Degradation of the mixture of ethyl formate, propionic aldehyde, and acetone by Aeromonas salmonicida: A novel microorganism screened from biomass generated in the citric acid fermentation industry.

Microorganisms play important roles in the degradation of volatile organic compounds. Aeromonas salmonicida strain (AEP-3) generated from biomass in the citric acid fermentation industry was screened and subjected to denaturing gradient gel electrophoresis (DGGE) fingerprinting and 16S rDNA gene sequencing. The growth conditions of AEP-3 in Luria-Bertani broth were optimized at 25 °C and approximately pH 7. AEP-3 was used to degrade ethyl formate, propionic aldehyde, or acetone alone and their mixture. The concentrations of ethyl formate, propionic aldehyde, and acetone were below 7500, 600, and 800 mg L-1, respectively, and their maximum degradation efficiencies were 100%, 92.41%, and 34.75%. AEP-3 first degraded acetone and propionic aldehyde in the mixture, followed by ethyl formate. The degradation pathways of these organic compounds in the mixture and their substrate interactions during degradation were explored. Propionic aldehyde was first converted into propionic acid in the metabolic process and was involved in the subsequent carboxylic acid cycle. By contrast, ethyl formate was first hydrolyzed into formic acid and ethanol. Then, formic acid participated in the cyclic metabolism of carboxylic acid, whereas, ethanol was hydrolyzed into acetaldehyde and acetic acid through alcohol and aldehyde dehydrogenase. Additionally, acetone directly interacted with nitrate in the medium under the action of hydrogen ions and produced carbon dioxide, water, and nitrogen. Overall, this study provides a new degrading bacterium biodegradability toward the exhaust gas of citric acid fermentation.

Simultaneous monitoring of the bioconversion from lysine to glutaric acid by ethyl chloroformate derivatization and gas chromatography-mass spectrometry.

Glutaric acid is a precursor of a plasticizer that can be used for the production of polyester amides, ester plasticizer, corrosion inhibitor, and others. Glutaric acid can be produced either via bioconversion or chemical synthesis, and some metabolites and intermediates are produced during the reaction. To ensure reaction efficiency, the substrates, intermediates, and products, especially in the bioconversion system, should be closely monitored. Until now, high performance liquid chromatography (HPLC) has generally been used to analyze the glutaric acid-related metabolites, although it demands separate time-consuming derivatization and non-derivatization analyses. To substitute for this unreasonable analytical method, we applied herein a gas chromatography - mass spectrometry (GC-MS) method with ethyl chloroformate (ECF) derivatization to simultaneously monitor the major metabolites. We determined the suitability of GC-MS analysis using defined concentrations of six metabolites (l-lysine, cadaverine, 5-aminovaleric acid, 2-oxoglutaric acid, glutamate, and glutaric acid) and their mass chromatograms, regression equations, regression coefficient values (R2), dynamic ranges (mM), and retention times (RT). This method successfully monitored the production process in complex fermentation broth.

Concurrent production of biodiesel and chemicals through wet in situ transesterification of microalgae.

This work addresses an unprecedented way of co-producing biodiesel (FAEE) and valuable chemicals of ethyl levulinate (EL), ethyl formate (EF) and diethyl ether (DEE) from wet in situ transesterification of microalgae. EL, EF, and DEE were significantly produced up to 23.1%, 10.3%, and 52.1% of the maximum FAEE mass with the FAEE yield higher than 90% at 125 °C. Experiments to elucidate a detailed route of EL and EF synthesis were fulfilled and it was found that its main route to the production of EL and EF was the acid hydrolysis of algal cells and esterification with ethanol. To investigate the effect of reaction variables on the products yields, comprehensive experiments were carried out with varying temperatures, solvent and alcohol volumes, moisture contents and catalyst amounts. Coproduction of DEE, EL, EF and FAEE can contribute to elevating the economic feasibility of microalgae-based biodiesel supply chain.

A colorimetric assay that specifically measures Granzyme B proteolytic activity: hydrolysis of Boc-Ala-Ala-Asp-S-Bzl.

