Whole‐cell studies of substrate and inhibitor specificity of isoprene monooxygenase and related enzymes

Abstract Co‐oxidation of a range of alkenes, dienes, and aromatic compounds by whole cells of the isoprene‐degrading bacterium Rhodococcus sp. AD45 expressing isoprene monooxygenase was investigated, revealing a relatively broad substrate specificity for this soluble diiron centre monooxygenase. A range of 1‐alkynes (C2–C8) were tested as potential inhibitors. Acetylene, a potent inhibitor of the related enzyme soluble methane monooxygenase, had little inhibitory effect, whereas 1‐octyne was a potent inhibitor of isoprene monooxygenase, indicating that 1‐octyne could potentially be used as a specific inhibitor to differentiate between isoprene consumption by bona fide isoprene degraders and co‐oxidation of isoprene by other oxygenase‐containing bacteria, such as methanotrophs, in environmental samples. The isoprene oxidation kinetics of a variety of monooxygenase‐expressing bacteria were also investigated, revealing that alkene monooxygenase from Xanthobacter and soluble methane monooxygenases from Methylococcus and Methylocella, but not particulate methane monooxygenases from Methylococcus or Methylomicrobium, could co‐oxidise isoprene at appreciable rates. Interestingly the ammonia monooxygenase from the nitrifier Nitrosomonas europaea could also co‐oxidise isoprene at relatively high rates, suggesting that co‐oxidation of isoprene by additional groups of bacteria, under the right conditions, might occur in the environment.


