1,2,3-Trichlorobenzene transformation by Methylosinus trichosporium OB3b expressing soluble methane monooxygenase

 Transformation of 1,2,3- and 1,2,4-trichlorobenzene in the presence of 20 mM sodium formate, by the methanotrophic bacterium Methylosinus trichosporium OB3b, was studied using cells grown in batch and continuous culture. Only 1,2,3-trichlorobenzene was transformed and transformation was strictly co-metabolic, only catalysed in the presence of the soluble form of methane monooxygenase. The kinetics of transformation could be described by simple first-order kinetics (0.00193 l min^-1 g^-1). Also the kinetics of transformation were found to be linearly proportional to cell density. No chloride ion release was observed during the reaction and the products of transformation (2,3,4- and 3,4,5-trichlorophenol) were identified by gas chromatography/mass spectroscopy and ¹H-NMR and a 1.84:1 ratio of products in favour of para hydroxylation was observed. It was also observed that the relationship between mass of substrate transformed and cell density was linear giving a transformation capacity of 88.8±11.8 μmol g^-1, after which the transformation of 1,2,3-trichlorobenzene was inhibited. This inhibition was not due to O₂ limitation, co-substrate (CHOONa) limitation or product inhibition. Recovery and washing of the cells did not reverse this inhibition, indicating that inhibition was irreversible. During transformation a substantial decrease in the endogenous and formate-dependent O₂ consumption rates was observed, although the methanol-dependent O₂ consumption rate varied little between fresh cell samples and samples that had been used to transform 1,2,3-trichlorobenzene. Received: 22 June 1995 / Received last revision: 26 October 1995 / Accepted: 30 October 1995     

Conversion of chlorinated propanes by Methylosinus trichosporium OB3b expressing soluble methane monooxygenase

Chlorinated propanes are important pollutants that may show persistent behaviour in the environment. The biotransformation of 1-chloropropane, 1,2-dichloropropane, 1,3-dichloropropane and 1,2,3-trichloropropane was studied using resting cell suspensions of Methylosinus trichosporium OB3b expressing soluble methane monooxygenase. The transformation followed first-order kinetics. The rate constants were in the order 1-chloropropane > 1,3-dichloropropane > 1,2-dichloropropane > 1,2,3-trichloropropane, and varied from 0.07 to 1.03 ml min⁻¹ mg of cells⁻¹ for 1,2,3-trichloropropane and 1-chloropropane respectively. Turnover-dependent inactivation occurred for all of the chloropropanes tested. The inactivation constants were lower for 1-chloropropane and 1,2-dichloropropane than for 1,2,3-trichloropropane and 1,3-dichloropropane. Not all the chloride was released during cometabolic transformation of the chlorinated propanes and production of monochlorinated- and dichlorinated propanols was found by gas chromatography. The reaction pathway of 1,2,3-trichloropropane conversion was studied by mass spectrometric analysis of products formed in ²H₂O, which indicated that 1,2,3-trichloropropane was initially oxidized to 2,3-dichloropropionaldehyde and 1,3-dichloroacetone, depending on whether oxygen insertion occurred on the C-3 or C-2 carbon of 1,2,3,-trichloropropane, followed by reduction to the corresponding propanols. The results show that chloropropanes are susceptible to cometabolic oxidation by methanotrophs, but that the transformation kinetics is worse than with cometabolic conversion of trichloroethylene. Received: 27 November 1997 / Received revision: 27 February 1998 / Accepted: 27 February 1998     

The metal centers of particulate methane monooxygenase from Methylosinus trichosporium OB3b

