Formation and splitting of esters in subterminal oxidation of dodecane by Fusarium lini

Summary Extracellular oxidation products having the same number of carbon atoms as the alkane that was oxidized were isolated from a Fusarium lini culture broth grown on n-dodecane. They were secondary isomeric alcohols, corresponding isomeric ketones and isomeric esters with 12 carbon atoms.Esterase activity in cell-free extracts of the fungus which was incubated on a p-nitrophenyl-acetate substrate increased with increasing temperatures and pH-values in the ranges 20–40°C and pH 6.0 to 8.0 respectively. The activity, when incubated on p-nitrophenyl-acetate,-laurate and-palmitate substrates, decreased with decreasing fatty acid chain lengths. When incubated with isomeric esters consisting of 12 carbon atoms, it was influenced by the ester linkage position in the chain. When the alcohol chain length in the ester increased from one to six carbon atoms, the esterase activity decreased. The same effect was observed when the chain length of the acid increased from two to six carbon atoms. Minimum esterase activity was reached when both the alcohol and the acid had a chain length of six carbon atoms.The view that all ketones produced during subterminal oxidation of alkanes by Fusarium lini and perhaps other members of Moniliales are further metabolized via ester intermediates is supported. A probable non-specific esterase or lipase catalyses the hydrolysis of the isomeric esters which are formed from the ketones.     

Degradation of long chain alkanes by bacilli

Summary Five different species of bacilli tested at 30°C degrade hydrocarbons by cooxidation. Subterminal degradation pathway of these bacilli was proved by estimating isomeric esters, developed from Baeyer-Villiger oxidation of ketones, and also by finding diols, ketols, and diketones. Comparing the composition of fatty acids in lipids of bacilli grown with and without alkane as well as growing them on labeled n-tridecane suggested a subterminal, partly a di-subterminal pathway.It was also shown that Bacillus stearothermophilus cannot grow on alkane as sole carbon source even in a thermophilic temperature range and that oxidation products are formed in amounts twenty times less when grown on cooxidation medium at 50°C, compared to product formation on the same medium at 30°C.     

Degradation of long chain n-alkanes by disrupted cells ofChlorella vulgaris

Summary The alkane oxidation byChlorella vulgaris is improved by disruption of the cells. Although living cells are not able to attack n-dodecane, disrupted cells produced detectable amounts of oxidation products. The amount of isomeric alcohols and ketones of n-tridecane was nearly double the sum found in living cells, whereas the equilibrium was shifted to the ketones. With n-tetradecane and n-pentadecane only the amount of ketones increased.     

Enzymatic evaluation of different Aspergillus strains by biotransformation of cyclic ketones

The potential of different Aspergillus strains in carrying out the biotransformation of cyclic ketones was investigated. All the strains employed showed alcohol dehydrogenase and Baeyer–Villiger monooxygenase activities. trans-2-Methylcyclohexanol and trans-4-methylcyclohexanol were prepared in a single isomeric form by the use of Aspergillus terreus SSP 1498 and the corresponding ketones. Baeyer–Villiger oxidation of cyclic ketones by all the fungi Aspergillus led to chiral lactones in good enantioselectivity.     

Terminale und subterminale Oxidation von n-Alkanen durch Schimmelpilze

Zusammenfassung Der Abbau von n-Alkanen durch cunninghamella echinulata, Absidia glauca und Mucor sp. erfolgt durch monoterminale Oxidation ohne Freisetzung von primären oder sekundären Alkoholen, Ketonen und Aldehyden. Das Fettsäuremuster der cellulären Lipide zeigt eine deutliche Abhängigkeit vom gegebenen n-Alkan. Aspergillus-, Penicillium- und Verticillium-Arten oxidieren n-Alkane subterminal unter Bildung verschiedener isomerer sekundärer Alkohole und Ketone. Das Fettsäuremuster zeigt keine deutliche Abhängigkeit vom gegebenen n-Alkan. —Die Abbauwege werden diskutiert.

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Terminal and subterminal oxidation of n-alkanes by molds

Summary Degradation of n-alkanes by Cunninghamella echinulata, Absidia glauca and Mucor sp. is effected by monoterminal oxidation of the alkane chain and production of a cellular fatty-acid pattern that depends on the given n-alkane. Primary and secondary alcohols, ketones and aldehydes were not excreted in course of the oxidation. Subterminal attack on the alkane chain is found in members of the genus Aspergillus, Penicillium and Verticillium with the concomitant release of various isomeric secondary alcohols and ketones, and production of a cellular fatty-acid pattern which does not clearly depend on the given n-alkane. — The degradation pathways are discussed.