INTRODUCTION
Isoprene (2-methyl-1,3-butadiene) is an important biogenic volatile organic compound (BVOC) emitted to the atmosphere in quantities similar to that of methane ($500 Tg(C) per year) (Guenther et al., 2012).The vast majority (>90%) is produced by terrestrial plants, possibly in response to heat and oxidative stress (Sharkey et al., 2008) with lesser contributions from bacteria, animals, biomass burning, and industrial processes, reviewed by McGenity et al. (2018).Because of its volatility (boiling point of 34 C) and reactivity caused by the presence of two carbon-carbon double bonds, isoprene has major but multifaceted effects on atmospheric chemistry and therefore on climate (Carlton et al., 2009;Pacifico et al., 2009).
Isoprene-degrading bacteria can grow aerobically on isoprene as sole carbon and energy source and are a substantial sink for isoprene, removing an estimated 20 Tg of isoprene from the atmosphere per year (Cleveland & Yavitt, 1997;Gray et al., 2015;Pegoraro et al., 2005).Cultivation-dependent and cultivation-independent studies have highlighted that isoprene degradation is a widespread trait across particular representatives of many bacterial genera, including Rhodococcus, Mycobacterium, Nocardioides, Gordonia, Sphingopyxis, and Variovorax, and isoprene degraders have been isolated from terrestrial, freshwater, coastal and marine environments including soils and the leaves of trees, reviewed by Dawson et al. (2023).Rhodococcus sp.AD45 is a Gram-positive isoprene-degrading Actinobacterium isolated from freshwater sediment (van Hylckama Vlieg et al., 1998), and Actinobacteria are often the dominant isoprene degraders in soils or estuarine sediments (El Khawand et al., 2016;Johnston et al., 2017).Proteobacterial isoprene degraders, including Variovorax, Sphingopyxis, and Pseudomonas, have been identified in other environments (Dawson et al., 2020;Gibson et al., 2021;Larke-Mejía et al., 2019;Srivastva et al., 2015).All characterised isoprene-degrading bacteria (with the exception of an Alcaligenes strain described by Uttarotai et al. (2022)) contain 11 core iso genes clustered together.An isoprene monooxygenase (IsoMO) is encoded by iso-ABCDEF while the gene products of isoGHIJ and aldH are required for subsequent metabolic steps (Dawson et al., 2022;van Hylckama Vlieg et al., 2000).In Rhodococcus sp.AD45 and Variovorax sp.WS11, iso genes for isoprene metabolism are located on megaplasmids (Crombie et al., 2015;Dawson et al., 2020) suggesting that the ability to grow on isoprene may be acquired via horizontal gene transfer.
Co-oxidation is the transformation of a non-growth substrate which, due to a lack of substrate specificity, can nevertheless serve as substrate for an enzymatic process during normal metabolism (Dalton et al., 1982).For example, bacterial SDIMOs typically co-oxidise a wide array of alternative substrates.Dawson et al. (2020) investigated the substrate range of IsoMO in Variovorax sp.WS11 and showed that this isoprene degrader could co-oxidise a broad range of alkenes, with preference for those with alkyl side chains.sMMO from methanotrophs transforms an extensive range of over 100 non-growth substrates including C 2 -C 9 alkanes and C 2 -C 5 alkenes, halogenated hydrocarbons, and aromatics such as styrene and toluene (Colby et al., 1977;Jiang et al., 2010;Smith & Dalton, 2004).Alkene monooxygenase from X. autotrophicus Py2 can catalyse the epoxidation of a range of alkenes, including trichloroethylene, and aromatics, but not alkanes (van Ginkel et al., 1987;Zhou et al., 1999).Some SDIMO-containing strains, including methanotrophs, Actinobacteria and Gram-negative toluene degraders, oxidise isoprene or 1,3-butadiene, which is structurally similar to isoprene (McClay et al., 2000;Patel et al., 1982;van Ginkel & de Bont, 1986), suggesting that some nonisoprene-degraders may contribute to isoprene consumption in the environment.
While co-oxidative processes are of biotechnological interest for their ability to generate high-value compounds including desirable enantiomers (Smith & Dalton, 2004), and although bacteria growing on isoprene probably constitute the major biological sink for isoprene, due to the ubiquity of sMMO, pMMO, and AMO-containing microorganisms in the environment, their potential contribution to the microbial sink for isoprene via co-oxidation needs to be addressed.The use of specific inhibitors can aid these studies.For example, sMMO, pMMO, and AMO are inhibited by acetylene (Hynes & Knowles, 1982;Prior & Dalton, 1985), alkene monooxygenases from Xanthobacter autotrophicus Py2 and Rhodococcus rhodochrous B276 are inhibited by propyne (Ensign et al., 1992;Fosdike et al., 2005), and toluene monooxygenases are inhibited by aromatic alkynes with terminal acetylene groups (Keener et al., 2001).Previous data from our lab showed that, compared to acetylene, IsoMO from Variovorax sp.WS11 was inhibited more by longer chain 1-alkynes such as 1-octyne (Dawson et al., 2020), which may provide a starting point for identification of a specific inhibitor of IsoMO.
This study aimed to characterize the whole-cell substrate specificity of IsoMO from Rhodococcus sp.AD45 and the effects of potential inhibitors such as C 2 -C 8 linear 1-alkynes.We wished to compare these to the inhibition profiles of Variovorax sp.WS11 and a methanotroph expressing sMMO, Methylococcus capsulatus strain Bath.Furthermore, we determined the rates of isoprene oxidation by methanotrophs (Methylocella, Methylococcus, Methylomicrobium) and ammonia oxidisers (Nitrosomonas), and the kinetic parameters of isoprene oxidation by Rhodococcus sp.AD45, Variovorax sp.WS11 and X. autotrophicus Py2 in comparison with the kinetics of propylene uptake in order to expand our knowledge of the substrate range of isoprene monooxygenase, its inhibitors, and the potential for co-oxidation of isoprene by non-isoprenedegraders.

Strains and growth media
Rhodococcus sp.AD45 and Variovorax sp.WS11 were grown in their respective media in batch mode in a 4-L working volume fermentor as previously described (Crombie et al., 2015;Dawson et al., 2020).Xanthobacter autotrophicus Py2 and Rhodococcus sp.AD45 (for epoxyisoprene product formation) were grown in wash culture in minimal medium as described (Crombie et al., 2015) and supplied with isoprene (approx.1% v/v) or propylene (10% v/v) in the headspace.Methylococcus capsulatus (Bath) was grown in nitrate mineral salts medium (Whittenbury et al., 1970) as previously described (Dawson et al., 2020) and Methylomicrobium alcaliphilum in modified P medium (Akberdin et al., 2018) in 50 mL volumes in 250 mL Quickfit conical flasks, shaking (180 rpm) at 30 C and supplied with methane (20% v/v).For expression of sMMO, Methylococcus capsulatus was grown in copper-free medium, and the naphthalene assay was used to verify sMMO expression (Brusseau et al., 1990).Nitrosomonas europaea ATCC19718 was grown in modified Skinner and Walker medium as described (Wright et al., 2020).
Cells were harvested by centrifugation (except for Nitrosomonas europaea which was harvested by filtration as described; Wright et al., 2020), washed and resuspended in the buffer specified below and either used fresh or drop-frozen in liquid nitrogen and stored at À80 C.

