文献类型：期刊文献

英文题名：Identification and Characterization of a Novel Gentisate 1,2-Dioxygenase Gene from a Halophilic Martelella Strain

作者：Huang, Ling[1];Hu, Haiyang[2,3];Tang, Hongzhi[2,3];Liu, Yongdi[1];Xu, Ping[2,3];Shi, Jie[1];Lin, Kuangfei[1];Luo, Qishi[4];Cui, Changzheng[1]

机构：[1]E China Univ Sci & Technol, Sch Resources & Environm Engn, State Environm Protect Key Lab Environm Risk Asse, Shanghai 200237, Peoples R China;[2]Shanghai Jiao Tong Univ, State Key Lab Microbial Metab, Shanghai 200240, Peoples R China;[3]Shanghai Jiao Tong Univ, Sch Life Sci & Biotechnol, Shanghai 200240, Peoples R China;[4]Shanghai Inst Design & Res Environm Engn Co Ltd, Shanghai Engn Res Ctr Contaminated Sites Remediat, Shanghai 200232, Peoples R China

年份：2015

卷号：5

外文期刊名：SCIENTIFIC REPORTS

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000361594700001)】；

基金：This research was supported by the National Natural Science Foundation of China (41301329 and 51378208), the Chinese National Science Foundation for Excellent Young Scholars (31422004), the Shanghai Municipal Natural Science Foundation (13ZR1410900) and the Open Funding Project of the State Key Laboratory of Bioreactor Engineering.

语种：英文

摘要：Halophilic Martelella strain AD-3, isolated from highly saline petroleum-contaminated soil, can efficiently degrade polycyclic aromatic hydrocarbons (PAHs), such as phenanthrene and anthracene, in 3-5% salinity. Gentisic acid is a key intermediate in the microbial degradation of PAH compounds. However, there is little information on PAH degradation by moderately halophilic bacteria. In this study, a 1,077-bp long gene encoding gentisate 1,2-dioxygenase (GDO) from a halophilic Martelella strain AD-3 was cloned, sequenced, and expressed in Escherichia coli. The recombinant enzyme GDO was purified and characterized in detail. By using the O-18 isotope experiment and LC-MS analysis, the sources of the two oxygen atoms added onto maleylpyruvate were identified as H2O and O-2, respectively. The K-m and k(cat) values for gentisic acid were determined to be 26.64 mu M and 161.29 s(-1), respectively. In addition, optimal GDO activity was observed at 30 degrees C, pH 7.0, and at 12% salinity. Site-directed mutagenesis demonstrated the importance of four highly conserved His residues at positions 155, 157, 167, and 169 for enzyme activity. This finding provides new insights into mechanism and variety of gentisate 1,2-dioxygenase for PAH degradation in high saline conditions.