Particulate methane monooxygenase (pMMO) is a membrane-bound metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. The nature of the pMMO active site and the overall metal content are controversial, with spectroscopic and crystallographic data suggesting the presence of a mononuclear copper center, a dinuclear copper center, a trinuclear center, and a diiron center or combinations thereof. Most studies have focused on pMMO from Methylococcus capsulatus (Bath). pMMO from a second organism, Methylosinus trichosporium OB3b, has been purified and characterized by spectroscopic and crystallographic methods. Purified M. trichosporium OB3b pMMO contains approximately 2 copper ions per 100 kDa protomer. Electron paramagnetic resonance (EPR) spectroscopic parameters indicate that type 2 Cu(II) is present as two distinct species. Extended X-ray absorption fine structure (EXAFS) data are best fit with oxygen/nitrogen ligands and reveal a Cu-Cu interaction at 2.52 A. Correspondingly, X-ray crystallography of M. trichosporium OB3b pMMO shows a dinuclear copper center, similar to that observed previously in the crystal structure of M. capsulatus (Bath) pMMO. There are, however, significant differences between the pMMO structures from the two organisms. A mononuclear copper center present in M. capsulatus (Bath) pMMO is absent in M. trichosporium OB3b pMMO, whereas a metal center occupied by zinc in the M. capsulatus (Bath) pMMO structure is occupied by copper in M. trichosporium OB3b pMMO. These findings extend previous work on pMMO from M. capsulatus (Bath) and provide new insight into the functional importance of the different metal centers.     

Role of iron in particulate methane monooxygenase from Methylosinus trichosporium OB3b

The effect of iron ions on particulate methane monooxygenase was studied by using the EDTA-treated membranes from Methylosinus trichosporium OB3b. When the membrane was treated with EDTA the activity remained 82% of the as-isolated membranes, and the activity of the EDTA-treated membranes was strongly influenced by the addition of metal ions. Among the metal ions, ferric, ferrous and cupric ions stimulated the activity, indicating those ions were needed for the activity. When propargylamine was added, pMMO activity decreased and also the iron ESR signal decreased. As the ESR signal involves the ferrous nitrosyl complex in EDTA-treated membranes, the active site of pMMO may contain a mononuclear non-heme iron.     

Degradation of chlorinated aliphatic hydrocarbons by Methylosinus trichosporium OB3b expressing soluble methane monooxygenase

Degradation of trichloroethylene (TCE) by the methanotrophic bacterium Methylosinus trichosporium OB3b was studied by using cells grown in continuous culture. TCE degradation was a strictly cometabolic process, requiring the presence of a cosubstrate, preferably formate, and oxygen. M. trichosporium OB3b cells degraded TCE only when grown under copper limitation and when the soluble methane monooxygenase was derepressed. During TCE degradation, nearly total dechlorination occurred, as indicated by the production of inorganic chloride, and only traces of 2,2,2-trichloroethanol and trichloroacetaldehyde were produced. TCE degradation proceeded according to first-order kinetics from 0.1 to 0.0002 mM TCE with a rate constant of 2.14 ml min-1 mg of cells-1. TCE concentrations above 0.2 mM inhibited degradation in cell suspensions of 0.42 mg of cells ml-1. Other chlorinated aliphatics were also degraded by M. trichosporium OB3b. Dichloromethane, chloroform, 1,1-dichloroethane, and 1,2-dichloroethane were completely degraded, with the release of stoichiometric amounts of chloride. trans-1,2-Dichloroethylene, cis-1,2-dichloroethylene, and 1,2-dichloropropane were completely converted, but not all the chloride was released because of the formation of chlorinated intermediates, e.g., trans-2,3-dichlorooxirane, cis-2,3-dichlorooxirane, and 2,3-dichloropropanol, respectively. 1,1,1-Trichloroethane, 1,1-dichloroethylene, and 1,3-dichloropropylene were incompletely converted, and the first compound yielded 2,2,2-trichloroethanol as a chlorinated intermediate. The two perchlorinated compounds tested, carbon tetrachloride and tetrachloroethylene, were not converted.     

