Effect of various co-substrates on 1,2-epoxypropane formation from propene by ethene-utilizing Mycobacteria

Summary Ethanol, ethylacetate and ethene were tested as no-substrates to improve prolonged 1,2-epoxypropane formation from propene by immobilized cells of three strains (Eu1, 2W, E3) of ethene-utilizing Mycobacteria. Cells grown either on ethene or on both ethene and ethanol were immobilised on lava and the effect of co-substrates on 1,2-epoxypropane formation from propene was recorded in a gas/solid bioreactor. The rate of 1,2-epoxypropane formation by immobilized cells of strains Eu1 and 2W was significantly enhanced when ethanol or ethylacetate were supplied simultaneously with propene, while biocatalysis during a prolonged period of time was achieved in the presence of ethylacetate. Co-substrates had no beneficial effect on 1,2-epoxypropane formation by strain E3. It was possible to increase the 1,2-epoxypropane formation rate by immobilized cells over a period of three weeks of operation by supplying propene and ethene alternately.     

Synthesis of optically pure 1,2-epoxypropane by microbial asymmetric reduction of chloroacetone

A number of bacteria and yeast was screened for asymmetric reduction of prochiral chloroacetone into chiral 1-chloro-2-propanol, which is chemically convertible into chiral 1,2-epoxypropane. In this way Rhodotorula glutinis produced optically pure S-1,2-epoxypropane with 98% enantiomeric excess and in a relatively high final concentration. The enzyme that catalysed the asymmetric reduction was an NAD(P)H-dependent alcohol dehydrogenase. Reduction of racemic 3-chloro-2-butanone resulted in mixtures of cis and trans-2,3-epoxybutane, indicating that no enantioselective reduction of this haloketone occurred. Correspondence to: C. A. G. M. Weijers     

Production of epoxides from gaseous alkenes by resting-cell suspensions and immobilized cells of alkene-utilizing bacteria

Summary Newly isolated and already available strains of alkene-utilizing bacteria were able to oxidize ethene, propene or 1-butene to the respective 1,2-epoxides. Resting-cell suspensions of organisms isolated on propene and butene, when grown on these substrates converted ethene quantitatively to epoxyethane. Some, but not all ethene-utilizing strains accumulated 1,2-epoxypropane or 1,2-epoxybutane when propene or butene was supplied, although not quantitatively because the epoxides produced were partially further metabolized. Suitable epoxide producers which eventually may be employed as biocatalysts in a biotechnological process were used for immobilization in calcium alginate and K-carrageenan; after immobilization, 60%–100% activity for epoxide production was retained.     

Gaseous alkene biotransformation and enantioselective epoxyalkane formation by Nocardioides sp. strain JS614

Enantiopure epoxides are valuable intermediates in the synthesis of optically pure biologically active fine chemicals (e.g., pharmaceuticals) that are often difficult to produce by chemical approaches. An attractive alternative is biological synthesis by microorganisms expressing stereoselective enzymes. In this study, we investigated the ability of ethene-grown Nocardioides sp. strain JS614 to produce highly enantio-enriched epoxyalkanes via stereoselective monooxygenase-mediated alkene epoxidation. Ethene-grown JS614 cells transformed propene, 1-butene, and trans-2-butene to their corresponding epoxyalkanes at rates ranging from 27.1 to 44.0 nmol/min mg protein. Chiral gas chromatography analysis revealed that R-1,2-epoxypropane, R-1,2-epoxybutane, and trans-2R,3R-epoxybutane were produced in enantiomeric excess (e.e.) of 98%, 74%, and 82%, respectively. Ethene-grown JS614 cells also preferentially transformed trans-2S,3S-epoxybutane from a racemic mixture, but could not resolve racemic 1,2-epoxypropane. Glucose facilitated increased epoxyalkane production by ethene-grown JS614 cells. However, after 22 h of propene biotransformation with 20 mM glucose, 84% of ethene-grown JS614 cells lost membrane integrity and the remaining live cells were not viable. Propene biotransformation by JS614 was extended beyond 22 h and 54% more epoxypropane was produced when cells were resuspended in fresh buffer + glucose at 8-h intervals. We conclude that JS614 is a promising new biocatalyst for applications that involve enantiopure epoxide production.     

