典型超微细菌降解邻苯二甲酸二丁酯的途径及水合酶的表达纯化研究

典型超微细菌菌株PAE-UM属于β-变形菌门丛毛单胞菌属(Curvibacter sp.),是革兰氏阴性菌株,可利用DBP作为唯一碳源。利用流式细胞仪检测细菌生长情况,并采用高效液相色谱测定DBP的浓度,结果显示该菌对DBP的降解率为94%,菌株最大生长速率为0.237 2 h-1。已经完成了该菌株的全基因组测序,Gen Bank登录号为LKCX00000000。通过全基因组数据分析及文献对比发现该菌在降解邻苯二甲酸二丁酯的途径中,4-oxalomesaconate水合酶(OMH)是原儿茶酸间位裂解代谢途径中的关键酶,具有催化4-oxalomesaconate(OMA)降解为4-carboxy-4-hydroxy-2-oxoadipate(CHA)的能力,该酶是降解邻苯二甲酸酯过程中的关键酶之一,也是芳香化合物降解过程中的重要降解酶。利用蛋白结晶技术及分子生物学手段,对关键酶(OMH)进行分子构建,表达、纯化、结晶的筛选,应用X-ray衍射技术对关键酶的晶体进行衍射,获得晶体的衍射数据,进行关键酶的三维结构解析。Curvibacter sp.PAE-UM菌株中的OMH蛋白在大肠杆菌中得到表达,经多种方法纯化后筛选获得了纯度大于95%的蛋白,并经试剂盒筛选获得高质量的蛋白质晶体。在上海光源同步辐射(SSRF)BL~(-1)9U线站收集数据,通过X射线衍射实验收集到分辨率达到2.00的衍射数据,并使用软件HKL2000进行了数据处理与修正,该OMH的结构解析为2.00。后续将通过该关键酶的三维结构的解析进一步阐明该菌降解PAEs的分子机理。

Microbial degradation pathway of dibutylphthalate by a typical ultramicrobacterium and the expression and purification of 4-oxalomesaconate hydratase

A typical ultramicrobacterium, Curvibacter sp. strain PAE-UM, capable of degrading dibutyl phthalate was isolated. It is a Gram negative strain. Flow cytometer was used to detect the bacterial growth and the HPLC-MS was applied to analyze the concentration of DBP. The result showed that this strain can degrade DBP with a degradation rate of 94% and the maximum growth rate of strain was 0.237 2 h^-1. Its draft genome has been sequenced in our previous study. The complete genome sequence has been deposited in GenBank under the accession number LKCX00000000. In this study, we proposed the degradation pathway of dibutyl phthalate and the key enzymes involved based on the genome analysis. The results showed that 4-oxalomesaconate hydratase(OMH) was the key enzyme in the meta-cleavage of protocatechuic acid in PAEs degradation pathway. It is also the key enzyme in the aromatic compounds degradation pathway. It has the ability to catalyze the 4-oxalomesaconate to 4-carboxy-4-hydroxy-2-oxoadipate. The gene encoding OMH was expressed in E. coli. The protein was then purified by a variety of methods. Protein crystals with high resolution were obtained. The crystal data was then collected by X-ray diffraction experiment in Shanghai Synchrotron Radiation Facility. The data was processed by the HKL2000 software and the resolution was 2.0 Å. These results could make an important contribution to the study of OMH functional mechanism. Furthermore, the results could further clarify the molecular mechanism of the PAEs degradation by ultramicrobacteria.