Graduate Thesis Or Dissertation
 

Cooxidation by three butane-grown bacteria and mechanism-based inactivation of butane monooxygenase

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  • Butane-grown cells of Pseudomonas butanovora, Nocardioides sp. CF8 and Mycobacterium vaccae JOB5 were tested for their ability to cooxidize methane, ammonia and ethylene. Less than 10 nmol of methane were degraded by each of the bacteria (0.17-0.35 mg protein) in 30 minutes. Hydroxylamine and nitrite accumulated when Nocardioides CF8 and P. butanovora were incubated with ammonia, while M. vaccae JOB5 accumulated only nitrite. The butane monooxygenase (BMO) was implicated in the formation of hydroxylamine and possibly nitrite as the presence of acetylene or butane decreased the production of hydroxylamine and nitrite and the addition of butyrate enhanced hydroxylamine and nitrite production. Oxygen was also required for ammonia oxidation. All three strains oxidized ethylene to ethylene oxide. This reaction was inhibited by acetylene and enhanced by butyrate. Production of ethylene oxide in P. butanovora stopped after 20 minutes, while proceeding at a constant rate for 2 hours in M. vaccae and Nocardioides CF8. Further tests indicated inactivation of butane oxidizing activity by ethylene oxide in P. butanovora. The characteristics of ethylene oxide inactivation of butane monooxygenase (BMO) in P. butanovora were investigated. BMO was found to be irreversibly inactivated by ethylene oxide in a time and concentration dependent manner. Butane protected BMO from inactivation and 0₂ was required for inactivation implying turnover was required. Other epoxides were found to inactivate BMO including epoxypropane, 1,2-epoxybutane and 1,2-epoxyhexane. Cis and trans-2,3-epoxybutane did not inactivate. Other bacterial monooxygenases were tested for sensitivity to ethylene oxide including ammonia monooxygenase in N. europaea, toluene-2-monooxygenase in Burkholderia cepacia G4 and alkane monooxygenases in M. vaccae JOB5, Nocardioides CF8 and Pseudomonas oleovorans. Of these, only alkane monooxygenases in Nocardioides sp. CF8 and M. vaccae JOB5 exhibited ethylene oxide sensitivity. The results presented here provide strong evidence that ethylene oxide is a mechanism-based inactivator of BMO in P. butanovora.
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