Biocatalysts for production of chiral epoxides

Summary A number of bacterial strains, representing a range of genera, were isolated in pure culture with ethene or propene as the sole source of carbon and energy. The organisms included Aerococcus, Alcaligenes, Micrococcus and Staphylococcus spp. and a variety of Gram-negative, Gram-positive and Gram-variable mesophilic rods/ coccobacilli not yet identified. This suggests that the ability to utilize gaseous olefins is more widespread in nature than previously recognised. All 18 organisms tested stereospecifically formed R-1,2-epoxypropane (enantiomeric excess, ee=90–96%), R-1,2-epoxybutane (ee=90–98%) and trans-(2R,3R)-epoxybutane (ee=64–88%) from the corresponding olefins. In addition to Micrococcus sp. M90C, the substrate specificities of six other organisms were studied. The pattern of reactivity for the group of four ethene (M26, M90C, M93A, M186)- and two propene (M142, M156)-utilizers differed from that found with peracids, whereas the chemical reactivity of the substrate appeared to affect enzymatic epoxidations in Staphylococcus sp. M97B. Offprint requests to: M. Mahmoudian     

Epoxidation of alkenes by alkene-grown Xanthobacter spp.

Summary Newly isolated Xanthobacter spp. were able to grow on the gaseous alkenes like ethene, propene, 1-butene and 1,3-butadiene. Resting-cell suspensions of propene-, 1-butene- or 1,3-butadiene-grown Xanthobacter Py10 accumulated 1,2-epoxyethane from ethene. Ethene-grown Xanthobacter Py10 did not produce any 1,2-epoxyalkane from the alkenes tested. Furthermore, propenegrown Xanthobacter Py2 accumulated 2,3-epoxybutane from trans-butene and cis-butene but did not form epoxides from other substrates tested.     

Inactivation of alkene oxidation by epoxides in alkene-and alkane-grown bacteria

Summary The oxidation of propene by resting-cells of ethene-grown Mycobacterium E3 was inactivated by 1,2-epoxypropane. Inactivation increased with increasing epoxide concentrations with 50% inactivation at approximately 30 mM epoxide. Other lower epoxides as epoxyethane and 1,2-epoxybutane also inactivated oxidation of propene as well as of other alkenes. Propene oxidation by resting-cells of ethane-grown Mycobacterium E20 and resting-cells of methane-grown Methylosinus trichosporium OB3b was inactivated for 50% at much lower 1,2-epoxypropane concentrations of approximately 1 and 3 mM respectively. It was demonstrated that in vivo the predominant effect of 1,2-epoxypropane was on the epoxidizing enzyme, i.e. alkene mono-oxygenase (strain E3), alkane mono-oxygenase (strain E20) and methane mono-oxygenase (methylotroph) and that the effect of the epoxide on the alkene mono-oxygenase was irreversible.     

Production of chiral epoxides by an ethene-utilisingMicrococcus sp.

Summary An ethene-utilising bacterium was isolated in pure culture from soil and was tentatively identified as aMicrococcus sp. The organism accumulated epoxyalkanes (0.2–13 mM) from internal, terminal, cyclic and aryl-substituted olefins and exhibited a substrate specificity which was different from that expected on the basis of the chemical reactivity pattern in peracid epoxidations. Epoxyalkanes were hydrolysed at a much slower rate than the epoxidation step which allowed them to accumulate. Ethene-grown cells catalysed the stereospecific formation of R-1,2-epoxypropane (enantiomeric excess: e.e.=96%), R-1,2-epoxybutane (e.e.=94%) andtrans-(2R,3R)-epoxybutane (e.e.=84%). An ethene monooxygenase was implicated in the production of chiral epoxides in cell-free extracts of the bacterium. The (2S,3S)-enantiomer of racemictrans-2,3-epoxybutane was stereoselectively hydrolysed to completion resulting in an enrichment in the (2R,3R)-enantiomer. Further hydrolysis of 1,2-epoxyalkanes (C₃-C₄), however, occurred via complete destruction of both stereoisomers.     

Mouse liver and lung glutathione S-epoxide transferase: Effects of phenobarbital and 3-methylcholanthrene administration

The effects of 3-methylcholanthrene (3MC) and phenobarbital (PB) administration on the levels of glutathione-S-epoxide transferase activity in supernatant preparations of liver and lung were studied in a number of different strains of mice, C57Bl/6, C3H, C3H_f^?, Balb/c^?, A⁺ and DBA/2⁺. Three epoxide substrates, 3MC-11,12-oxide, styrene oxide (SO) and 3,3,3-trichloro-1,2-epoxypropane (TCPO), were employed in this investigation. PB administration (75 mg/kg body weight for 3 days) resulted in 13–57% increases in enzyme activity in the liver supernatant but was ineffective in inducing activity in lung. 3MC administration (40 mg/kg body weight for 2 days) on the other hand was without any effect on glutathione-S-epoxide transferase activity in both liver and lung.     