     

Degradation of long chain n-alkanes by Mucorales

Summary The degradation pathways on n-dodecane and n-tridecane were studied in seven representative strains of five families of the order Mucorales. Using thin-layer chromatographic, gas-liquid chromatographic and mass spectrometric methods, extracellular oxidation products of the relevant n-alkanes could be isolated and identified. All strains tested exhibited a formation of isomeric primary and secondary alcohols, isomeric ketones and monoic acids with chain length equivalent to the n-alkanes in the substrates, proving that in the order Mucorales both a terminal and a subterminal oxidation pathway are realized.     

Location of double bonds and cyclopropane rings in fatty acids by mass spectrometry

Electron-impact mass spectrometric procedures for locating the position of double bonds and cyclopropane rings in long-chain fatty acids are reviewed. Since unsaturation is not located directly by mass spectrometry, the properties of suitable derivatives are summarized. Epoxides are readily prepared from double bonds and on opening of the ring with various reagents useful derivatives are obtained, the most promising to date being hydroxymethoxy esters whose trimethylsilyl ethers give good mass spectra. Trimethylsilyl ethers of vicinal diols, prepared by direct hydroxylation, are recommended for the analysis of polyunsaturated fatty acid esters using combined gas chromatography-mass spectrometry. Oxymercuration-demercuration techniques are very convenient and one particular procedure can specifically locate unsaturation up to five carbons distant from the carboxyl group. An alternative approach enables the location of double bonds and cyclopropane rings in fatty acids by direct mass spectrometry of pyrrolidides. Cyclopropane rings can be positively located in fatty acid esters by mass spectrometry of isomeric ketones or methoxy derivatives prepared by chromium trioxide oxidation on poron trifluoride-catalysed methoxylation, respectively. A variety of other procedures are also considered and some guidelines are given for choosing a method to suit a particular unsaturated acid.     

Reactive oxygen species contribute to the release of volatile organic compounds from Chlamydomonas reinhardtii during programmed cell death

Acetic acid at pH 5.0 can induce programmed cell death (PCD) in Chlamydomonas reinhardtii cells, and abundant volatile organic compounds (VOCs) were released during the process. In this study, the caspase‐3‐like activity was determined during the PCD, and it was increased significantly after 1 h. During the PCD, the dynamic release of VOCs from the cells was analyzed, and the emissions of total VOCs were raised markedly and reached the highest level at 2 h. Among the seven types of VOCs, such as alkanes, alkenes, terpenoids, alcohols, aldehydes, ketones and esters, three oxygenated compounds (aldehydes, ketones and esters) showed the most significant increase. O₂^‐· and H₂O₂ were rapidly accumulated to high levels in the cells at the beginning of the PCD, but their content was reduced during the process. The activities of antioxidant enzymes were reduced gradually and even disappeared completely, demonstrating that the reduction of reactive oxygen species (ROS) may not be scavenged by the antioxidant enzyme system. ROS have an intense oxidation and scavenging ability to volatile compounds, and the oxidation results in the production of oxygenated compounds. Therefore, the abundant production of oxygenated compounds indicated that ROS may play an important role in the dynamic release of VOCs from C. reinhardtii cells during PCD.     

Microbial oxidation of oleic acid.

Resting cells of Saccharomyces cerevisiae (baker's yeast, type II; Sigma) were used to convert oleic acid into 10-hydroxyoctadecanoic acid with a 45% yield. Nocardia aurantia (ATCC 12674), Nocardia sp. (NRRL 5646), and Mycobacterium fortuitum (UI 53378) all converted oleic acid into 10-oxo-octadecanoic acid with 65, 55, and 80% yields, respectively. Structures of all metabolites were suggested by 1H and 13C nuclear magnetic resonance and by infrared and mass spectrometry. Structures of isomeric hydroxystearate and oxostearate derivatives and the stereochemical purity of hydroxystearates are difficult to prove unambiguously unless authentic standard compounds are available for spectral comparison. We describe the use of the chemical Baeyer-Villiger oxidation technique with 10-oxo-octadecanoic acid followed by mass spectral analysis of neutral extracts as a simple method to confirm the position of oxo-functional groups in the structures of fatty acid ketones. We further introduce a simple method based on 1H nuclear magnetic resonance analysis of diastereomeric S-(+)-O-acetylmandelate esters of hydroxystearates as a means of ascertaining stereochemical purities of hydroxy fatty acids.     

Microbial oxidation of oleic acid.