Rhodococcus sp. AD45 substrate range
Substrate-induced oxygen uptake was quantified using a Clark-type oxygen electrode, as previously described (Dawson et al., 2020).Potential substrates were prepared as aqueous solutions in sealed flasks.In the case of gaseous substrates, aqueous concentrations were calculated from the headspace concentration using values of the Henry's law constants taken from Sander (2015).Cell suspension (1.5 mg dry weight of cells; dw)) in a volume of 3 mL air-saturated phosphate buffer (50 mM, pH 7.0) was added to the reaction cell, maintained at 30 C. After recording the endogenous rate of oxygen uptake, substrates (5-150 μL) were injected into the chamber to a final concentration of 100 μM.The substrate-induced rate was calculated by subtracting the endogenous rate from the rate in the presence of substrate.

Inhibition of IsoMO with terminal alkynes
The inhibition of IsoMO by 1-alkynes was quantified as in Dawson et al. (2020).Briefly, approximately 0.25 mg (dw) of Rhodococcus sp.AD45 cells were resuspended in 1 mL phosphate buffer (50 mM, pH 7.0) in a 25 mL flask, sealed with a rubber stopper, and incubated at 30 C in a shaking water bath.Isoprene (300 ppmv) was added by injection through the septum as previously described (Crombie et al., 2015) and left to equilibrate for 3 min.Isoprene was quantified from headspace samples every 5 min.After 15 min, inhibitors were added to a final concentration of 50 μM.Gaseous 1-alkyne inhibitors (C 2 -C 4 ) were added directly to assay vials using a gas-tight syringe and the resultant aqueous concentration calculated using the Henry's law coefficient as described above.C 5 -C 8 (liquid) alkynes were dissolved in DMSO and added in a volume of 10 μL, having previously established that this volume of DMSO alone had no effect on isoprene oxidation by IsoMO.After allowing 5 min for the reaction to stabilise, a further five headspace samples were taken at 5 min intervals.Rates of isoprene uptake in the presence or absence of alkyne inhibitors were quantified by injection of 50 μL of headspace gas into an Agilent 7820A gas chromatograph fitted with a Porapak Q column (Supelco) coupled to a flame ionisation detector.The injector, oven, and detector temperatures were 150, 125, and 200 C, respectively.Headspace isoprene concentration was quantified using standards prepared from a known quantity of isoprene in air.The percentage inhibition was calculated by comparing the change in isoprene uptake before and after the addition of inhibitor.Control experiments with no inhibitor added or with addition of DMSO alone were used to account for any reduction in rate due to the decreasing isoprene concentration.

Kinetics of isoprene and propylene uptake
Frozen cells of Rhodococcus sp.AD45 or X. autotrophicus Py2 (0.25-3.5 mg dw) were resuspended in 1-2 mL phosphate buffer (50 mM, pH 7.0) in 25 mL vials, sealed with rubber stoppers and incubated at 30 C in a shaking water bath.For isoprene uptake by X. autotrophicus Py2, pyruvate (10 mM) was added as a reductant.Isoprene or propylene (140-40,000 ppmv) was added to the headspace and uptake measured by injection of headspace gas (10-100 μL) into a fast isoprene sensor (Hills Scientific, Boulder, CO, USA) every 3-15 min, against standards prepared in the range 12-40,000 ppmv.Corresponding liquid phase concentrations were calculated using the Henry's law constants as described above.Data were analysed using QtiPlot (v 5.6.1) and gas uptake rates plotted against substrate concentration.Fermentor-grown cells of Variovorax sp.WS11 (0.25 mg dw) were transferred to 30 mL vials, sealed and isoprene vapour was added to the headspace.The rate of isoprene uptake was measured for 15 min using a gas chromatograph as described above.The apparent K m and V max for isoprene or propylene uptake, together with associated standard error and 95% confidence interval, were calculated by non-linear regression using Hyper32 (v.1.0) software (University of Liverpool, UK).