Growth-substrate dependent dechlorination of 1,2-dichloroethane by a homoacetogenic bacterium

A rod shaped, gram positive, non sporulating Acetobacterium strain was isolated that dechlorinated 1,2-dichloroethane (1,2-DCA) to ethene at a dechlorination rate of up to 2 nmol Cl^- min^-1 mg^-1 of protein in the exponential growth phase with formate (40 mM) as the substrate. Although with other growth substrates such as pyruvate, lactate, H₂/CO₂, and ethanol higher biomass productions were obtained,the dechlorination rate with these substrates was more than 10-fold lower compared with formate growing cells. Neither cell extracts nor autoclaved cells of the isolatedAcetobacterium strain mediated the dechlorination of 1,2-DCA at significant rates. The addition of 1,2-DCA to the media did not result in increased cell production. No significant differences in corrinoid concentrations could be measured in cells growing on several growth-substrates. However, these measurements indicated that differences in corrinoid structure might cause the different dechlorination activity. The Acetobacterium sp. strain gradually lost its dechlorination ability during about 10 transfers in pure culture, probably due to undefined nutritional requirements. 16S rDNA analysis of the isolate revealed a 99.7% similarity with Acetobacterium wieringae. However, the type strains of A. wieringae and A. woodii did not dechlorinate 1,2-DCA.     

Aliphatic and chlorinated alkenes and epoxides as inducers of alkene monooxygenase and epoxidase activities in Xanthobacter strain Py2.

The inducible nature of the alkene oxidation system of Xanthobacter strain Py2 has been investigated. Cultures grown with glucose as the carbon source did not contain detectable levels of alkene monooxygenase or epoxidase, two key enzymes of alkene and epoxide metabolism. Upon addition of propylene to glucose-grown cultures, alkene monooxygenase and epoxidase activities increased and after an 11-h induction period reached levels of specific activity comparable to those in propylene-grown cells. Addition of chloramphenicol or rifampin prevented the increase in the enzyme activities. Comparison of the banding patterns of proteins present in cell extracts revealed that polypeptides with molecular masses of 43, 53, and 57 kDa accumulate in propylene-grown but not glucose-grown cells. Pulse-labeling of glucose-grown cells with [35S]methionine and [35S]cysteine revealed that the 43-, 53-, and 57-kDa proteins, as well as two additional polypeptides with molecular masses of 12 and 21 kDa, were newly synthesized upon exposure of cells to propylene or propylene oxide. The addition to glucose-grown cells of a variety of other aliphatic and chlorinated alkenes and epoxides, including ethylene, vinyl chloride (1-chloroethylene), cis- and trans-1,2-dichloroethylene, 1-chloropropylene, 1,3-dichloropropylene, 1-butylene, trans-2-butylene, isobutylene, ethylene oxide, epichlorohydrin (3-chloro-1,2-epoxypropane), 1,2-epoxybutane, cis- and trans-2,3-epoxybutane, and isobutylene oxide stimulated the synthesis of the five propylene-inducible polypeptides as well as increases in alkene monooxygenase and epoxidase activities. In contrast, acetylene, and a range of aliphatic and chlorinated alkanes, did not stimulate the synthesis of the propylene-inducible polypeptides or the increase in alkene monooxygenase and epoxidase activities.     

SmoXYB1C1Z of Mycobacterium sp. Strain NBB4: a Soluble Methane Monooxygenase (sMMO)-Like Enzyme,Active on C2 to C4 Alkanes and Alkenes