Redox behavior of copper in particulate methane monooxygenase from Methylosinus trichosporium OB3b

The redox properties of the copper in particulate methane monooxygenase from Methylosinus trichosporium OB3b were investigated. The ESR spectrum of the pMMO-containing membranes from M. trichosporium OB3b indicated a typical type II copper (II) signal (g = 2.24, A = 18.4 mT, g = 2.06, ₂= 0.84). By anaerobic addition of excess amounts of duroquinol, an optimum reductant of pMMO, the ESR spectra indicated that the copper cluster in membranes was reduced and successively oxidized by dioxygen, a substrate of pMMO. The result suggests that the copper is the active site of pMMO or an electron carrier. During the titration, the intensity of the type II copper signal decreased with decreasing potential and the multiple hyperfine structure at g = 2.06 appeared clearly. Although the copper signal did not change by treatment of the EDTA-treated membranes with duroquinol and dioxygen, the copper signal intensity decreased with decreasing potential in the redox titration. These results suggest that some redox mediators play a role as an electron carrier between the active site and a reductant, and the presence of at least two types of copper sites in pMMO- containing membranes. On the basis of the ESR spectra of the EDTA-treated membranes and the as-isolated membranes, it is concluded that one type of the copper sites functions as the active site of pMMO (A-site), and the other type of copper sites plays a role as an electron carrier (E-site)     

Batch cultivation of Methylosinus trichosporium OB3b. I: Production of soluble methane monooxygenase

Methanotrophs have promising applications in bioremediation and in the production of fuel-related chemicals due to their nonspecific enzyme, methane monooxygenase (MMO). The optimal conditions for cell growth and production of the soluble from of MMO (sMMO) were determined from batch cultivations of an obligatory methanotrophs, Methylosinus trichosporium OB3b, in shake flasks and a 5-L bioreactor. It was confirmed that a copper deficiency is essential for the formation of the cytoplasmic sMNO. Optimum cell growth without added copper was observed at pH 6.0-7.0, temperature of 30-34 degrees C, and phosphate concentration of 10-40 mM. In the bioreactor experiments, external CO(2) addition eliminated the long lag period observed in the absence of added CuSO(4), i.e., prior to the exponential cell growth phase. When methane was continuously supplied, the profile of the cell growth showed two different phases depending on the availability of nitrate, an initial fast exponential growth phase (specific growth rate, mu = 0.08 h(-1)) and a later slow growth phase (mu = 0.008 h(-1)). The cell density at the transition from a fast to a slow growth rate was proportional to the initial medium nitrate concentration in the range 5-20 mM and cell yield was estimated to be 7.14 g dry cell wt/g N. Whole-cell sMNO activity remained essentially constant regardless of the growth rate unit cell growth stopped. With an initial medium iron concentration below 40 mM, an abrupt decrease in sMNO activity was observed. The lower sMNO activity could be restored by supplying additional iron to the bioreactor culture. Cell yield on iron was estimated to be 1.3 x 10(3) g dry cell wt/g Fe.     

Dichloromethane and trichloroethylene inhibition of methane oxidation by the membrane-associated methane monooxygenase of Methylosinus trichosporium OB3b

Whole-cell assays were used to measure the effect of dichloromethane and trichloroethylene on methane oxidation by Methylosinus trichosporium OB3b synthesizing the membrane-associated or particulate methane monooxygenase (pMMO). For M. trichosporium OB3b grown with 20 μM copper, no inhibition of methane oxidation was observed in the presence of either dichloromethane or trichloroethylene. If 20 mM formate was added to the reaction vials, however, methane oxidation rates increased and inhibition of methane oxidation was observed in the presence of dichloromethane and trichloroethylene. In the presence of formate, dichloromethane acted as a competitive inhibitor, while trichloroethylene acted as a noncompetitive inhibitor. The finding of noncompetitive inhibition by trichloroethylene was further examined by measuring the inhibition constants K _iE and K _iES. These constants suggest that trichloroethylene competes with methane at some sites, although it can bind to others if methane is already bound. Whole-cell oxygen uptake experiments for active and acetylene-treated cells also showed that provision of formate could stimulate both methane and trichloroethylene oxidation and that trichloroethylene did not affect formate dehydrogenase activity. The finding that different chlorinated hydrocarbons caused different inhibition patterns can be explained by either multiple substrate binding sites existing in pMMO or multiple forms of pMMO with different activities. The whole-cell analysis performed here cannot distinguish between these models, and further work should be done on obtaining active preparations of the purified pMMO. Received: 3 November 1998 / Accepted: 1 March 1999     

Degradation of chlorinated aliphatic hydrocarbons by Methylosinus trichosporium OB3b expressing soluble methane monooxygenase.

