Purification and characterization of β-agarase from seaweed decomposing bacterium Microbulbifer sp. Strain CMC-5
2011
Jonnadula, Ravichand, Goa University, Goa, India | Ghadi, Sanjeev C., Goa University, Goa, India
Microbulbifer strain CMC-5 was isolated from decomposing seaweeds, and was found to degrade agar, alginate, carboxymethyl cellulose, carrageenan, xylan, and chitin. The extracellular agarase enzyme from strain CMC-5 was purified 103-fold by ultrafiltration, ion-exchange chromatography, using diethylaminoethyl sepharose FF, and gel filtration, using sephacryl S-300HR, with a yield of 6.7%. Zymogram and protein staining of the purified agarase on a SDS-polyacrylamide gel revealed a single band, with an apparent molecular weight of 59 kDa. The purified enzyme was endo-type β-agarase, as it was able to hydrolyze the β-1, 4 glycosidic linkages of agarose, releasing neoagarotetraose and neoagarohexaose as the end products. The optimum pH and temperature of agarase were 7 and 50℃, respectively. Thermal stability studies indicated that the agarase retained 62% of its activity after incubating at 50℃ for 30 min. Treatment with EDTA reduced the agarase activity by 54%. The agarase activity was stimulated by the presence of Ca²+ and Mg²+ ions; whereas, Zn²+, Hg²+, Cu²+, Fe²+, and Co²+ abolished the activity. Further, the presence of NaCl at concentrations lower than 100 mM caused a decrease in the agarase activity; whereas, the activity was enhanced up to a concentration of 500 mM.
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