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is the most studied granzyme in humans and mice and therefore, researchers need specific and reliable tools to study its function and role in pathophysiology. This especially necessitates assays that do not recognize proteases such as caspases or other granzymes that are structurally or functionally related. Here, we apply GzmB's preference for cleavage after aspartic acid residues in a colorimetric assay using the peptide thioester Boc-Ala-Ala-Asp-S-Bzl. GzmB is the only mammalian serine protease capable of cleaving this substrate. The substrate is cleaved with similar efficiency by human, mouse and rat GzmB, a property not shared by other commercially available peptide substrates, even some that are advertised as being suitable for this purpose. This protocol is demonstrated using unfractionated lysates from activated NK cells or CTL and is also suitable for recombinant proteases generated in a variety of prokaryotic and eukaryotic systems, provided the correct controls are used. This assay is a highly specific method to ascertain the potential pro-apoptotic activity of cytotoxic molecules in mammalian lymphocytes, and of their recombinant counterparts expressed by a variety of methodologies.

Formate dehydrogenase--a biocatalyst with novel applications in organic chemistry.

In the field of industrial biocatalysis, formate dehydrogenase (FDH) is well established, in particular for its broad application in cofactor regeneration. Further applications have been limited by the enzyme's narrow range of substrates. These restrictions have been overcome now by the finding, that the enzyme is capable of selectively cleaving formic acid esters to the respective alcohol. Five homologous alkyl formates and phenyl formate as an aromatic ester were converted quantitatively by FDH from Candida boidinii in a batch reaction within 3 to 5 h. The substrates were turned irreversibly into carbon dioxide and the respective alcohol through hydride abstraction from the formyl group with full conversion. The mechanism shows parallels to hydrolysis reactions of the A(AC)1-type. K(M)-values and reactions rates of the tested formic acid esters display a tendency to higher conversion rates with increasing chain length. FDH emerged to be a superior deformylation catalyst compared to hydrolases as well as classical catalysts, as was shown by the selective deformylation of 1-acetoxy-4-formoxy butane (92%) and 1,3-bis(3-formoxypropyl)tetramethyldisiloxane. In particular its capability to distinguish between formic acid esters and non-formic acid esters renders the method particularly suitable for protective group chemistry. Furthermore the completeness of deformylation allows for converting substrates highly incompatible with aqueous media like siloxanes within a few hours.

Metabolism and cometabolism of cyclic ethers by a filamentous fungus, a Graphium sp.

The filamentous fungus Graphium sp. (ATCC 58400) grows on gaseous n-alkanes and diethyl ether. n-Alkane-grown mycelia of this strain also cometabolically oxidize the gasoline oxygenate methyl tert-butyl ether (MTBE). In this study, we characterized the ability of this fungus to metabolize and cometabolize a range of cyclic ethers, including tetrahydrofuran (THF) and 1,4-dioxane (14D). This strain grew on THF and other cyclic ethers, including tetrahydropyran and hexamethylene oxide. However, more vigorous growth was consistently observed on the lactones and terminal diols potentially derived from these ethers. Unlike the case in all previous studies of microbial THF oxidation, a metabolite, gamma-butyrolactone, was observed during growth of this fungus on THF. Growth on THF was inhibited by the same n-alkenes and n-alkynes that inhibit growth of this fungus on n-alkanes, while growth on gamma-butyrolactone or succinate was unaffected by these inhibitors. Propane and THF also behaved as mutually competitive substrates, and propane-grown mycelia immediately oxidized THF, without a lag phase. Mycelia grown on propane or THF exhibited comparable high levels of hemiacetal-oxidizing activity that generated methyl formate from mixtures of formaldehyde and methanol. Collectively, these observations suggest that THF and n-alkanes may initially be oxidized by the same monooxygenase and that further transformation of THF-derived metabolites involves the activity of one or more alcohol dehydrogenases. Both propane- and THF-grown mycelia also slowly cometabolically oxidized 14D, although unlike THF oxidation, this reaction was not sustainable. Specific rates of THF, 14D, and MTBE degradation were very similar in THF- and propane-grown mycelia.