Degradation of trichloroethylene (TCE) by the methanotrophic bacterium Methylosinus trichosporium OB3b was studied by using cells grown in continuous culture. TCE degradation was a strictly cometabolic process, requiring the presence of a cosubstrate, preferably formate, and oxygen. M. trichosporium OB3b cells degraded TCE only when grown under copper limitation and when the soluble methane monooxygenase was derepressed. During TCE degradation, nearly total dechlorination occurred, as indicated by the production of inorganic chloride, and only traces of 2,2,2-trichloroethanol and trichloroacetaldehyde were produced. TCE degradation proceeded according to first-order kinetics from 0.1 to 0.0002 mM TCE with a rate constant of 2.14 ml min-1 mg of cells-1. TCE concentrations above 0.2 mM inhibited degradation in cell suspensions of 0.42 mg of cells ml-1. Other chlorinated aliphatics were also degraded by M. trichosporium OB3b. Dichloromethane, chloroform, 1,1-dichloroethane, and 1,2-dichloroethane were completely degraded, with the release of stoichiometric amounts of chloride. trans-1,2-Dichloroethylene, cis-1,2-dichloroethylene, and 1,2-dichloropropane were completely converted, but not all the chloride was released because of the formation of chlorinated intermediates, e.g., trans-2,3-dichlorooxirane, cis-2,3-dichlorooxirane, and 2,3-dichloropropanol, respectively. 1,1,1-Trichloroethane, 1,1-dichloroethylene, and 1,3-dichloropropylene were incompletely converted, and the first compound yielded 2,2,2-trichloroethanol as a chlorinated intermediate. The two perchlorinated compounds tested, carbon tetrachloride and tetrachloroethylene, were not converted.     

Effect of copper speciation on whole-cell soluble methane monooxygenase activity in Methylosinus trichosporium OB3b

Soluble methane monooxygenase (sMMO) activity in Methylosinus trichosporium OB3b was found to be more strongly affected as copper-to-biomass ratios changed in a newly developed medium, M2M, which uses pyrophosphate for metal chelation, than in nitrate mineral salts (NMS), which uses EDTA. When M2M medium was amended with EDTA, sMMO activity was similar to that in NMS medium, indicating that EDTA-bound copper had lower bioavailability than pyrophosphate-bound copper. EDTA did not limit the association of copper with the cells; rather, copper was sequestered in a form which did not affect sMMO activity.     

Haloalkene oxidation by the soluble methane monooxygenase from Methylosinus trichosporium OB3b: mechanistic and environmental implications

The soluble, three-protein component methane monooxygenase purified from Methylosinus trichosporium OB3b is capable of oxidizing chlorinated, fluorinated, and brominated alkenes, including the widely distributed ground-water contaminant trichloroethylene (TCE). The oxidation rates for the chloroalkenes were observed to be comparable to that for methane, the natural substrate, and up to 7000-fold higher than those reported for other well-defined biological systems. The competitive inhibitor tetrachloroethylene was found to be the only chlorinated ethylene not turned over. However, this appears to be due to steric effects rather than electronic effects or the lack of an abstractable proton because chlorotrifluoroethylene was efficiently oxidized. As evidenced by the formation of diagnostic adducts with 4-(p-nitrobenzyl)pyridine, the halogenated alkenes were oxidized predominantly by epoxidation. Stable acidic products resulting from subsequent hydrolysis were identified as the major products. However, additional aldehydic products resulting from intramolecular halide or hydride migration were observed in 3-10% yield during the oxidation of TCE, vinylidene chloride, trifluorethylene, and tribromoethylene. Product analysis of the hydrolysis reaction of authentic TCE epoxide showed little or no 2,2,2-trichloroacetaldehyde (chloral) formation, indicating that atomic migration occurred prior to product dissociation from the enzyme. The occurrence of atomic migration products shows that an intermediate in the substrate to product conversion carries significant cationic character. Such a species could be generated through interaction with a highly electron-deficient activated oxygen in the active site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of the membranes containing the particulate methane monooxygenase from Methylosinus trichosporium OB3b

