Expression,purification and partial characterization of a xanthine oxidase (XOD) in Arthrobacter sp. |
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Authors: | Yu Xin Hailin Yang Xiaole Xia Ling Zhang Yuran Zhang Chen Cheng Wu Wang |
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Affiliation: | Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China |
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Abstract: | A xanthine oxidase (XOD) was expressed, purified and partially characterized from Arthrobacter sp. with a negative immune protocol. To determine the optimal inducer for XOD, xanthine, hypoxanthine and uric acid were added into the medium of cultivation. The results revealed that with the inducement of about 14 mM xanthine, the highest XOD activity could be detected. To separate XOD from Arthrobacter sp., the cells were first cultured without any inducement; then the total proteins of the collected cells were extracted and immunized to rabbits for the polyclonal antibodies. These antibodies were then coupled with sepharose CL 6 B, and the medium was further employed to deplete most of the cells’ back ground proteins. Began with ~20 mg crude protein from disrupted cells was subjected to the antibody medium, and ~1.45 mg protein was detected in unbinding fractions with ~92.0% of activity. The extracted xanthine oxidase was ~85% pure with native-PAGE analysis, and ~90% pure with SDS-PAGE analysis, the yield of protein was ~7.4%. The specific activity of the enzyme was 36.0 U/mg. The native enzyme should be a dimer (~280 kDa) of a protein composed with two different peptides with the mass of approximately 55.5 and 85.5 kDa, respectively. The optimal pH and temperature of this enzyme were determined at about pH 7 and 50 °C. Furthermore, EDTA revealed almost no influences on the activity. |
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