Cao Bin, Geng Anli, Loh Kai-Chee
Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore, 117576, Singapore.
Appl Microbiol Biotechnol. 2008 Nov;81(1):99-107. doi: 10.1007/s00253-008-1728-3. Epub 2008 Oct 4.
The degradation pathways of benzoate at high concentration in Pseudomonas putida P8 were directly elucidated through mass spectrometric identification of some key catabolic enzymes. Proteins from P. putida P8 grown on benzoate or succinate were separated using two-dimensional gel electrophoresis. For cells grown on benzoate, eight distinct proteins, which were absent in the reference gel patterns from succinate-grown cells, were found. All the eight proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as catabolic enzymes involved in benzoate degradation. Among them, CatB (EC5.5.1.1), PcaI (EC2.8.3.6), and PcaF (EC2.3.1.174) were the enzymes involved in the ortho-cleavage pathway; DmpC (EC1.2.1.32), DmpD (EC3.1.1.-), DmpE (EC4.2.1.80), DmpF (EC1.2.1.10), and DmpG (EC4.1.3.-) were the meta-cleavage pathway enzymes. In addition, enzyme activity assays showed that the activities of both catechol 1,2-dioxygenase (C12D; EC1.13.11.1) and catechol 2,3-dioxygenase (C23D; EC1.13.11.2) were detected in benzoate-grown P. putida cells, undoubtedly suggesting the simultaneous expression of both the ortho- and the meta-cleavage pathways in P. putida P8 during the biodegradation of benzoate at high concentration.
通过对一些关键分解代谢酶进行质谱鉴定，直接阐明了恶臭假单胞菌P8中高浓度苯甲酸的降解途径。使用二维凝胶电泳分离在苯甲酸或琥珀酸上生长的恶臭假单胞菌P8的蛋白质。对于在苯甲酸上生长的细胞，发现了八种不同的蛋白质，而在琥珀酸生长细胞的参比凝胶图谱中不存在这些蛋白质。通过基质辅助激光解吸/电离飞行时间质谱法鉴定出所有这八种蛋白质均为参与苯甲酸降解的分解代谢酶。其中，CatB（EC5.5.1.1）、PcaI（EC2.8.3.6）和PcaF（EC2.3.1.174）是参与邻位裂解途径的酶；DmpC（EC1.2.1.32）、DmpD（EC3.1.1.-）、DmpE（EC4.2.1.80）、DmpF（EC1.2.1.10）和DmpG（EC4.1.3.-）是间位裂解途径的酶。此外，酶活性测定表明，在苯甲酸生长的恶臭假单胞菌细胞中检测到了儿茶酚1,2-双加氧酶（C12D；EC1.13.11.1）和儿茶酚2,3-双加氧酶（C23D；EC1.13.11.2）的活性，这无疑表明在高浓度苯甲酸生物降解过程中，恶臭假单胞菌P8中同时表达了邻位和间位裂解途径。