N-ethoxycarbonylation combined with (S)-1-phenylethylamidation for enantioseparation of amino acids by achiral gas chromatography and gas chromatography-mass spectrometry.

N-ethoxycarbonylation was combined with (S)-1-phenylethylamidation for enantioseparation of amino acids by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) on achiral capillary columns. The method provided complete enantioseparations of 12 amino acids as diastereomeric N-ethoxycarbonyl/(S)-1-phenylethylamides with exceptional resolutions for proline (R(s) > or = 9.9) and pipecolic acid (R(s) > or = 10.2). GC-MS analysis in selected ion monitoring mode employing standard addition method, facilitated quantitation of D-pipecolic acid in kidney bean (0.95 microg/10 mg) and adzuki bean (0.14 microg/10 mg). The peak area ratios indicated that they had the identical chiral composition at 2.5% for D-pipecolic acid and 97.5% for L-pipecolic acid.

Synthesis and activity of 2-oxoamides containing long chain beta-amino acids.

2-Oxoamides based on long chain beta-amino acids were synthesized. 1-Benzyl substituted long chain amines, needed for such synthesis, were synthesized starting from Boc-phenylalaninol. The oxidative conversion of a phenyl group to a carboxyl group was used as the key transformation synthetic step. The compounds synthesized were studied for their activity against GIVA PLA(2), and were proven to be weak inhibitors.

Alcohol dehydrogenases that catalyse methyl formate synthesis participate in formaldehyde detoxification in the methylotrophic yeast Candida boidinii.

Methyl formate synthesis during growth on methanol by methylotrophic yeasts has been considered to play a role in formaldehyde detoxification. An enzyme that catalyses methyl formate synthesis was purified from methylotrophic yeasts, and was suggested to belong to a family of alcohol dehydrogenases (ADHs). In this study we report the gene cloning and gene disruption analysis of three ADH-encoding genes in the methylotrophic yeast Candida boidinii (CbADH1, CbADH2 and CbADH3) in order to clarify the physiological role of methyl formate synthesis. From the primary structures of these three genes, CbAdh1 was shown to be cytosolic and CbAdh2 and CbAdh3 were mitochondrial enzymes. Gene products of CbADH1, CbADH2 and CbADH3 expressed in Escherichia coli showed both ADH- and methyl formate-synthesizing activities. The results of gene-disruption analyses suggested that methyl formate synthesis was mainly catalysed by a cytosolic ADH (CbAdh1), and this enzyme contributed to formaldehyde detoxification through glutathione-independent formaldehyde oxidation during growth on methanol by methylotrophic yeasts.

Growth of Candida boidinii on methanol and the activity of methanol-degrading enzymes as affected from formaldehyde and methylformate.

Formaldehyde and methylformate affect the growth of Candida boidinii on methanol and the activity of methanol-degrading enzymes. The presence of both intermediates in the feeding medium caused an increase in biomass yield and productivity and a decrease in the specific rate of methanol consumption. In the presence of formaldehyde, the activity of formaldehyde dehydrogenase and formate dehydrogenase was essentially increased, whereas the activity of methanol oxidase was decreased. On the contrary, the presence of methylformate caused an increase of the activity of methanol oxidase and a decrease of the activity of formaldehyde dehydrogenase and formate dehydrogenase. Interpretations concerning the yeast behavior in the presence of intermediate oxidation products were considered and discussed.

Ester synthesis by NAD(+)-dependent dehydrogenation of hemiacetal: production of methyl formate by cells of methylotrophic yeasts.

A water-soluble ester, methyl formate, was detected as a metabolite in the culture medium of methylotrophic yeasts. Methyl formate synthase, which catalyses NAD(+)-dependent dehydrogenation of the hemiacetal adduct of methanol and formaldehyde, catalyses the ester synthesis. The enzyme activity was induced on a methanol medium and was increased further by the addition of formaldehyde. In the reaction system using intact cells of Pichia methanolica AKU 4262, 135 mM (8.1 g/liter) methyl formate was produced from 2 M methanol. This is a new biological process for ester synthesis that couples spontaneous formation of hemiacetal and alcohol dehydrogenase.