Resting cells of Saccharomyces cerevisiae (baker's yeast, type II; Sigma) were used to convert oleic acid into 10-hydroxyoctadecanoic acid with a 45% yield. Nocardia aurantia (ATCC 12674), Nocardia sp. (NRRL 5646), and Mycobacterium fortuitum (UI 53378) all converted oleic acid into 10-oxo-octadecanoic acid with 65, 55, and 80% yields, respectively. Structures of all metabolites were suggested by 1H and 13C nuclear magnetic resonance and by infrared and mass spectrometry. Structures of isomeric hydroxystearate and oxostearate derivatives and the stereochemical purity of hydroxystearates are difficult to prove unambiguously unless authentic standard compounds are available for spectral comparison. We describe the use of the chemical Baeyer-Villiger oxidation technique with 10-oxo-octadecanoic acid followed by mass spectral analysis of neutral extracts as a simple method to confirm the position of oxo-functional groups in the structures of fatty acid ketones. We further introduce a simple method based on 1H nuclear magnetic resonance analysis of diastereomeric S-(+)-O-acetylmandelate esters of hydroxystearates as a means of ascertaining stereochemical purities of hydroxy fatty acids.     

The Isomeric Composition of Hydroperoxides Formed by Autoxidation of Unsaturated Triglycerides and Vegetable Oils

Trioleoylglycerol (TO), trilinoleoylglycerol (TL), and trilinolenoylglycerol (TLN)were autoxi-dized in the dark at 37°C. Monohydroperoxides (MHP), the primary products, were isolated by preparative thin-layer chromatography (TLC). The isomeric compositions of their hydroperoxy fatty acid components were determined by gas chromatography-mass spectrometry (GC-MS) as follows—TO: the 8-, 9-, 10-, and 11-isomers; TL: the 9-, and 13-isomers; and TLN: the 9-, 12-, 13-, and 16-isomers. The proportions of isomers in each MHP did not vary with the oxidation time. The isomeric compositions of hydroperoxy fatty acid components obtained from autoxidized soybean and olive oils indicated that each unsaturated fatty acyl group of triacylglycerol (TG) in vegetable oils produced isomeric hydroperoxides during autoxidation in a way similar to the corresponding fatty acid methyl esters. The proportions of the isomers obtained from autoxidized oils changed with the level of oxidation. Isomers coming from linolenic acid in soybean oil and those from linoleic acid in olive oil decreased remarkably at a high level of oxidation.     

3, 4-Dihydroxyphenylalanine is oxidized by phenoxyl radicals of hydroxycinnamic acid esters in leaves ofVicia faba L

Mechanisms of oxidation of 3,4-dihydroxyphenylalanine (dopa) in leaves ofVicia faba have not yet been elucidated in details. The author hypothesized its oxidation by radicals of hydroxycinnamic acid esters that were generated by a peroxidase-dependent reaction in vacuoles. The results obtained in this study were followings. 1) Vacuolar peroxidase isolated from the leaves oxidized dopa more slowly than 4-coumaric and caffeic acid esters isolated from the leaves. 2) The hydroxycinnamic acid esters enhanced peroxidase-dependent oxidation of dopa and dopa suppressed their oxidation. 3) Degree of the enhancement was roughly correlated with rates of the oxidation of hydroxycinnamic acid esters. 4) The hydroxycinnamic acid esters increased levels of dopa radical in the presence of peroxidase. 5) In protoplasts of mesophyll cells ofV. faba, hydrogen peroxide-induced oxidation of dopa was faster than that of 4-coumaric acid and caffeic acid esters. These results support the above hypothesis that dopa in vacuoles is oxidized by phenoxyl radicals of hydroxycinnamic acid esters that are generated by vacuolar peroxidase.     

Research into the oxidation of abietic acid–derived enone with atmospheric oxygen

This work presents results of methyl 7-oxoabiet-13(14)-en-18-oate ( 3 ) self-oxidation with air-oxygen in the presence of various bases such as triethylamine or sodium t-butoxide. While under aerobic conditions, the use of sodium t-butoxide as a base results in the formation of four isomeric alcohols, an addition of triethylamine into reaction medium directs the enone 3 oxidation to hydroperoxides. To clarify this base dependence and to obtain more in-depth information about this reaction additional studies with cyclohexenone as a reference enone have been undertaken. Their results demonstrated the predisposition of abietane hydroperoxides to oxidize α,β-unsaturated ketones to epoxides in the presence of t-butoxide while reducing the hydroperoxide group to hydroxyl. This ability of hydroperoxides to epoxidize conjugated double bonds and confirmed by the present study intermolecular course allowed proposing a plausible mechanism for this reaction.     

Cloning,expression and characterization of a Baeyer-Villiger monooxygenase from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> KT2440

The gene encoding a Baeyer-Villiger monooxygenase and identified in Pseudomonas putida KT2440 was cloned and functionally expressed in Escherichia coli. The highest yield of soluble protein could be achieved by co-expression of molecular chaperones. In order to determine the substrate specificity, biocatalyses were performed using crude cell extract, growing and resting cells. Examination of aromatic, cyclic and aliphatic ketones revealed a high specificity towards short-chain aliphatic ketones. Interestingly, some open-chain ketones were converted to the alkylacetates, while for others formation of the ester products with oxygen on the other side of the keto group could also be detected yielding the corresponding methyl or ethyl esters.     