The particulate methane monooxygenase (pMMO) from Methylosinus trichosporium OB3b was partiallypurified and characterized by measuring the effects of reducing agents and additives, and the stability ofpMMO was studied. Duroquinol was a suitable reducing agent, and pMMO was stabilized by bovine serumalbumin (BSA). Among the additivies, the copper (II) ion stimulated pMMO at low concentration andinhibited at high concentration. The optimum conditions for pMMO activity were as follows: 45 ° C, pH 6.5and 55 mM 3-morpholinopropanesulfonic acid (MOPS) buffer, and the rate of propene epoxide formationwas 13.6 nmol min mg protein. ESR spectra indicate that the copper cluster in the membrane fraction isreduced by duroquinol and oxidized by dioxygen. The result suggests that the copper cluster is containedin the active site of pMMO.     

Trichloroethylene and chloroform degradation by a recombinant pseudomonad expressing soluble methane monooxygenase from Methylosinus trichosporium OB3b.

Soluble methane monooxygenase (sMMO) from Methylosinus trichosporium OB3b can degrade many halogenated aliphatic compounds that are found in contaminated soil and groundwater. This enzyme oxidizes the most frequently detected pollutant, trichloroethylene (TCE), at least 50 times faster than other enzymes. However, slow growth of the strain, strong competition between TCE and methane for sMMO, and repression of the smmo locus by low concentrations of copper ions limit the use of this bacterium. To overcome these obstacles, the 5.5-kb smmo locus of M. trichosporium OB3b was cloned into a wide-host-range vector (to form pSMMO20), and this plasmid was electroporated into five Pseudomonas strains. The best TCE degradation results were obtained with Pseudomonas putida F1/pSMMO20. The plasmid was maintained stably, and all five of the sMMO proteins (alpha, beta, and gamma hydroxylase proteins, reductase, and component B) were observed clearly by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblotting. TCE degradation rates were quantified for P. putida F1/pSMMO20 with a gas chromatograph (Vmax = 5 nmol per min per mg of protein), and the recombinant strain mineralized 55% of the TCE (10 microM) as indicated by measuring chloride ion concentrations with a chloride ion-specific electrode. The maximum TCE degradation rate obtained with the recombinant strain was lower than that of M. trichosporium OB3b but greater than other TCE-degrading recombinants and most well-studied pseudomonads. In addition, this recombinant strain mineralizes chloroform (a specific substrate for sMMO), grows much faster than M. trichosporium OB3b, and degrades TCE without competitive inhibition from the growth substrate.     

Methylosinus trichosporium OB3b whole-cell methane monooxygenase activity in a biphasic matrix

 Reconstituted whole-cell preparations of lyophilized Methylosinus trichosporium OB3b were used to demonstrate soluble methane monooxygenase activity in a two-phase (biphasic) matrix consisting of a buffered aqueous phase and 2,2,4-trimethylpentane (isooctane). The rate of conversion of gaseous propylene to propylene oxide, a non-metabolized liquid, was used as the primary measure of enzyme activity. Appreciable soluble methane monooxygenase activity was detected when the volume of the aqueous phase represented at least 1% of the total volume, although the initial rate of product formation did increase as the volume of the aqueous phase increased. In comparison to the aqueous system, the specific rate and yields in the biphasic system were much less sensitive to increases in the concentrations of formate and protein (the methane monooxygenase). However, there was some evidence that the enzyme system was more stable in the biphasic matrix, since the rate of propylene oxide formation remained linear for an extended period of time. V _(app.) in the biphasic system decreased by a factor of 0.6 relative to the same parameter in the aqueous system. Conversely, K _m(app.) for propylene was 1.6 times greater in the biphasic system. Hence, the apparent catalytic efficiency in the aqueous system was four times that in the biphasic system, as indicated by a decrease in the corresponding ratios of V _(app.) to K _m(app.). Received: 21 July 1995/Received last revision: 1 February 1996/Accepted: 5 December 1996     

Enhancement of biomass production and soluble methane monooxygenase activity in continuous cultures of Methylosinus trichosporium OB3b

Summary Using cell recycle, the concentration of M. trichosporium OB3b was increased to more than 12g/L in continuous culture and biomass productivity was enhanced up to 0.708g/L/h. The optimal temperature and pH for soluble MMO activity were 35°C and 6.2–6.4. The Optimal concentrations of methanol and sodium formate, as cofactor regenerators, were 0.5mM and 10 mM, respectively, at which the soluble MMO activity was about 8 times enhanced.     