Purification and properties of methyl formate synthase, a mitochondrial alcohol dehydrogenase, participating in formaldehyde oxidation in methylotrophic yeasts.

Methyl formate synthase, which catalyzes methyl formate formation during the growth of methylotrophic yeasts, was purified to homogeneity from methanol-grown Candida boidinii and Pichia methanolica cells. Both purified enzymes were tetrameric, with identical subunits with molecular masses of 42 to 45 kDa, containing two atoms of zinc per subunit. The enzymes catalyze NAD(+)-linked dehydrogenation of the hydroxyl group of the hemiacetal adduct [CH2(OH)OCH3] of methanol and formaldehyde, leading to the formation of a stoichiometric amount of methyl formate. Although neither methanol nor formaldehyde alone acted as a substrate for the enzymes, they showed simple NAD(+)-linked alcohol dehydrogenase activity toward aliphatic long-chain alcohols such as octanol, showing that they belong to the class III alcohol dehydrogenase family. The methyl formate synthase activity of C. boidinii was found in the mitochondrial fraction in subcellular fractionation experiments, suggesting that methyl formate synthase is a homolog of Saccharomyces cerevisiae Adh3p. These results indicate that formaldehyde could be oxidized in a glutathione-independent manner by methyl formate synthase in methylotrophic yeasts. The significance of methyl formate synthase in both formaldehyde resistance and energy metabolism is also discussed.

Solid-supported syntheses of 3-thio-1,2,4-triazoles.

Two solid-supported synthesis strategies for the preparation of 3-thio-1,2,4-triazoles are described. In the first, Rink amide resin is combined with Fmoc-protected omega-amino acids, acid hydrazides, and alkyl halides to provide diverse sets of starting materials from which numerous triazoles may be prepared. The second employs t-alkylcarbamate resin (Boc resin) which permits the use of additional pools of starting materials, including isothiocyanates and alpha- and omega-amino esters, resulting in triazoles with patterns of functional groups that are not possible from the initial route. The combination of multiple resins and resin attachment sites allows the preparation of a diverse library based upon the 3-thio-1,2,4-triazole scaffold and avoids the pitfall of having a single linker functionality present at the same position in all library members. General synthetic procedures and representative products from each route are presented. A similarity analysis of representative sublibraries from each synthesis strategy concludes that variation of the solid-phase linker chemistry and attachment site can enhance molecular diversity of the combined triazole library.

Trout liver slices for metabolism and toxicity studies.

Aquatic species are increasingly used in metabolism and toxicity studies, both from the perspective of potential for chemical exposure and usefulness as nonmammalian model systems. In the present study, trout liver slices were compared with freshly isolated trout hepatocytes with regard to metabolic capabilities and biochemical indices of cell health. Liver slices were also used to discern toxicant-induced changes in liver cell histology. Levels of ATP and glutathione were similar between liver slice and isolated hepatocyte preparations. The cytochrome P450-dependent rate of formation of biphenyl metabolites was 0.48 +/- 0.04 nmol/min/mg protein in slices and 0.43 +/- 0.06 nmol/min/mg protein in isolated cells. 7-Ethoxycoumarin metabolism was also comparable between preparations (1.36 vs. 1.22 nmol/min/mg protein). For conjugative metabolism, glucuronidation of 7-hydroxycoumarin or 1-naphthol did not differ in the two in vitro systems. However, neither slices nor isolated hepatocytes sulfated 7-hydroxycoumarin, whereas 1-naphthylsulfate represented as much as 20% of total 1-naphthol metabolites in both preparations. Histological evaluation of control liver slices after a 24-hr incubation indicated only minor changes. Response to the hepatotoxicants allyl formate and allyl alcohol was evaluated in slices only. Both compounds, after a 4-hr treatment and at concentrations between 0.1 and 1.0 mM, caused extensive depletion of glutathione, but ATP levels were unchanged. Histopathological damage was seen in slices incubated for 24 hr with either toxicant, but was most pronounced with allyl alcohol. These data indicate that liver slices are an excellent in vitro model for metabolism and toxicity studies in aquatic species.

