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

作　　者：沈继红 阚光锋 史翠娟 雷振环 解秋菊 钱文佳 

机构地区：[1]Key Laboratory of Marine Bio-active Substances, State Oceanic Administration [2]School of the Ocean, Harbin Institute of Technology at Weihai

出　　处：《Chinese Journal of Oceanology and Limnology》2011年第5期1086-1092,共7页中国海洋湖沼学报（英文版）

基　　金：Supported by the National High Technology Research and Development Program of China (863 Program) (No. 2007AA091905);the Natural Science Foundation of Shandong Province (No. ZR2010DQ010);the Fundamental Research Funds for the Central Universities (No. HIT.IBRSEM.2009148)

摘　　要：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-β-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 β-glucan, but not cellobiose, xylan and p-Nitrophenyl-β-D-glucopyranoside. The optimal temperature and pH for the β-glucanase activity of AN-1 were determined to be at 30℃ and pH 6.0, respectively. AN-1 was stable at acidic solutions of pH 5.0-6.5 and temperatures below 30℃ for 1 h. Moreover, the specific activity was enhanced by Ca2＋ and Mg2., and inhibited by Cu2＋. The kinetic parameters Michaelis constant （Km） and maximum velocity （Vmax） of AN-1 were 3.96 mg/mL and 6.06×10-2 mg/（min.mL）, respectively.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-β-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 β-glucan, but not cellobiose, xylan and p-Nitrophenyl-β-D-glucopyranoside. The optimal temperature and pH for the β-glucanase activity of AN-1 were determined to be at 30°C and pH 6.0, respectively. AN-1 was stable at acidic solutions of pH 5.0-6.5 and temperatures below 30°C for 1 h. Moreover, the specific activity was enhanced by Ca2+ and Mg2+, and inhibited by Cu2+. The kinetic parameters Michaelis constant (Km) and maximum velocity (Vmax) of AN-1 were 3.96 mg/mL and 6.06×10-2 mg/(min·mL), respectively.

关 键 词：PSEUDOALTEROMONAS endo-l 4-β-glucanase cold-active enzyme Antarctic sea ice stability 

分 类 号：Q814[生物学—生物工程]