Detection of epoxyisoprene by GC-MS
Cells were resuspended in buffer (5 mL), at the growth pH for each strain, to a final OD 540 between 0.25 and 6.0 in 25 mL glass vials, sealed with rubber stoppers.To inhibit monooxygenase activity, the cells were preincubated with alkynes for 30 min at their respective growth temperatures.For sMMO and CuMMOs, acetylene was added to a concentration of 0.04 mM and, for the alkene MO and IsoMO, 1-propyne (9.2 mM) and 1-octyne (0.05 mM), respectively.The cells were then washed twice and resuspended in alkyne-free buffer with additional reductant if required (20 mM formate for methanotrophs or 0.6 mM hydrazine for N. europaea).For R. sp.AD45, epoxyhexane (1 mM) was added to inhibit subsequent metabolism of epoxyisoprene (van Hylckama Vlieg et al., 1998).An initial sample (0.4 mL) was taken, isoprene vapour was added to a liquid concentration of approximately 130 μM and vials were incubated with shaking.Cell suspension (0.4 mL) was removed with a syringe through the septum every 15 min and transferred to a 1.5 mL tube.Epoxyisoprene was extracted into diethyl ether alongside standards prepared from known concentrations (0.002-4.0 mM) of commercial epoxyisoprene (Merck, UK) in diethyl ether and quantified by gas chromatography (GC)-MS as previously described (Rix et al., 2023).

RESULTS AND DISCUSSION
The substrate specificity of Rhodococcus sp.AD45 Rhodococcus sp.AD45 was grown in batch culture on either isoprene or succinate, conditions under which IsoMO is either expressed or repressed, respectively (Crombie et al., 2015).Rates of alkene-induced oxygen uptake were quantified using a Clark-type oxygen electrode.Substrates were added to 100 μM, or to the limit of solubility for less soluble longer-chain alkenes (as indicated in Figure 1).With the exception of the compounds mentioned below, oxygen uptake was not stimulated by alkenes in succinate-grown Rhodococcus sp.AD45 cells (Figure 1).However, IsoMOexpressing cells oxidised a wide range of alkenes without a distinct relationship between the length or structure of the alkene and the resultant rate.The specific rate of oxygen uptake in response to isoprene was 23.1 ± 0.4 nmol min À1 mg dw À1 , whereas 1,3-butadiene, which relative to isoprene lacks only a methyl group, induced oxygen uptake at half the rate of isoprene (10.4 ± 0.3 nmol min À1 mg dw À1 ) (Figure 1B).1-octene induced a near identical rate to that of isoprene, by cells expressing IsoMO (23.1 ± 0.8 nmol min À1 mg dw À1 ), but there was also a considerable rate of 1-octeneinduced oxygen uptake by succinate-grown cells (14.2 ± 1.6 nmol min À1 mg dw À1 ).Similarly, methylcyclohexene and 3-methyl-1,4-pentadiene also induced oxygen uptake by succinate-grown cells, suggesting that these compounds may also be substrates for a different, constitutively expressed oxygenase.Apart from IsoMO, the genome of the metabolically versatile Rhodococcus sp.AD45 encodes numerous oxygenases and other enzymes involved in the metabolism of aliphatic and aromatic hydrocarbons (including several cytochrome P450), some of which may be expressed during growth on succinate (Crombie et al., 2015).The data (Figure 1A) show that these are inactive with isoprene, but some could be active with alternative compounds tested here.Also, note that bona-fide isoprene degraders will consume more oxygen per mole isoprene metabolized than organisms only able to complete the initial oxygenation since the latter will be unable to further oxidize the resultant epoxide.Thus, the stoichiometry and relative rates of oxygen and isoprene uptake might differ between these two groups, possibly resulting in a slight underestimation of the relative rate of isoprene consumption by a co-oxidiser.

