Oxidation Of N-alkanes Research Articles

Isolation and characterization of a novel oxygenase that catalyzes the first step of n-alkane oxidation in Acinetobacter sp. strain M-1.

In the Finnerty pathway for n-alkane, oxidation in Acinetobacter sp., n-alkanes are postulated to be attacked by a dioxygenase and the product, n-alkyl hydroperoxide, is further metabolized to the corresponding aldehyde via the peroxy acid [W. R. Finnerty, P. 184-188, in A. H. Applewhite (ed.), Proceedings of the World Conference on Biotechnology for the Fats and Oil Industry, 1988]. However, no biochemical evidence regarding the first-step reaction is available. In this study, we found a novel n-alkane-oxidizing enzyme that requires only molecular oxygen, i.e., not NAD(P)H, in our isolate, Acinetobacter sp. strain M-1, and purified it to apparent homogeneity by gel electrophoresis. The purified enzyme is a homodimeric protein with a molecular mass of 134 kDa, contains 1 mol of flavin adenine dinucleotide per mol of subunit, and requires CU2+ for its activity. The enzyme uses n-alkanes ranging in length from 10 to 30 carbon atoms and is also active toward n-alkenes (C12 to C20) and some aromatic compounds with substituted alkyl groups but not toward a branched alkane, alcohol, or aldehyde. Transient accumulation of n-alkyl hydroperoxide was detected in the course of the reaction, and no oxygen radical scavengers affected the enzyme activity. From these properties, the enzyme is most probably a dioxygenase that catalyzes the introduction of two atoms of oxygen to the substrate, leading to the formation of the corresponding n-alkyl hydroperoxide. The enzymatic evidence strongly supports the existence of an n-alkane oxidation pathway, which is initiated by a dioxygenase reaction, in Acinetobacter spp.

Inhibition of Growth of a Graphium sp. on Gaseous n-Alkanes by Gaseous n-Alkynes and n-Alkenes

The growth of a filamentous fungus, a Graphium sp., on n-alkanes (C(inf2) to C(inf4)) was inhibited by low concentrations of acetylene, propyne, 1-butyne, ethylene, and propylene. Acetylene and other unsaturated hydrocarbons had no effect on the growth of the Graphium sp. on potato dextrose broth, ethanol, or acetate. Our results suggest that n-alkynes and n-alkenes are selective inhibitors of a nonspecific monooxygenase enzyme responsible for the initial oxidation of n-alkanes.

A non-conventional dissimilation pathway for long chain n-alkanes in Acinetobacter sp. M-1 that starts with a dioxygenase reaction

n-Alkane oxidation in a long chain n-alkane utilizer, Acinetobacter sp. M-1, was investigated. In Acinetobacter, n-alkanes have been postulated to be converted to the acid via a non-conventional oxidation pathway: n-alkane→ n-alkyl hydroperoxide→aldehyde→acid (Finnerty, W.R.: Lipids of Acinetobacter. In Proceedings of the World Conference on Biotechnology for the Fats and Oils Industry, 184–188, 1988). However, there is little biochemical information on the enzymes involved in the postulated pathway, particularly the enzyme catalyzing the first step. In this study, we purified and n-alkane-oxidizing enzyme to apparent homogeneity by SDS-PAGE. The enzyme was a flavoprotein, and required molecular oxygen and Cu 2+ for its activity, but did not require a reduced coenzyme such as NAD(P)H. A hydroperoxide was detected as a product of the enzyme reaction. We assume that the n-alkane-oxidizing enzyme is a dioxygenase. In addition, as a fatty alcohol does not appear to be an intermediate, fatty alcohol dehydrogenase is assumed not to participate in the n-alkane oxidation. The validity of the postulated pathway is supported by the following observations: (i) n-alkane monooxygenase activity was not detected, (ii) fatty alcohol dehydrogenase activities were low, and (iii) NAD(P)H-dependent long chain fatty aldehyde dehydrogenase activities were strongly induced in n-alkane-grown cells. NAD(P)H-dependent fatty aldehyde reductase activity was also found in n-alkane-grown cells, which may contribute to the formation of waxes that are cell reserve substances in n-alkane-utilizing Acinetobacter.

