Purification and characterization of cold-active endo-1,4-β-glucanase produced by Pseudoalteromonas sp AN545 from Antarctica

A bacterium hydrolyzing carboxymethylcellulose, isolated from Antarctic sea ice, was identified as Pseudoalteromonas sp. based on 16S rDNA gene sequences and named as Pseudoalteromonas sp. AN545. The extracellular endo-1,4-beta-glucanase AN-1 was purified successively by ammonium sulfate precipitation, DEAE-Sepharose ion exchange chromatography and Sephadex G-75 gel filtration chromatography. The molecular mass of AN-1 was estimated to be 47.5 kDa utilizing SDS-PAGE and gel chromatography analysis. AN-1 could hydrolyze caboxymethylcellulose, avicel and beta-glucan, but not cellobiose, xylan and p-Nitrophenyl-beta-D-glucopyranoside. The optimal temperature and pH for the beta-glucanase activity of AN-1 were determined to be at 30A degrees C and pH 6.0, respectively. AN-1 was stable at acidic solutions of pH 5.0-6.5 and temperatures below 30A degrees C for 1 h. Moreover, the specific activity was enhanced by Ca2+ and Mg2+, and inhibited by Cu2+. The kinetic parameters Michaelis constant (K (m)) and maximum velocity (V (max)) of AN-1 were 3.96 mg/mL and 6.06x10(-2) mg/(min center dot mL), respectively.