Monooxygenase (MO) enzymes initiate the aerobic oxidation of alkanes and alkenes in bacteria. A cluster of MO genes (smoXYB1C1Z) of thus-far-unknown function was found previously in the genomes of two Mycobacterium strains (NBB3 and NBB4) which grow on hydrocarbons. The predicted Smo enzymes have only moderate amino acid identity (30 to 60%) to their closest homologs, the soluble methane and butane MOs (sMMO and sBMO), and the smo gene cluster has a different organization from those of sMMO and sBMO. The smoXYB1C1Z genes of NBB4 were cloned into pMycoFos to make pSmo, which was transformed into Mycobacterium smegmatis mc²-155. Cells of mc²-155(pSmo) metabolized C₂ to C₄ alkanes, alkenes, and chlorinated hydrocarbons. The activities of mc²-155(pSmo) cells were 0.94, 0.57, 0.12, and 0.04 nmol/min/mg of protein with ethene, ethane, propane, and butane as substrates, respectively. The mc²-155(pSmo) cells made epoxides from ethene, propene, and 1-butene, confirming that Smo was an oxygenase. Epoxides were not produced from larger alkenes (1-octene and styrene). Vinyl chloride and 1,2-dichloroethane were biodegraded by cells expressing Smo, with production of inorganic chloride. This study shows that Smo is a functional oxygenase which is active against small hydrocarbons. M. smegmatis mc²-155(pSmo) provides a new model for studying sMMO-like monooxygenases.     

Analysis of bacterial degradation pathways for long-chain alkylphenols involving phenol hydroxylase, alkylphenol monooxygenase and catechol dioxygenase genes

Eighteen 4-t-octylphenol-degrading bacteria were isolated and screened for the presence of degradative genes by polymerase chain reaction method using four designed primer sets. The primer sets were designed to amplify specific fragments from multicomponent phenol hydroxylase, single component monooxygenase, catechol 1,2-dioxygenase and catechol 2,3-dioxygenase genes. Seventeen of the 18 isolates exhibited the presence of a 232 bp amplicon that shared 61-92% identity to known multicomponent phenol hydroxylase gene sequences from short and/or medium-chain alkylphenol-degrading strains. Twelve of the 18 isolates were positive for a 324 bp region that exhibited 78-95% identity to the closest published catechol 1,2-dioxygenase gene sequences. The two strains, Pseudomonas putida TX2 and Pseudomonas sp. TX1, contained catechol 1,2-dioxygenase genes also have catechol 2,3-dioxygenase genes. Our result revealed that most of the isolated bacteria are able to degrade long-chain alkylphenols via multicomponent phenol hydroxylase and the ortho-cleavage pathway.     

The synthesis and the 1h- and 13c-nuclear magnetic resonance spectroscopy of the cyclic sulfites of some sugars

Treatment of methyl β-D-ribofuranoside with thionyl chloride in hexamethyl-phosphoric triamide gives two diastereoisomeric methyl 5-chloro-5-deoxy-β-D-ribo-furanoside 2,3-cyclic sulfites. Similar cyclic sulfites are formed from benzyl β-D-ribofuranoside and 1,4-anhydro-DL-ribitol. If acetonitrile is substituted for hexa-methylphosphoric triamide, the cyclic sulfites are the main products, and only traces of the chlorinated sugars are formed. ¹H- and ¹³C-n.m.r.-spectral analysis of these reactions demonstrated that one of the diastereomers preponderates. The structure of these cyclic sulfites was established by comparison of the ¹H-n.m.r. spectra with those of the propylene sulfites. Treatment of 1,2-O-isopropylidene-α-D-glucofuranose (14) with thionyl chloride in hexamethylphosphoric triamide yields 3-chloro-3-deoxy-1,2-O-isopropylidene-α-D-allofuranose 5,6-cyclic suffite. In contrast to the 2,3-cyclic suffites, which are stable, the cyclic sulfites derived from 14 slowly decompose at room temperature.     

Trans-1,2-bis(diphenylphosphino)ethene as bridging ligand in thione-S-ligated dimeric copper(I) chloride complexes

Several mixed-ligand complexes have been prepared by treatment of copper(I) chloride with equimolar amounts of trans-1,2-bis(diphenylphosphino)ethene (trans-dppen) in acetonitrile followed by the addition of a methanolic solution of one equivalent of a heterocyclic thione (L). The novel complex compounds have been characterized by single-crystal X-ray diffraction, ¹H NMR and IR spectroscopy as well as by elemental analyses and melting points. The X-ray structures of three examples confirm that the compounds are homobimetallic dimers of type [CuCl(μ₂-trans-dppen)(L)]₂ containing two tetrahedral coordination units joined by two trans-dppen bridges.     