Bacterial survival and n-alkane degradation within Omani crude oil and a mousse

A mixed community of eight bacteria able to degrade crude oil was isolated from contaminated sands around the Gulf of Oman. When the community was introduced into 50% oil-water mousse, it demonstrated long-term survival of one isolate identified as Pseudomonas aeruginosa. In 50 and 75% oil-water mousse, cfu's of P. aeruginosa remained at the same levels over 60 days, but a 90% oil-water mixture failed to form a mousse and by day 10 there was no bacterial survival. Respirometry comparing oxygen uptake of P. aeruginosa from a 75% mousse with the same bacteria grown in a nutrient broth gave higher oxidation and greater reduction of n-alkanes for the former, when both were grown on crude oil. The significance of environmental adaptations of bacteria and the reduction of specific alkanes by a single bacterial species are discussed briefly.

N-Alkane oxidation in soil. Formation of internal monoalkenes

The biodegradation of pure eicosane (nC 20) in rendzina soil samples was studied in a laboratory experiment at 20°C. After 8 weeks, 25% of eicosane was mineralized. Terminal oxidation led to C 20:0 acid, with eicosan-1-ol as probable intermediate. An interesting dehydrogenation process occurred, leading to eicos-9-ene which was oxidized into eicos-9-enoic and eicos-11-enoic acids. This process was also observed with C 19 and C 22 hydrocarbons. Hydrolysis of the complex lipid fraction demonstrated the incorporation of eicosane-derived molecules in this fraction.

Cloning and heterologous expression of a gene encoding an alkane-induced extracellular protein involved in alkane assimilation from Pseudomonas aeruginosa

Pseudomonas aeruginosa PG201 produces a 16-kDa extracellular protein in media containing n-hexadecane as a carbon source but not in media containing glycerol or glucose. This protein was purified, and the N-terminal amino acid sequence was determined. The amino acid composition of the protein was found to be very similar to that of the so-called protein-like activator for n-alkane oxidation (PA) from P. aeruginosa S7B1. This extracellular protein was previously characterized (K. Hisatsuka, T. Nakahara, Y. Minoda, and K. Yamada, Agric. Biol. Chem. 41:445-450, 1977) and found to stimulate the growth of P. aeruginosa on n-hexadecane and to possess emulsifying activity. To study the role(s) of the PA protein and to make it accessible for possible future applications, we have cloned the PA-encoding (pra) gene and determined its nucleotide sequence. This analysis revealed a protein-coding region of 162 amino acids, with the first 25 residues being reminiscent of those of a typical bacterial signal sequence. The pra gene was inactivated by insertional mutagenesis, and the resulting strain was found to lack extracellular PA protein and to be retarded in its growth in n-hexadecane-containing media. These results are consistent with the growth stimulatory role of the PA protein. The pra gene was expressed in Escherichia coli, and substantial amounts of the recombinant protein were found in the extracellular growth medium. The recombinant protein was purified by metal chelate affinity chromatography. The ability to produce secreted PA protein by E. coli provides a simple and safe means to analyze its function(s) in alkane assimilation in the future.

Bioconversion of n-octane to octanoic acid by a recombinant Escherichia coli cultured in a two-liquid phase bioreactor.

The alk genes from the catabolic OCT plasmid of Pseudomonas oleovorans, which encode the enzymes involved in the oxidation of n-alkanes to carboxylic acids, were introduced into E. coli W3110. The resulting recombinant converts n-octane in a two-liquid phase medium into the corresponding alkanoate and excretes this compound into the aqueous phase. The rate of octanoic acid production by the recombinant E. coli is equal to or better than the alkane oxidation rate of P. oleovorans, suggesting that two-liquid phase fermentations with E. coli might have future industrial applications.

