected from decaying part of Sargassum fusiforme.According to physiological and biochemical properties and the analysis of sequence of 16SrDNA,strain X8 was identified as Acinetobacter junii.There are many factors that influence the alginate lyase production of X8,so it’s enzyme production under liquid fermentation was investigated.According to results of single factor experiments and orthogonal experiments of fermentation medium,the optimized liquid fermentation medium（w/v） were:0.6%alginate,0.7%peptone,1.5% NaCl,0.3%K₂HPO₄·3H₂O.The optimized fermentation conditions were:initial pH value 7.0,culture temperature 25℃,rotation speed 150 r/min,culture time16 h,liquid volume 50mL in 250mL flask under which condition the yield of alginate lyase was up to 157.32 U/mL,which was 2.59 times than that under the ordinary conditions.During the progress of isolation-purification,the crude alginate lyase produced by the strain X8 is extacted by ammonium sulfate precipitation,purified by DEAE-Sepharose Fast Flow chromatography, and Sephacryl S-100 gel filtration.Molecular weights of the enzyme by SDS-PAGE were estimated to be 32 kDa.The recovery rate and purification value of this enzyme was 22.74%and 5.17,respectively.Studies on some basic properties of this alginate lyase shows that the optimal temperature and pH of enzyme degredation were respectively 30℃and 8.8,under which condition the K_m and V_max were 6.68 mg/min and 960.89μg/mL/min respectively.The stable temperature in 30 min and 5 days were below 40℃and 4℃respectively,and the stable pH value ranges from 6.9 to 9.8.Finally,the enzyme activity was activated by Mg²⁺、K⁺,while it was inhibited by Mn²⁺、Ca²⁺、Fe²⁺、Cu²⁺、Zn²⁺ and Pb²⁺,of which Cu²⁺、Zn²⁺ and Pb²⁺ gave complete suppression.