Degradation 1,2-dimethylbenzene by Corynebacterium strain C125

In an attempt to obtain bacteria growing on 1,2-dimethylbenzene as sole carbon and energy source two different strains were isolated. One was identified as an Arthrobacter strain, the other as a Corynebacterium strain. Corynebacterium strain C125 was further investigated. The organism was not capable to grow on 1,3- and 1,4-dimethylbenzene. cis-1,2-Dihydroxycyclohexa-3,5-diene oxidoreductase and 3,4-dimethylcatechol-2,3-dioxygenase activity was found in cell extracts. When 3,4-dimethylcatechol was added to cell extract of 1,2-dimethylbenzene-grown cells, first a compound with the spectral properties of 2-hydroxy-5-methyl-6-oxo-2,4-heptadienoate was formed and subsequently acetate was produced. It is proposed that dioxygenases are involved in the initial steps of 1,2-dimethylbenzene degradation, and ring opening proceeds via meta-cleavage.     

Rapid purification of an active recombinant His-tagged 2,3-dihydroxybiphenyl 1,2-dioxygenase from Pseudomonas putida OU83

2,3-Dihydroxybiphenyl 1,2-dioxygenase (2,3-DBPD) is an extradiol-type dioxygenase that catalyzes the aromatic ring fission of 2,3-dihydroxybiphenyl, the third step in the biphenyl degradation pathway. The nucleotide sequence of the Pseudomonas putida OU83 gene bphC, which encodes 2,3-DBPD, was cloned into a plasmid pQE31. The His-tagged 2,3-DBPD produced by a recombinant Escherichia coli strain, SG13009(pREP4)(pAKC1), and purified with a Ni-nitrilotriacetic acid resin affinity column using the His-bind Qiagen system. The His-tagged 2,3-DBPD construction, carrying a single 6×His tail on the N-terminal of the polypeptide, was active. SDS-PAGE analysis of the purified active 2,3-DBPD gave a single band of 34 kDa; this is in agreement with the size of the bphC coding region. The K_m for 2,3-dihydroxybiphenyl was 14.5±2 μM. The enzyme activity was enhanced by ferrous ion but inhibited by ferric ion. The enzyme activity was inhibited by thiol-blocking reagents and heavy metals HgCl₂, CuSO₄, NiSO₄, and CdCl₂. The yield was much higher and the time required to purify recombinant 2,3-DBPD from clone pAKC1 was faster than by the conventional chromatography procedures.     

Degradation of trans-1,2-dichloroethene by mixed and pure cultures of methanotrophic bacteria

Summary Out of seven chlorinated aliphatic hydrocarbons tested, only trans-1,2-dichloroethene was relatively non-toxic for a mixed methanotrophic culture. The compound was degraded at a rate of 0.4 mol/mg protein·h^-1 and liberation of inorganic chloride was observed. Trans-2,3-dichlorooxirane was formed as an intermediate which was converted further only by chemical transformation with a half life of 31 h. From the consortium, a pure culture was isolated and found to be capable of degradation of trans-1,2-dichloroethene when grown in the presence of methane or methanol. The ability of cometabolic degradation of this compound was not specific for this isolate, since Methylomonas methanica NCIB11130 and Methylosinus trichosporium OB3b also showed degradation of trans-1,2-dichloroethene when grown with methane as sole carbon source.Abbreviations t12DCE trans-1,2-dichloroethene - t23DCO trans-2,3-dichlorooxirane - c23DCO cis-2,3-dichlorooxirane - ¹H-NMR proton nuclear magnetic resonance     

Conversion of 2-Fluoromuconate to cis-Dienelactone by Purified Enzymes of Rhodococcus opacus 1cp

The present study describes the ¹⁹F nuclear magnetic resonance analysis of the conversion of 3-halocatechols to lactones by purified chlorocatechol 1,2-dioxygenase (ClcA2), chloromuconate cycloisomerase (ClcB2), and chloromuconolactone dehalogenase (ClcF) from Rhodococcus opacus 1cp grown on 2-chlorophenol. The 3-halocatechol substrates were produced from the corresponding 2-halophenols by either phenol hydroxylase from Trichosporon cutaneum or 2-hydroxybiphenyl 3-mono-oxygenase from Pseudomonas azelaica. Several fluoromuconates resulting from intradiol ring cleavage by ClcA2 were identified. ClcB2 converted 2-fluoromuconate to 5-fluoromuconolactone and 2-chloro-4-fluoromuconate to 2-chloro-4-fluoromuconolactone. Especially the cycloisomerization of 2-fluoromuconate is a new observation. ClcF catalyzed the dehalogenation of 5-fluoromuconolactone to cis-dienelactone. The ClcB2 and ClcF-mediated reactions are in line with the recent finding of a second cluster of chlorocatechol catabolic genes in R. opacus 1cp which provides a new route for the microbial dehalogenation of 3-chlorocatechol.