Role of superoxide dismutase in the oxidation of N-alkanes by yeasts

Yeast microorganisms from Candida genus are investigated for their superoxide dismutase (SOD) and catalase activity during cultivation on N-alkanes. The later caused a considerable increase of Cu/Zn SOD activity of yeast cells in comparison with glucose. A correlation between SOD and catalase activity existed. It is further observed that cells of Candida lipolytica 68–72 which contain a high level of Cu/Zn SOD were more resistant to lethality of exogenous O 2 −. An over-production of Cu/Zn SOD during the assimilation of N-alkanes by yeasts is also connected to their considerable resistance to increased concentrations of Cu 2+ and Zn 2+ ions in the nutrient medium. The results are consistent with the assumption that the enhanced resistance of yeast cells to O 2 − and high concentrations of Cu 2+ and Zn 2+-ions are due to the increased activity of Cu/Zn SOD and that SOD is involved in the protection of some cellular components. Polyacrylamide gel electrophoresis of Candida lipolutica cell-free extracts revealed the same chromatic bands of SOD activity under growth on glucose and N-alkanes. The type of the carbon source used from yeast cells as a single source of carbon and energy had no influence on the SOD profile of the cell.

Significance of Photosensitized Oxidation of Alkanes During the Photochemical Degradation of Petroleum Hydrocarbon Fractions in Seawater

Abstract A crude oil hydrocarbon fraction was photooxidized as a surface film on seawater under simulated environmental conditions. After irradiation, gas chromatography and gas chromatography/mass spectrometry analyses allowed to demonstrate the presence of relatively high quantities of compounds deriving from the photosensitized oxidation of n-alkanes, pristane and phytane, being among the non-acidic photoproducts of this fraction. The results suggest that the photochemical degradation of alkanes should be considered in studies of the fate of petroleum crudes in the marine environment.

Degradation of long-chain n-alkanes by the yeast Candida maltosa

The yeast Candida maltosa precultivated on liquid n-alkanes utilized different solid n-alkanes (especially C20−C25) in the presence of pristane as an organic phase with rates comparable to, or somewhat larger than, those of liquid n-alkanes. Analysis of cellular fatty acids indicated an assimilation of solid n-alkanes via monoterminal oxidation. The resulting fatty acids with substrate chain length were chain-shortened by C2 units down to an optimal range of chain length from C16 to C18 and incorporated into cellular, lipids directly or after desaturation. The intermediates of chain-shortening with numbers of carbon atoms higher than C18, as well as the unusually long-chain fatty acids of substrate chain length, were detected in trace amounts only. Even-carbon-numbered and odd-numbered fatty acids predominated in experiments with evenchain and odd-chain n-alkanes, respectively. Studies with cerulenin indicated that de novo synthesis of fatty acids was negligible. Oxidation of solid n-alkanes by the yeast C. maltosa yielded fatty acid patterns similar to those of cells grown on liquid n-alkanes.

Comments on Azide and Mercuric salts as seawater preservatives, and alkenes in the gulf of Mexico

A homologous series of volatile low molecular weight 1-alkenes was found in the surface waters of the Gulf of Mexico. Previous workers in the Gulf and other tropical waters did not report these compounds but they had poisoned their samples at sea for analysis later. The efficacy of azide and mercuric salts to preserve a representative alkene, 1-heptene, and heptane was tested. Neither salt significantly prevented loss of the hydrocarbons from spiked seawater. The 1-alkenes are most likely the abiotic products of photochemical oxidation and fragmentation of n-alkanes.

Features of the oxidation of n-alkanes in the presence of metal-complex catalysts

Features of the oxidation of n-alkanes in the presence of metal-complex catalysts