Crystal structure of the hydroxylase component of methane monooxygenase from Methylosinus trichosporium OB3b.

Methane monooxygenase (MMO), found in aerobic methanotrophic bacteria, catalyzes the O2-dependent conversion of methane to methanol. The soluble form of the enzyme (sMMO) consists of three components: a reductase, a regulatory "B" component (MMOB), and a hydroxylase component (MMOH), which contains a hydroxo-bridged dinuclear iron cluster. Two genera of methanotrophs, termed Type X and Type II, which differ markedly in cellular and metabolic characteristics, are known to produce the sMMO. The structure of MMOH from the Type X methanotroph Methylococcus capsulatus Bath (MMO Bath) has been reported recently. Two different structures were found for the essential diiron cluster, depending upon the temperature at which the diffraction data were collected. In order to extend the structural studies to the Type II methanotrophs and to determine whether one of the two known MMOH structures is generally applicable to the MMOH family, we have determined the crystal structure of the MMOH from Type II Methylosinus trichosporium OB3b (MMO OB3b) in two crystal forms to 2.0 A resolution, respectively, both determined at 18 degrees C. The crystal forms differ in that MMOB was present during crystallization of the second form. Both crystal forms, however, yielded very similar results for the structure of the MMOH. Most of the major structural features of the MMOH Bath were also maintained with high fidelity. The two irons of the active site cluster of MMOH OB3b are bridged by two OH (or one OH and one H2O), as well as both carboxylate oxygens of Glu alpha 144. This bis-mu-hydroxo-bridged "diamond core" structure, with a short Fe-Fe distance of 2.99 A, is unique for the resting state of proteins containing analogous diiron clusters, and is very similar to the structure reported for the cluster from flash frozen (-160 degrees C) crystals of MMOH Bath, suggesting a common active site structure for the soluble MMOHs. The high-resolution structure of MMOH OB3b indicates 26 consecutive amino acid sequence differences in the beta chain when compared to the previously reported sequence inferred from the cloned gene. Fifteen additional sequence differences distributed randomly over the three chains were also observed, including D alpha 209E, a ligand of one of the irons.     

Optimization of trichloroethylene degradation using soluble methane monooxygenase of Methylosinus trichosporium OB3b expressed in recombinant bacteria

By complementing cell-free extracts of Pseudomonas putida F1/pSMMO20 with purified soluble methane monooxygenase (sMMO) components of Methylosinus trichosporium OB3b, the low cloned-gene sMMO activity in the recombinant strain was found to be due to incomplete activity of the hydroxylase component. To address this incomplete activity, additional sMMO-expressing strains were formed by transferring mmo-containing pSMMO20 and pSMMO50 into various bacterial species including pseudomonads and alpha-2 subdivision strains such as methanotrophs, methylotrophs, Agrobacterium tumefaciens A114, and Rhizobium meliloti 102F34 (11 new strains screened); sMMO activity was detected in the last two strains. To increase plasmid segregational stability, the hok/sok locus originally from Escherichia coli plasmid R1 was inserted downstream of the mmo locus of pSMMO20 (resulting in pSMMO40) and found to enhance plasmid stability in P. putida F1 and R. meliloti 102F34 (first report of hok/sok in Rhizobium). To further increase sMMO activity, a modified Whittenbury minimal medium was selected from various minimal and complex media based on trichloroethylene (TCE) degradation and growth rates and was improved by removing the sMMO-inhibiting metal ions [Cu(II), Ni(II), and Zn(II)] and chloramphenicol from the medium and by supplementing with an iron source (3.6 muM of ferrous ammonium sulfate). Using chemostat-grown P. putida F1/pSMMO40, it was found that sMMO activity was higher for cells grown at higher dilution rates. These optimization efforts resulted in a twofold increase in the extent of TCE degradation and more consistent sMMO activity. (c) 1996 John Wiley & Sons, Inc.     

