Oxidative Biotransformation Research Articles

Cytochrome P450 monooxygenase of Acanthamoeba castellanii participates in resistance to polyhexamethylene biguanide treatment.

Acanthamoeba spp. are free-living parasites that can cause severe infections such as granulomatous amoebic encephalitis (GAE) and amoebic keratitis (AK). Polyhexamethylene biguanide (PHMB) is a topical application for AK treatment. However, PHMB is not entirely effective against all Acanthamoeba strains or isolates. The mechanisms by which Acanthamoeba protects itself against extreme drug conditions without encystation are still unknown. According to a previous study, cytochrome P450 monooxygenase (CYP450MO) plays an important role in the oxidative biotransformation of numerous drugs related to metabolism. In this study, a CYP450MO fragment was inserted into the pGAPDH-EGFP vector and transfected into Acanthamoeba castellanii. We found that CYP450MO-overexpressing Acanthamoeba had higher survival rates than those of the control cells after PHMB treatment. Moreover, we also found that encystation-related genes such as cellulose synthase I (CSI), encystation-mediating serine proteinase (EMSP), and autophagy-related protein 8 (ATG8) expression levels were not significantly different between Acanthamoeba transfected by pGAPDH-EGFP or pGAPDH-EGFP-CYP450MO. We suggest that Acanthamoeba transfected by pGAPDH-EGFP-CYP450MO may not induce encystation-related genes to resist PHMB treatment. In conclusion, these findings indicate that CYP450MO may be an additional target when PHMB is used for treatment of amoebic keratitis.

A tunable l-arabinose-inducible expression plasmid for the acetic acid bacterium Gluconobacter oxydans

The acetic acid bacterium (AAB) Gluconobacter oxydans incompletely oxidizes a wide variety of carbohydrates and is therefore used industrially for oxidative biotransformations. For G. oxydans, no system was available that allows regulatable plasmid-based expression. We found that the l-arabinose-inducible PBAD promoter and the transcriptional regulator AraC from Escherichia coli MC4100 performed very well in G. oxydans. The respective pBBR1-based plasmids showed very low basal expression of the reporters β-glucuronidase and mNeonGreen, up to 480-fold induction with 1% l-arabinose, and tunability from 0.1 to 1% l-arabinose. In G. oxydans 621H, l-arabinose was oxidized by the membrane-bound glucose dehydrogenase, which is absent in the multi-deletion strain BP.6. Nevertheless, AraC-PBAD performed similar in both strains in the exponential phase, indicating that a gene knockout is not required for application of AraC-PBAD in wild-type G. oxydans strains. However, the oxidation product arabinonic acid strongly contributed to the acidification of the growth medium in 621H cultures during the stationary phase, which resulted in drastically decreased reporter activities in 621H (pH 3.3) but not in BP.6 cultures (pH 4.4). These activities could be strongly increased quickly solely by incubating stationary cells in d-mannitol-free medium adjusted to pH 6, indicating that the reporters were hardly degraded yet rather became inactive. In a pH-controlled bioreactor, these reporter activities remained high in the stationary phase (pH 6). Finally, we created a multiple cloning vector with araC-PBAD based on pBBR1MCS-5. Together, we demonstrated superior functionality and good tunability of an AraC-PBAD system in G. oxydans that could possibly also be used in other AAB.Key points• We found the AraC-PBADsystem from E. coli MC4100 was well tunable in G. oxydans.• In the absence of AraC orl-arabinose, expression from PBADwas extremely low.• This araC-PBADsystem could also be fully functional in other acetic acid bacteria.

Relating Metatranscriptomic Profiles to the Micropollutant Biotransformation Potential of Complex Microbial Communities.