Lipids of aquatic organisms as potential contributors to lacustrine sediments—II

The relationship between the lipid composition of organisms in the water column of an eutrophic lake and the lipid composition of underlying sediments, previously examined for n-alkanols and steroids, is now reported for hydrocarbons, ketones and carboxylic acids. The n-C 7 alkane and alkenoic acids from two primary sources are rapidly metabolized in the water column and surficial sediment. Bacterial biomarkers, including hopenes and i ai - branched fatty acids, were detected in the photosynthetic bacterial layer occurring just above the sediment-water interface. Within the sediment the apparent conversion of free n-alkanes, alkan-2-ones and ω-hydroxy acids to the corresponding bound form is noted; microbiological oxidation of n-alkanes to alkan-2-ones is supported by the detection of the intermediate alkan-2-ols with a distribution similar to that of the ketones. The geochemistry of sediment deposited c. 1900, prior to biological study of the site, was interpreted from stable biomarkers and the diagenetic changes recognised in the study of contemporary deposition. A qualitative difference in algal input to the older sediment is inferred from the low Δ 7-sterol content and presence of 2,6,10-trimethyl-7-(3-methylbutyl)-dodecane. However, there was still significant dinoflagellate input, as indicated by the presence of 4α-methylsterols. A difference in higher-plant input to the older sediment, indicated from the n-alkane, alkene and triterpenoid ketone distributions, is consistent with the recent development of tree cover.

Characterization of the OCT plasmid encoding alkane oxidation and mercury resistance in Pseudomonas putida.

Transformation of Pseudomonas putida and analysis for plasmid DNA revealed that both n-alkane oxidation and mercury resistance are encoded on a single 220-megadalton OCT plasmid molecule. Derivatives of OCT having lost the mercury resistance function could be readily isolated and contained a smaller plasmid estimated to be 170 megadaltons. The results show that segregation of the mercury resistance property occurs not by loss of a separate MER plasmid as previously thought but by a deletion in the OCT plasmid.

Respiratory activities in intact cells and spheroplasts of hydrocarbon-utilizing yeasts.

The respiratory activities of Candida rugosa JF-101 and C. lipolytica No. 6-20 grown on glucose were studied in relation to the n-alkanes (n-C9H20-n-C18H38). Both yeasts readily oxidized n-decane, n-undecane and n-dodecane. Cells of both yeasts grown on glucose showed a lag in n-alkane oxidation, whereas the cells grown on n-alkane did not. n-Hexadecane was oxidized by cells grown on the same n-alkane better than by cells grown on n-decane and glucose. The respiratory action of each yeast on n-hexadecane tended to be induced and the activity on n-decane was constitutive. The glucose oxidation rates of the spheroplasts from C. rugosa and C. lipolytica were, respectively, 70-80% and 32-34% as high as those of the intact cells and the n-decane oxidation rates of the spheroplasts were 4-8% and 0% as high as the respective intact cells. Addition of the non-ionic surfactant Tween 80 restored the action of spheroplasts on n-decane to 10-19% as high as the intact cells, except for the spheroplasts from the glucose grown cells of C. lipolytica.

Production of Methyl Ketones from Secondary Alcohols by Cell Suspensions of C 2 to C 4 n -Alkane-Grown Bacteria

Nineteen new C(2) to C(4)n-alkane-grown cultures were isolated from lake water from Warinanco Park, Linden, N.J., and from lake and soil samples from Bayway Refinery, Linden, N.J. Fifteen known liquid alkane-utilizing cultures were also found to be able to grow on C(2) to C(4)n-alkanes. Cell suspensions of these C(2) to C(4)n-alkane-grown bacteria oxidized 2-alcohols (2-propanol, 2-butanol, 2-pentanol, and 2-hexanol) to their corresponding methyl ketones. The product methyl ketones accumulated extracellularly. Cells grown on 1-propanol or 2-propanol oxidized both primary and secondary alcohols. In addition, the activity for production of methyl ketones from secondary alcohols was found in cells grown on either alkanes, alcohols, or alkylamines, indicating that the enzyme(s) responsible for this reaction is constitutive. The optimum conditions for in vivo methyl ketone formation from secondary alcohols were compared among selected strains: Brevibacterium sp. strain CRL56, Nocardia paraffinica ATCC 21198, and Pseudomonas fluorescens NRRL B-1244. The rates for the oxidation of secondary alcohols were linear for the first 3 h of incubation. Among secondary alcohols, 2-propanol and 2-butanol were oxidized at the highest rate. A pH around 8.0 to 9.0 was found to be the optimum for acetone or 2-butanone formation from 2-alcohols. The temperature optimum for the production of acetone or 2-butanone from 2-propanol or 2-butanol was rather high at 60 degrees C, indicating that the enzyme involved in the reaction is relatively thermally stable. Metal-chelating agents inhibit the production of methyl ketones, suggesting the involvement of a metal(s) in the oxidation of secondary alcohols. Secondary alcohol dehydrogenase activity was found in the cell-free soluble fraction; this activity requires a cofactor, specifically NAD. Propane monooxygenase activity was also found in the cell-free soluble fraction. It is a nonspecific enzyme catalyzing both terminal and subterminal oxidation of n-alkanes.

