Purification and Characterization of Alginate Lyase from Locally Isolated Marine Pseudomonas Stutzeri MSEA04
An alginate lyase with high specifc enzyme activity was purifed from Pseudomonas stutzeri MSEA04, isolated from marine brown algae. The alginate lyase was purifed by precipitation with ammonium sulphate, acetone and ethanol individually. 70% ethanol fraction showed maximum specifc activity (133.3 U/mg). This fraction was re-purifed by anion exchange chromatography DEAE- Cellulose A-52. The loaded protein was separated into 3 peaks. The second protein peak was the major one which contained 48.2% of the total protein recovered and 79.4% of the total recovered activity. The collected fractions of this peak were subjected to further purifcation by re-chromatography on Sephadex G-100. Alginate lyase activity was fractionated in the Sephadex column into one major peak, and the specifc activity of this fraction reached 116 U/mg. The optimal substrate concentration, pH and temperature for alginate lyase activity were 8 mg/ml, pH 7.5 and 37 °C, respectively. While, Km and Vmax values were 1.07 mg alginate/ ml and 128.2 U/mg protein, respectively. The enzyme was partially stable below 50 °C, and the activity of the enzyme was strongly enhanced by K+, and strongly inhibited by Ba+2, Cd+2, Fe+2 and Zn+2. The purifed enzyme yielded a single band on SDS-PAGE with molecular weight (40.0 kDa).
KeywordsPseudomonas sp. Alginate lyase Enzyme purifcation Enzyme properties
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