Inhibition of oxygenase activity by alkynes
Triple-bonded compounds used in metalloenzyme inhibition studies include acetylene and longer-chain alkynes.For example, acetylene inhibits many metalloenzymecatalysed microbial processes including N 2 fixation, denitrification, nitrification, methanotrophy, methanogenesis, nitrate assimilation, and H 2 metabolism [reviewed in Hyman and Daniel (1988)] and acts as a suicide substrate for sMMO, pMMO, and AMO (Hyman & Wood, 1985;Hynes & Knowles, 1982;Prior & Dalton, 1985).However, for sMMO, longer chain-length alkynes including propyne and 1-butyne are not as inhibitory as acetylene (Dalton & Whittenbury, 1976;Stirling & Dalton, 1977).The sensitivity of Rhodococcus sp.AD45 to C 2 -C 8 linear 1-alkynes was tested and compared to previously published data for Variovorax sp.WS11 and sMMO-expressing M. capsulatus (Dawson et al., 2020).Isoprene was added to a liquid-phase concentration of 5 μM and uptake from the headspace was measured by GC.All alkynes were added to a final concentration of 50 μM, with C 5 -C 8 alkynes dissolved in DMSO, which alone at the concentration used had no inhibitory effect on oxygenase activity in the strains reported here.
Acetylene had no inhibitory effect on isoprene degradation by Rhodococcus sp.AD45, whereas C 4 -C 8 1-alkynes had a significant inhibitory effect and 1-octyne inhibited isoprene uptake by 74% (Figure 2A).This is similar to the data reported for Variovorax sp.WS11, which was inhibited by acetylene and 1-octyne by 6.5% and 95%, respectively (Dawson et al., 2020), Figure 2B.In contrast, isoprene oxidation by the methanotroph Methylococcus capsulatus, expressing sMMO, was inhibited by 99% and 5.7% by acetylene and 1-octyne, respectively (Dawson et al., 2020), Figure 2C.The increasingly potent inhibitory effect of longer chain alkynes, as compared to acetylene, was also found for growth of alkene MOcontaining Rhodococcus rhodochrous B276 (Fosdike et al., 2005;Gallagher et al., 1997), and propyne was found to be an effective inhibitor of alkene MO from X. autotrophicus Py2, whereas acetylene had no effect (Ensign et al., 1992).For toluene 4-monooxygenase, which is closely related to IsoMO (Crombie et al., 2015), acetylene was ineffective compared with aliphatic or aromatic alkynes (Yeager et al., 1999).It would be interesting to test the effects of aromatic alkynes on IsoMO activity, as differences in cellular architecture and physiology may cause variations in the effectiveness of alkyne inhibitors.

The production of epoxyisoprene from isoprene by monooxygenases
We assessed whether other monooxygenases, including the membrane-bound copper CuMMO, could oxidise isoprene during whole-cell activity assays by measuring epoxyisoprene formation from isoprene.Methane oxidisers and the nitrifier N. europaea, expressing AMO, were supplemented with formate or hydrazine, respectively, as external reductant.For the isoprene degrader Rhodococcus sp.AD45, epoxyhexane, an irreversible inhibitor of the glutathione Stransferase IsoI, was added to cell suspensions to prevent the subsequent metabolism of the epoxyisoprene generated (van Hylckama Vlieg et al., 1998).The rate of epoxyisoprene production was quantified using GC-MS (Table 1).
The rates of epoxyisoprene production from isoprene by cells expressing SDIMOs were similar (1.38-1.88nmol min À1 mg dw À1 ) but contrasted with methanotrophs expressing pMMO (Table 1), which were 20-fold less for both M. alcaliphilum and M. capsulatus.Curiously, AMO from N. europaea (a CuMMO similar to pMMO of methanotrophs) had the fastest rate of all the oxygenases tested, approximately three times faster than IsoMO from Rhodococcus sp.AD45.However, since epoxides are highly reactive and toxic to the cell, it is likely that the accumulation of epoxyisoprene would prevent high rates being sustained for longer periods in non-isoprene degraders.Epoxyisoprene production by the different bacterial strains was efficiently inhibited by the alkynes specific to the oxygenase expressed.For sMMO and CuMMOs acetylene was used, for Rhodococcus sp.AD45 and X. autotrophicus Py2 1-octyne and 1-propyne, respectively (Table 1).

Isoprene and propylene uptake kinetics by whole cells
To examine the specificity of IsoMO and alkene MO for isoprene and propylene, uptake kinetic parameters were determined for whole cells of the isoprene degraders Rhodococcus sp.AD45 and Variovorax sp.WS11, and the propylene degrader X. autotrophicus Py2, grown on isoprene or propylene, respectively, by measuring the rate of depletion of headspace gas.Isoprene and propylene uptake by Rhodococcus sp.AD45, Variovorax sp.WS11 and X. autotrophicus Py2 followed Michaelis-Menten type saturation kinetics.The K m(app) and V max(app) for isoprene determined here for Rhodococcus sp.AD45 were 4.0 μM and 47.2 nmol min À1 mg dw À1 , respectively (Table 2), in reasonably good agreement with the results of van Hylckama Vlieg et al. (1998).Cells of Variovorax sp.WS11 had a four-fold lower affinity (10.5 μM) but a similar maximum rate, comparable with values reported for the isoprene degrader Sphingopyxis sp.OPL5 (Larke-Mejía et al. (2020), Table 2).Interestingly, Rhodococcus sp.AD45 had a similar affinity and maximum oxidation rate for its non-growth substrate, propylene, as for isoprene (K m(app) (propylene) and V max(app) (propylene) of 2.0 μM and 36.6 nmol min À1 mg dw À1 respectively) (Table 2).X. autotrophicus Py2 had a similar affinity for propylene (K m(app) 5.0 μM), its growth substrate, as Rhodococcus sp.AD45 and only a moderately higher maximum rate, (V max(app) 97.2 nmol min À1 mg dw À1 ) (Table 2).However, its affinity for isoprene was more than 10-fold lower (K m(app) 55.1 μM), consistent with the fact that isoprene is not its natural substrate.