Morphogenetic effects of some volatile,organic compounds on Pestalotia rhododendri

Summary Some volatile alcohols, acids, esters, aldehydes and ketones influenced the morphogenesis of Pestalotia rhododendri when added through the gas phase. Linear growth, formation of aerial hyphae and production of conidial spores were determined. Mostly, increasing amounts of test substance inhibited linear growth and only one case of stimulation was noticed, viz. by nonenal.Many substances stimulated formation of aerial hyphae, particularly allyl alcohol, acetaldehyde and nonanal, but inhibitions were more common.Spore production was promoted by compounds from all the chemical classes investigated. The activity decreased as follows; alcohols > esters > acids, aldehydes > ketones. Inhibitions of spore production were also observed.The effects were reproducible on two different media. Pestalotia sydowiana exhibited the same main responses as the other Pestalotia species, but only two substances, furfural and n-propanol, were observed to promote spore formation of this species.     

Studies on the BF3·Et2O catalyzed Baeyer-Villiger reaction of spiroketalic steroidal ketones

During reactions of 23-oxosapogenins and the corresponding isomeric 22-oxo-23-spiroketals with MCPBA in the presence of BF₃·Et₂O, equilibration occurs between the ketones. The Baeyer-Villiger type oxidation is followed by fragmentation to the dinorcholanic lactones and 3-methylbutyrolactone. The mechanistic aspects of these reactions in the 25R and 25S series are discussed.     

Biocatalytic properties of a recombinant aldo-keto reductase with broad substrate spectrum and excellent stereoselectivity

In the screening of 11 E. coli strains overexpressing recombinant oxidoreductases from Bacillus sp. ECU0013, an NADPH-dependent aldo-keto reductase (YtbE) was identified with capability of producing chiral alcohols. The protein (YtbE) was overexpressed, purified to homogeneity, and characterized of biocatalytic properties. The purified enzyme exhibited the highest activity at 50°C and optimal pH at 6.5. YtbE served as a versatile reductase showing a broad substrate spectrum towards different aromatic ketones and keto esters. Furthermore, a variety of carbonyl substrates were asymmetrically reduced by the purified enzyme with an additionally coupled NADPH regeneration system. The reduction system exhibited excellent enantioselectivity (>99% ee) in the reduction of all the aromatic ketones and high to moderate enantioselectivity in the reduction of α- and β-keto esters. Among the ketones tested, ethyl 4,4,4-trifluoroacetoacetate was found to be reduced to ethyl (R)-4,4,4-trifluoro-3-hydroxy butanoate, an important pharmaceutical intermediate, in excellent optical purity. To the best of our knowledge, this is the first report of ytbE gene-encoding recombinant aldo-keto reductase from Bacillus sp. used as biocatalyst for stereoselective reduction of carbonyl compounds. This study provides a useful guidance for further application of this enzyme in the asymmetric synthesis of chiral alcohol enantiomers.     

Some recent developments in the use of enzyme catalysed reactions in organic synthesis.

The following processes are discussed in this article: enzyme-catalysed hydrolyses of carboxylic acid esters and amides, phosphate esters, nitriles and epoxides; esterification and inter-esterification reactions catalysed by enzymes; reduction of ketones to secondary alcohols using whole-cell systems or isolated dehydrogenases; oxidation of alicyclic and aromatic substrates using mono-oxygenases and dioxygenases in bacteria and fungi including enzyme-catalysed Baeyer-Villiger oxidations; aldol reactions, formation of optically active cyanohydrins and enzyme-catalysed acyloin type reactions. The use of these biocatalytic methods for the stereo-controlled preparation of important target structures is reviewed and some of the future directions for the biotransformation area are discussed.     

Differences in Protein Structure and Similarities in Catalytic Function of Two l-Stereoselective Carbonyl Reductases from Bakers’ Yeast

We purified and studied two l-stereoselective carbonyl reductases from bakers’ yeast (Saccharomyces cerevisiae). One catalyzed exclusively the enantioselective reduction of carbonyl compounds such as β-keto esters and the other acted on α-acetoxy ketones and β-keto esters. The enzymes had identical molecular weights and catalyzed the l-stereoselective reduction of various carbonyl compounds with similar substrate specificity, but they were different proteins coded by different genes.     

Control of chemoselectivity of microbial reaction with resin adsorbent: enhancement of Baeyer–Villiger oxidation over reduction

Amberlite XAD-7, a hydrophobic polymer, was used to change microbial reaction of ketones from reduction to Baeyer–Villiger (BV) oxidation. Thus, D. magnusii NBRC 4600 and G. reessii NBRC 1112 could catalyze the BV reaction of ketones in the presence of the polymer while reduction of the substrates proceeded, and BV oxidation was scarcely found in the absence of the polymer.