A novel formaldehyde oxidation pathway in methylotrophic yeasts: methylformate as a possible intermediate.

A considerable amount of methylformate accumulated in the culture medium of methanol-grown methylotrophic yeasts. Methylformate is considered as an intermediate in a novel formaldehyde oxidation pathway. Through investigations with Pichia methanolica, methylformate formation was found to be catalysed by a new type of alcohol dehydrogenase, which was named methylformate synthase. When cells were grown on a relatively high concentration of methanol or exposed to a high concentration of formaldehyde, formation of methylformate was enhanced and the level of methylformate synthase in the cells increased. How methylformate synthase is involved in formaldehyde oxidation and formaldehyde detoxification is discussed.

Development of potent and selective CCK-A receptor agonists from Boc-CCK-4: tetrapeptides containing Lys(N epsilon)-amide residues.

A series of Boc-CCK-4 derivatives represented by the general structure Boc-Trp-Lys(N epsilon-COR)-Asp-Phe-NH2, where R is an aromatic, heterocyclic, or aliphatic group, are potent and selective CCK-A receptor agonists. These amide-bearing compounds complement the previously described urea-based tetrapeptides (Shiosaki et al. J. Med. Chem. 1991, 34, 2837-2842); structure-activity studies revealed parallel as well as divergent trends between these two series. A significant correlation was observed between pancreatic binding affinity and the resonance constant R of the phenyl substituent in one particular series of derivatives. Sulfation of phenolic amides appended onto the epsilon-amino group of the lysine did not affect affinity for the CCK-A receptor in contrast to the 500-fold increase in binding potency observed upon sulfation of CCK-8, suggesting that the lysine appendage and the sulfated tyrosine in CCK-8, both key structural elements that impart high affinity for the CCK-A receptor, are interacting differently with the receptor. The amide-bearing tetrapeptides are full agonists relative to CCK-8 in stimulating pancreatic amylase release while being partial agonists in eliciting phosphoinositide (PI) hydrolysis. Both effects were blocked by selective CCK-A receptor antagonists.

Partial purification and properties of an enzyme from Escherichia coli that catalyzes the conversion of glutamic acid and 10-formyltetrahydropteroylglutamic acid to 10-formyltetrahydropterol-gamma-glutamyglutamic acid.

An enzyme that catalyzes the conversion of L-glutamic acid and 10-formyl-H4folic acid (also known as 10-formyl-H4pteroylglutamic acid) to 10-formyl-H4pteroyl-gamma-glutamylglutamic acid has been purified by 74-fold from extracts of Escherichia coli. ATP, Mg-2+, and a monovalent cation (K+ or NH-4, but not Na+) are required for the enzyme to function. Radioactive and bioautographic analyses revealed the formation of a single product. This product was identified as 10-formyl-H-4pteroyl-gamma-glutamylglutamic acid from its spectral characteristics, its ability to be used effectively as a growth faster for Lactobacillus casei 7469, and from radioactive analysis that indicated the incorporation into the product of 1 mol glutamate/mol of 10-formyl-H-4pteroylglutamic acid utilized. The enzyme functions optimally at pH 9.0-9.8 and at 50 degrees. Its molecular weight is estimated at 42,000-43,000. The Km values are 180 muM for L-glutamic acid and less than 2 muM for (-) 10-formyl-H-4pteroylglutamic acid. The only other naturally occurring folate compounds with significant activity as substrate are H-4pteroylglutamic acid and 5,10-methylene-H-4pteroylglutamic acid; however, these compounds are not used as effectively (K-m values are 10-12 mu-M) as 10-formyl-H-4pteroylglutamic acid.