X-ray absorption spectroscopic studies of the methane monooxygenase hydroxylase component from Methylosinus trichosporium OB3b

The conversion from methane to methanol is catalyzed by methane monooxygenase (MMO) in methanotrophic bacteria. Earlier work on the crystal structures of the MMO hydroxylase component (MMOH) from Methylococcus capsulatus (Bath) at 4??°C and –160??°C has revealed two different core arrangements for the diiron active site. To ascertain the generality of these results, we have now carried out the first structural characterization on MMOH from Methylosinus trichosporium OB3b. Our X-ray absorption spectroscopic (XAS) analysis suggests the presence of two Fe-Fe distances of about 3?Å and 3.4?Å, which are proposed to reflect two populations of MMOH molecules with either a bis(μ-hydroxo)(μ-carboxylato)- or a (μ-hydroxo)(μ-carboxylato)diiron(III) core structure, respectively. The observation of these two different core structures, together with the crystallographic results of the MMOH from Methylococcus capsulatus (Bath), suggests the presence of an equilibrium that may reflect a core flexibility that is required to accommodate the various intermediates in the catalytic cycle of the enzyme. XAS studies on the binding of component B (MMOB) to the hydroxylase component show that MMOB does not perturb either this equilibrium or the gross structure of the oxidized diiron site in MMOH.     

Optimization and maintenance of soluble methane monooxygenase activity inMethylosinus trichosporium OB3b

Soluble methane monooxygenase (sMMO) maximization studies were carried out as part of a larger effort directed towards the development and optimization of an aqueous phase, multistage, membrane bioreactor system for treatment of polluted groundwater. A modified version of the naphthalene oxidation assay was utilized to determine the effects of methane:oxygen ratio, nutrient supply, and supplementary carbon sources on maximizing and maintaining sMMO activity inMethylosinus trichosporium OB3b.Methylosinus trichosporium OB3b attained peak sMMO activity (275–300 nmol of naphthol formed h^–1 mg of protein^–1 at 25°C) in early stationary growth phase when grown in nitrate mineral salts (NMS) medium. With the onset of methane limitation however, sMMO activity rapidly declined. It was possible to define a simplified nitrate mineral salts (NMS) medium, containing nitrate, phosphate and a source of iron and magnesium, which allowed reasonably high growth rates (_max 0.08 h^–1) and growth yields (0.4–0.5 g cells/g CH₄) and near maximal activities of sMMO. In long term batch culture incubations sMMO activity reached a stable plateau at approximately 45–50% of the initial peak level and this was maintained over several weeks. The addition of d-biotin, pyridoxine, and vitamin B₁₂ (cyanocobalamin) increased the activity level of sMMO in actively growing methanotrophs by 25–75%. The addition of these growth factors to the simplified NMS medium was found to increase the plateau sMMO level in long term batch cultures up to 70% of the original peak activity.Abbreviations sMMO soluble methane monooxygenase - pMMO particulate methane monooxygenase - NMS nitrate mineral salts - TCE trichloroethene - NADH reduced nicotinamide adenine dinucleotide     

Optimization of methanol biosynthesis from methane using Methylosinus trichosporium OB3b

Methylosinus trichosporium OB3b oxidized methane to methanol in the presence of a high concentration of Cu2+. Further oxidation of methanol to formaldehyde was prevented by adding 200 mM NaCl which acted as a methanol dehydrogenase H inhibitor. The bacterium, 0.6 mg dry cell ml(-1), in methane/air (1:4, v/v) at 25 degrees C in 12.9 mM phosphate buffer (pH 7) containing 20 mM sodium formate and 200 mM NaCl accumulated 7.7 mM methanol over 36 h.