Selective oxidation of n-alkanes with lead tetraacetate

Alkanes, when treated with lead tetraacetate under thermal or photochemical conditions, undero a slow but highly selective oxidation to form secondary acetates.

Perspectives for the genetic engineering of hydrocarbon oxidizing bacteria.

Hydrocarbons have many different structures, but these microbial substrates have two general characteristics which influence the nature of oxidation pathways. (1) Hydrocarbons are usually hydrophobic molecules, and oxidizing activities are consequently located in cellular membranes. (2) Hydrocarbons are composed of several basic structural units (such as aromatic rings and aliphatic chains). In order to break down these composite substrates, oxidation pathways are organized into segments that are conveniently labeled “throats” and “stomachs”. Throats contain the initial activities which convert a specific hydrocarbon substrate into one of a few common intermediates, such as fatty acids or catechols. Stomachs contain the subsequent activities which transform the intermediates into the organic acid substrates of central metabolic pathways. Typical stomachs include β-oxidation and catechol ring fission pathways. Detailed genetic analysis has been carried out for a few hydrocarbon oxidation pathways, including the plasmidborne alk and xyl systems for oxidation of n-alkanes and of toluene or xylenes. In both cases, there is duplication of plasmic-and chromosome-encoded activities at certain steps of the oxidation pathway. Induction specificities in both systems are often more important than enzyme specificities in determining growth phenotypes on different substrates. Oxidation of halogenated aromatic substrates, such as chlorobenzoic acid or the pesticide 24D, has been the subject of less detailed genetic study, but haloaromatic pathways illustrate the same basic principles as those for non-halogenated substrates: (i) the need to match throats and stomachs, and (ii) the importance of proper pathway regulation to ensure substrate catabolism. The use of broad host-range cloning vectors means that recombinant DNA methods are available for use with hydrocarbon-oxidizing gram-negative bacteria. However, detailed consideration of the possible rate-limiting steps in hydrocarbon oxidation indicates that cloning and overproduction of particular enzymes may often not be successful in strain improvement for specific biodegradations or bioconversions. A particular problem can be the toxicity of membrane hydrocarbon-oxidizing proteins when they are produced at very high levels.

Cytological responses of Cladosporium resinae when shifted from glucose to hydrocarbon medium

Cladosporium resinae was grown on glucose and then transferred to medium with glucose or with kerosene as the sole carbon source. Growth on hydrocarbon was associated with thinner cell walls in both hyphae and spores, with the presence of large vacuoles in cells, with the synthesis of microbodies, and with increased synthesis of catalase. Some vacuoles in hydrocarbon-grown cells contained small, spherical, electron-dense inclusions which were not observed in cells from glucose medium. Large, electron-dense bodies within vacuoles were observed in glucose-grown and in hydrocarbon-grown cells. A working model is proposed for oxidation of n-alkanes by C. resinae.

Microbial oxidation of gaseous hydrocarbons: Oxidation of lower n-alkanes and n-alkanes by resting cell suspensions of various methylotrophic bacteria and the effect of methane metabolites Hou, C.T., Patel, R.N., Laskin, A.I. and Barnable, N. FEMS microbiology letters, 9 (1980) 267–270

Microbial oxidation of gaseous hydrocarbons: Oxidation of lower n-alkanes and n-alkanes by resting cell suspensions of various methylotrophic bacteria and the effect of methane metabolites Hou, C.T., Patel, R.N., Laskin, A.I. and Barnable, N. FEMS microbiology letters, 9 (1980) 267–270