CONCLUSIONS
In summary, using whole-cell studies, we show that isoprene monooxygenase from the Gram-positive Rhodococcus sp.AD45 is a catalytically versatile member of the SDIMO enzyme family, which cooxidises a wide range of alkenes, dienes and even aromatic compounds, consistent with findings in the Gram-negative Variovorax.This demonstrates that isoprene monooxygenases from distinctly different groups of microorganisms, with relatively modest levels of sequence identity, are functionally similar in terms of their inhibition and co-oxidation profiles.IsoMO generates isoprene epoxide with a high enantiomeric excess (Dawson et al., 2023) and the biotechnological potential of this enzyme should be explored further for the production of bulk and high value compounds such as chiral epoxides.We demonstrate that particulate methane monooxygenase cannot cooxidise isoprene but that soluble methane monooxygenase, and surprisingly, ammonia monooxygenase from Nitrosomonas, can co-oxidise isoprene at significant rates.This has implications for alternative routes of isoprene removal/degradation by the multiple different monooxygenases represented in microbial communities.For example, peat uplands are significant sources of both methane and isoprene and methanotrophs are abundant in these environments (Chen et al., 2008;Faubert et al., 2011).Currently, however, the extent to which methanotrophs contribute to isoprene degradation (and vice versa) is unknown.Similarly, estuarine and coastal regions are considerable sources of isoprene, while harbouring diverse nitrifier communities (Bernhard & Bollmann, 2010;Exton et al., 2012), also with an unknown impact on isoprene biodegradation.This co-metabolic activity may also supply carbon to the wider community via hydrolysis of the relatively unstable epoxide (Gervasi & Longo, 1990).While the data presented here suggest that these effects may not be negligible, we also provide a toolkit for its quantification.We show that 1-octyne is a potent inhibitor of isoprene monooxygenase and that acetylene, which inhibits oxygenases such as methane and ammonia monooxygenase, does not significantly inhibit isoprene monooxygenase.Thus, parallel incubations of environmental samples, either unamended or following inhibition with either octyne or acetylene, provide a method to evaluate the contribution of these co-oxidisers to isoprene degradation.This has important implications for future environmental studies which seek to distinguish the relative roles of isoprene degradation by bona fide isoprene-utilising bacteria from co-oxidation by other groups such as methanotrophs and nitrifiers.
T A B L E 2 Isoprene and propylene uptake kinetics of isoprene degraders, in comparison with X. autotrophicus Py2.

Strain (oxygenase)
K m(app) (μM) V max(app) (nmol min À1 mg dw sp. AD45) oxidises many of the same substrates as Rhodococcus sp.AD45 and Variovorax sp.WS11, F I G U R E 1 Relative rates of substrate-induced oxygen uptake by whole-cell suspensions of isoprene-grown (dark grey bars) or succinategrown (light grey bars) Rhodococcus sp.AD45.Rates are presented as a percentage of the rate of isoprene-induced oxygen uptake by isoprene-grown cells.Oxygen uptake rate induced by (A) terminal alkenes and (B) dienes, cyclic alkenes, and aromatics.Error bars show standard error of the mean (n = 3).*, low-solubility substrate.
T A B L E 1 Initial rates of epoxyisoprene production from isoprene.Strain (oxygenase expressed)Rate of epoxyisoprene formation (nmol min À1 mg dw À1 ) Epoxyisoprene formation in the presence of the oxygenase inhibitor (in parentheses) The rates shown are the mean of three replicates ± standard error of the mean.Abbreviation: n.d., not detected.
À1) Reference The data from this study, (shown in bold font ± standard error (95% confidence interval), are compared with previous studies.
Note:a Based on substrate-induced oxygen uptake.b Converted from gas phase concentration.c Rate given as per mg protein.