Biotransformation of chemical contaminants is of importance in various natural and engineered systems. However, in complex microbial communities and with chemical contaminants at low concentrations, our current understanding of biotransformation at the level of enzyme-chemical interactions is limited. Here, we explored an approach to identify associations between micropollutant biotransformation and specific gene products in complex microbial communities, using association mining between chemical and metatranscriptomic data obtained from experiments with activated sludge grown at different solid retention times. We successfully demonstrate proportional relationships between the measured rate constants and associated gene transcripts for nitrification as a major community function, but also for the biotransformation of two nitrile-containing micropollutants (bromoxynil and acetamiprid) and transcripts of nitrile hydratases, a class of enzymes that we experimentally confirmed to produce the detected amide transformation products. As these results suggest that metatranscriptomic information can indeed be quantitatively correlated with low abundant community functions such as micropollutant biotransformation in complex microbial communities, we proceeded to explore the potential of association mining to highlight enzymes likely involved in catalyzing less well-understood micropollutant biotransformation reactions. Specifically, we use the cases of nitrile hydration and oxidative biotransformation reactions to show that the consideration of additional experimental evidence (such as information on biotransformation pathways) increases the likelihood of detecting plausible novel enzyme-chemical relationships. Finally, we identify a cluster of mono- and dioxygenase fourth-level enzyme classes that most strongly correlate with oxidative micropollutant biotransformation reactions in activated sludge.

Capsaicinoid metabolism by the generalist Helicoverpa armigera and specialist H. assulta: Species and tissue differences

Helicoverpa armigera and H. assulta are two of the few insects that can feed on hot pepper fruits. Capsaicin and dihydrocapsaicin (i.e., capsaicinoids) are the principal pungent compounds in hot peppers. To explore possible molecular mechanisms of adaptation that allow these two species to consume capsaicinoids, the capacity of the three detoxification tissues (fat body, midgut, and Malpighian tubule) of the two pests, to metabolically degrade capsaicin and dihydrocapsaicin, was compared. The results showed that capsaicin and dihydrocapsaicin were metabolized by crude enzyme preparations from all three tissues of the two pests. Five metabolites of capsaicin, and five metabolites of dihydrocapsaicin were identified. Tissue and species differences in the degree of capsaicin and dihydrocapsaicin metabolism were observed. The specialist H. assulta had an overall greater capacity to degrade the capsaicinoids compared to the generalist H. armigera. Further, the midgut was the most significant contributor to capsaicinoid metabolism. The notably high specific activity in Malpighian tubules of H. armigera also further highlights the significance of this organ in xenobiotic detoxification. Alkyl hydroxylation and dehydrogenation were the main pathways for the oxidative biotransformation of both capsaicin and dihydrocapsaicin by cytochrome P450s. This study provides evidence that enhanced metabolic decomposition of capsaicinoids may be an adaptation explaining dietary preferences for Capsicum fruits by these two pests.

Correction to "In Vitro Hepatic Oxidative Biotransformation of Trimethoprim".

Correction to "In Vitro Hepatic Oxidative Biotransformation of Trimethoprim".

Transcriptional profiling of cytochrome P450 genes in the liver of adult zebrafish, <i>Danio rerio</i>

Increasing use of zebrafish in biomedical, toxicological and developmental studies requires explicit knowledge of cytochrome P450 (CYP), given the central role of CYP in oxidative biotransformation of xenobiotics and many regulatory molecules. A full complement of CYP genes in zebrafish and their transcript expression during early development have already been examined. Here we established a comprehensive picture of CYP gene expression in the adult zebrafish liver using a RNA-seq technique. Transcriptional profiling of a full complement of CYP genes revealed that CYP2AD2, CYP3A65, CYP1A, CYP2P9 and CYP2Y3 are major CYP genes expressed in the adult zebrafish liver in both sexes. Quantitative real-time RT-PCR analysis for selected CYP genes further supported our RNA-seq data. There were significant sex differences in the transcript levels for CYP1A, CYP1B1, CYP1D1 and CYP2N13, with males having higher expression levels than those in females in all cases. A similar feature of gender-specific expression was observed for CYP2AD2 and CYP2P9, suggesting sex-specific regulation of constitutive expression of some CYP genes in the adult zebrafish liver. The present study revealed several "orphan" CYP genes as dominant isozymes at transcript levels in the adult zebrafish liver, implying crucial roles of these CYP genes in liver physiology and drug metabolism. The current results establish a foundation for studies with zebrafish in drug discovery and toxicology.

