The biodegradation of nitrobenzene was attempted by using Pseudomonas putida TB101 which possesses the hybrid pathway combining the tod and the tol pathway. Analysis of the metabolic flux of nitrobenzene through the hybrid pathway indicated that nitrobenzene was initially oxidized to cis-1,2-dihydroxy-3-nitrocyclohexa-3,5-diene by toluene dioxygenase in the tod pathway and then channeled into the tol pathway in the form of dihydrodiol. A crucial metabolic step connecting the tod and the tol pathways in the catabolism of nitrobenzene was determined by the studies on the activities of the enzymes involved in the tol pathway. From these studies, it was found that purified toluate-cis-glycol dehydrogenase could convert cis-1.2-dihydoxy-3-nitrocyclohexa-3,5-diene to catechol in the presence of $NAD^+$ with liberation of nitrite and NADH into the medium. Based on the kinetic analysis of the degradation of nitrobenzene, it was determined that a bottleneck step in the whole catabolism of nitrobenzene through the hybrid pathway is toluene dioxygenase. Furthermore, a lag period appeared prior to the degradation of nitrobenzene due to the delayed expression of the tod operon in P. putida TB101. Cloning of todC1C2BA genes encoding toluene dioxygenase on the broad-host-range plasmid, RSF1010, and the introduction of the resulting plasmid into P. putida mt-2 led to the construction of the second hybrid strain, P. putida TB103. P. puitda TB103 was found to possess the basically the same hybrid pathway as that of P. putida TB101, but showed enhanced activity of toluene dioxygenase. The biodegradation of nitrobenzene performed by using P. putida TB103 showed the increase in the degradation rate of nitrobenzene by about 3 fold compared with that of P. putida TB101. In addition, the lag period appeared prior to the degradation of nitrobenzene was not observed when P. putida TB103 was employed.
TOL 과 TOD 대사경로의 hybrid pathway를 가진 균주에 의한 nitrobenzene의 대사가능성과 그 대사경로를 밝히는 연구를 수행하였다. P.putida TB101 균주에 의하여 nitrobenzene은 TOD 대사경로중의 toluene dioxygenase에 의하여 cis-1,2-dihydroxy-3-nitrocyclohexa-3,5-dien 형태로 전환된 후 TOL 대사경로로 유입되어 대사 된다. 이러한 대사 과정중 TOD와 TOL 대사경로를 연결시켜 주는 핵심적인 대사단계를 TOL 대사경로 중의 효소들의 활성을 조사함으로써 밝혔다. 이러한 연구를 통하여 정제된 toluate-cis-glycol dehydrogenase는 $NAD^+$ 존재하에서 cis-1,2-dihydroxy-3-nitrocyclohexa-3,5-dien을 catechol로 전환시키며 nitrite와 NADH를 유리시킨다는 사실을 밝혔다. Nitrobenzene에 대한 대사의 연구를 통하여 hybrid pathway 전체의 대사속도를 경정하는 단계는 toluene dioxygenase의 단계임을 알 수 있었다. 또한 tod operon의 지연된 발현으로 인하여 대사시 유도기가 존재한다. Toluene dioxygenase의 유전자인 todC1C1BA의 클로닝 작업을 통해 해당효소의 활성을 증폭시켰으며 이렇게 생성된 균주인 P.putida TB103은 P.putida TB101과 같은 대사 경로를 가지고 있으나 P.putida TB101에 비해 nitrobenzene에 대한 높은 분해속도를 보이며 유도기 또한 관찰되지 않는다.
