Characterization of the Initial Reactions during the Cometabolic Oxidation of Methyl tert -Butyl Ether by Propane-Grown Mycobacterium vaccae JOB5

Abstract

The initial reactions in the cometabolic oxidation of the gasoline oxygenate, methyl tert-butyl ether (MTBE), by Mycobacterium vaccae JOB5 have been characterized. Two products, tert-butyl formate (TBF) and tert-butyl alcohol (TBA), rapidly accumulated extracellularly when propane-grown cells were incubated with MTBE. Lower rates of TBF and TBA production from MTBE were also observed with cells grown on 1-or 2-propanol, while neither product was generated from MTBE by cells grown on casein-yeast extract-dextrose broth. Kinetic studies with propane-grown cells demonstrated that TBF is the dominant (>80%) initial product of MTBE oxidation and that TBA accumulates from further biotic and abiotic hydrolysis of TBF. Our results suggest that the biotic hydrolysis of TBF is catalyzed by a heat-stable esterase with activity toward several other tert-butyl esters. Propane-grown cells also oxidized TBA, but no further oxidation products were detected. Like the oxidation of MTBE, TBA oxidation was fully inhibited by acetylene, an inactivator of short-chain alkane monooxygenase in M. vaccae JOB5. Oxidation of both MTBE and TBA was also inhibited by propane (K i ‫؍‬ 3.3 to 4.4 M). Values for K s of 1.36 and 1.18 mM and for V max of 24.4 and 10.4 nmol min ؊1 mg of protein ؊1 were derived for MTBE and TBA, respectively. We conclude that the initial steps in the pathway of MTBE oxidation by M. vaccae JOB5 involve two reactions catalyzed by the same monooxygenase (MTBE and TBA oxidation) that are temporally separated by an esterase-catalyzed hydrolysis of TBF to TBA. These results that suggest the initial reactions in MTBE oxidation by M. vaccae JOB5 are the same as those that we have previously characterized in gaseous alkane-utilizing fungi.Methyl tert-butyl ether (MTBE) is presently an important component of many gasoline formulations used in the United States and several other countries. Low concentrations of MTBE (Ͻ3% vol/vol) were first added to gasoline in the United States in the 1980s to enhance the octane rating of unleaded gasoline. More recently, MTBE has been added as one of several oxygenating compounds intended to reduce automobile emissions of carbon monoxide and smog-related air pollutants. For this purpose MTBE is added to gasoline at concentrations in excess of 10% (vol/vol). The widespread use of MTBE and its recent detection in many urban groundwater supplies (44) have prompted concerns over the human health effects of chronic exposure to this compound through contaminated water supplies (26). The U.S. Environmental Protection Agency presently classifies MTBE as a possible human carcinogen and has issued a drinking water advisory for MTBE of 20 to 40 g/liter (parts per billion) (48).Ether bonds (49) and branched hydrocarbon skeletons (3) are features found in many compounds that are persistent in the environment. Although initial studies suggested that MTBE was not subject to extensive biodegradation under anaerobic conditions (35,53), it is now known to be degraded, albeit slowly, under methanogenic (51), s...

n-Alkane assimilation and tert-butyl alcohol (TBA) oxidation capacity in Mycobacterium austroafricanum strains

“…Propane was a good inducer of TBA degradation activity in M. vaccae JOB5 (Fig. 3), as previously determined by Smith et al (2003).…”

“…Several grampositive microorganisms belonging to the Nocardia-Corynebacterium-Mycobacterium-Rhodococcus complex showed degradation capacity towards TBA. Among them, Mycobacterium vaccae JOB5, isolated for its ability to grow on 2-methylbutane (Ooyama and Foster 1965) oxidised MTBE and also TBA after growth on propane (Hyman et al 1998;Johnson et al 2004;Smith et al 2003;Steffan et al 1997), hexane or 2-methylbutane (Hyman and O'Reilly 1999). Smith et al (2003) showed at a physiological level that a propane-induced monooxygenase was responsible for both MTBE and TBA oxidation in this strain.…”

Evaluation of Intrinsic Bioremediation of Methyl Tert-butyl Ether (MTBE) Contaminated Groundwater

“…A mixed culture may be more suitable for treatment options than pure culture (Pruden et al, 2001). It could be the existence of many rea- (Francois et al, 2003;Smith et al, 2003;Ferreira et al, 2006). However, TBF was not detected in our study.…”

Genes involved in the methyl tert-butyl ether (MTBE) metabolic pathway of Mycobacterium austroafricanum IFP 2012

“…We recently detected 2-methyl 1,2-propanediol (2-M1,2-PD) as the product of TBA oxidation in M. austroafricanum IFP 2012and IFP 2015(Lopes Ferreira et al, 2005, and demonstrated its transformation to 2-hydroxyisobutyric acid (HIBA) in resting-cell experiments. 2-Methyl 1,2-propanediol (2-M1,2-PD) has been reported by Steffan et al (1997), using propane-oxidizing bacteria able to degrade MTBE by cometabolism, and by Smith et al (2003), using Mycobacterium vaccae JOB5 as the product of TBA degradation.…”