Cladosporin Derivatives Obtained by Biotransformation Provide Guidance for the Focused Derivatization of this Antimalarial Lead Compound.

Cladosporin, a natural product known for decades, has recently been discovered to display potent and selective antiplasmodial activity by inhibition of lysyl-tRNA synthetase. It was subjected to a panel of oxidative biotransformations with one fungal and two actinomycetes strains, as well as a triple mutant bacterial CYP102A1, yielding eight, mostly hydroxylated, derivatives. These new compounds covered a wide chemical space and contained two pairs of epimers in the tetrahydropyran ring. Although less potent than the parent compound, all analogues showed activity in a cell-based synthetase assay, thus demonstrating uptake and on-target activity in living cells with varying degrees of selectivity for the enzyme lysyl-tRNA synthetase from Plasmodium falciparum and highlighting sites suitable for synthesis of future cladosporin analogues. Compounds with adjacent hydroxy functions showed different MS/MS fragmentation that can be explained in terms of an, in some cases, regioselective loss of water followed by a retro-Diels-Alder reaction.

Global mRNA decay and 23S rRNA fragmentation in Gluconobacter oxydans 621H

BackgroundGluconobacter oxydans is a strictly aerobic Gram-negative acetic acid bacterium used industrially for oxidative biotransformations due to its exceptional type of catabolism. It incompletely oxidizes a wide variety of carbohydrates regio- and stereoselectively in the periplasm using membrane-bound dehydrogenases with accumulation of the products in the medium. As a consequence, only a small fraction of the carbon and energy source enters the cell, resulting in a low biomass yield. Additionally, central carbon metabolism is characterized by the absence of a functional glycolysis and absence of a functional tricarboxylic acid (TCA) cycle. Due to these features, G. oxydans is a highly interesting model organism. Here we analyzed global mRNA decay in G. oxydans to describe its characteristic features and to identify short-lived mRNAs representing potential bottlenecks in the metabolism for further growth improvement by metabolic engineering.ResultsUsing DNA microarrays we estimated the mRNA half-lives in G. oxydans. Overall, the mRNA half-lives ranged mainly from 3 min to 25 min with a global mean of 5.7 min. The transcripts encoding GroES and GroEL required for proper protein folding ranked at the top among transcripts exhibiting both long half-lives and high abundance. The F-type H+-ATP synthase transcripts involved in energy metabolism ranked among the transcripts with the shortest mRNA half-lives. RNAseq analysis revealed low expression levels for genes of the incomplete TCA cycle and also the mRNA half-lives of several of those were short and below the global mean. The mRNA decay analysis also revealed an apparent instability of full-length 23S rRNA. Further analysis of the ribosome-associated rRNA revealed a 23S rRNA fragmentation pattern exhibiting new cleavage regions in 23S rRNAs which were previously not known.ConclusionsThe very short mRNA half-lives of the H+-ATP synthase, which is likely responsible for the ATP-proton motive force interconversion in G. oxydans under many or most conditions, is notably in contrast to mRNA decay data from other bacteria. Together with the short mRNA half-lives and low expression of some other central metabolic genes it could limit intended improvements of G. oxydans’ biomass yield by metabolic engineering. Also, further studies are needed to unravel the multistep process of the 23S rRNA fragmentation in G. oxydans.

Trends in Micropollutant Biotransformation along a Solids Retention Time Gradient.

For many polar organic micropollutants, biotransformation by activated sludge microorganisms is a major removal process during wastewater treatment. However, our current understanding of how wastewater treatment operations influence microbial communities and their micropollutant biotransformation potential is limited, leaving major parts of observed variability in biotransformation rates across treatment facilities unexplained. Here, we present biotransformation rate constants for 42 micropollutants belonging to different chemical classes along a gradient of solids retention time (SRT). The geometric mean of biomass-normalized first-order rate constants shows a clear increase between 3 and 15 d SRT by 160% and 87%, respectively, in two experiments. However, individual micropollutants show a variety of trends. Rate constants of oxidative biotransformation reactions mostly increased with SRT. Yet, nitrifying activity could be excluded as primary driver. For substances undergoing other than oxidative reactions, i.e., mostly substitution-type reactions, more diverse dependencies on SRT were observed. Most remarkably, characteristic trends were observed for groups of substances undergoing similar types of initial transformation reaction, suggesting that shared enzymes or enzyme systems that are conjointly regulated catalyze biotransformation reactions within such groups. These findings open up opportunities for correlating rate constants with measures of enzyme abundance such as genes or gene products, which in turn should help to identify enzymes associated with the respective biotransformation reactions.

HPLC study on Fenton-reaction initiated oxidation of salicylic acid. Biological relevance of the reaction in intestinal biotransformation of salicylic acid

Fenton-reaction initiated in vitro oxidation and in vivo oxidative biotransformation of salicylic acid was investigated by HPLC-UV-Vis method. By means of the developed high performance liquid chromatography (HPLC) method salicylic acid, catechol, and all the possible monohydroxylated derivatives of salicylic acid can be separated. Fenton oxidations were performed in acidic medium (pH 3.0) with two reagent molar ratios: (1) salicylic acid: iron: hydrogen peroxide 1:3:1 and (2) 1:0.3:1. The incubation samples were analysed at different time points of the reactions. The biological effect of elevated reactive oxygen species concentration on the intestinal metabolism of salicylic acid was investigated by an experimental diabetic rat model. HPLC-MS analysis of the in vitro samples revealed presence of 2,3- and 2,5-dihydroxybenzoic acids. The results give evidence for nonenzyme catalysed intestinal hydroxylation of xenobiotics.

Biotransformation of Finerenone, a Novel Nonsteroidal Mineralocorticoid Receptor Antagonist, in Dogs, Rats, and Humans, In Vivo and In Vitro.

Mass balance and biotransformation of finerenone, a nonsteroidal mineralocorticoid receptor antagonist, were investigated in four healthy male volunteers following a single oral administration of 10 mg (78 μCi) of [14C]finerenone and compared with data from studies in dogs and rats. The total recovery of the administered radioactivity was 101% in humans, 94.7% in dogs, and 95.2% in rats. In humans, radioactivity was mainly excreted renally (80%); in rats, it was primarily the biliary/fecal route (76%); and in dogs, excretion was more balanced. Finerenone was extensively metabolized in all species by oxidative biotransformation, with minor amounts of unchanged drug in excreta (humans: 1%; dogs, rats: <9%). In vitro studies suggested cytochrome P450 3A4 was the predominant enzyme involved in finerenone metabolism in humans. Primary metabolic transformation involved aromatization of the dihydronaphthyridine moiety of metabolite M1 as a major clearance pathway with a second oxidative pathway leading to M4. These were both prone to further oxidative biotransformation reactions. Naphthyridine metabolites (M1-M3) were the dominant metabolites identified in human plasma, with no on-target pharmacological activity. In dog plasma, finerenone and metabolite M2 constituted the major components; finerenone accounted almost exclusively for drug-related material in rat plasma. For metabolites M1-M3, axial chirality was observed, represented by two atropisomers (e.g., M1a and M1b). Analysis of plasma and excreta showed one atropisomer (a-series, >79%) of each metabolite predominated in all three species. In summary, the present study demonstrates that finerenone is cleared by oxidative biotransformation, mainly via naphthyridine derivatives.

Cytochrome P450 CYP199A4 from Rhodopseudomonas palustris Catalyzes Heteroatom Dealkylations, Sulfoxidation, and Amide and Cyclic Hemiacetal Formation

The cytochrome P450 enzymes execute a range of selective oxidative biotransformations across many biological systems. The bacterial enzyme CYP199A4 catalyzes the oxidative demethylation of 4-methoxybenzoic acid. The benzoic acid moiety of the molecule binds in the active site of the enzyme such that the functional group at the para-position is held close to the heme iron. Therefore, CYP199A4 has the potential to catalyze alternative monooxygenase reactions with different para-substituted benzoic acid substrates such as thioethers and alkylamines. The oxidation of 4-methyl- and 4-ethyl-thiobenzoic acids by CYP199A4 resulted in sulfur oxidation. 4-Ethylthiobenzoic acid sulfoxidation and 4-ethylbenzoic acid hydroxylation by CYP199A4 occurred with high enantioselectivity (>74% enantiomeric excess). By way of contrast, CYP199A4 catalyzed exclusive oxidative N-demethylation over N-oxide formation with 4-methyl- and 4-dimethylaminobenzoic acids. Unexpectedly acetamide formation by CYP199A4 competes with dealkyla...

An immobilized and highly stabilized self‐sufficient monooxygenase as biocatalyst for oxidative biotransformations

AbstractBACKGROUNDThe requirement for expensive cofactors that must be efficiently recycled is one of the major factors hindering the wide implementation of industrial biocatalytic oxidation processes. In this research, a sustainable approach based on immobilized self‐sufficient Baeyer–Villiger monooxygenases is discussed.RESULTSA bifunctional biocatalyst composed of an NADPH‐dependent cyclohexanone monooxygenase (CHMO) fused to an NADP+‐accepting phosphite dehydrogenase (PTDH) catalyzes ϵ‐caprolactone synthesis from cyclohexanone, using phosphite as a cheap sacrificial substrate for cofactor regeneration. Several immobilized derivatives of the fused enzyme have been prepared with high immobilization yield (97%); the one obtained by affinity adsorption on Co‐IDA (Co: cobalt chelated; IDA: iminodiacetic acid) support has shown to be highly stable affording average yields of 80% after 18 reaction cycles.CONCLUSIONSThe immobilized self‐sufficient monooxygenase has demonstrated to be able to perform Baeyer–Villiger oxidation with efficient cofactor recovery and biocatalyst recycling. The proposed biocatalytic process offers access to valuable molecules with high atom economy and limited waste generation. © 2017 Society of Chemical Industry

Changes in CYP2D enzyme activity following induction of type 2 diabetes, and administration of cinnamon and metformin: an experimental animal study

1. Alterations in the activity of hepatic cytochrome P-450 isoenzymes result in changes in the pharmacokinetic behavior of drugs. This study was designed to explore the impact of type II diabetes, metformin and cinnamon on the activity of CYP2D isoenzyme.2. Streptozotocin-nicotinamide-induced diabetic and normal rats were gavaged by cinnamon and/or metformin for 14 days. Using isolated perfusion of rat livers, the metabolic activity of CYP2D in the study groups was evaluated based on the oxidative biotransformation of tramadol hydrochloride.3. The metabolic ratios of O-desmethyltramadol, the product of CYP2D-mediated metabolism of tramadol, in normal and diabetic control rats were found to be 0.33 ± 0.12 and 0.29 ± 0.07, respectively. Cinnamon significantly reduced the mentioned ratio in both normal and diabetic rats (0.13 ± 0.05 and 0.15 ± 0.04) and metformin increased the reduced activity in diabetic rats (0.37 ± 0.09 versus 0.29 ± 0.07).4. In conclusion, it is evident that this study has shown the significant inhibitory effect of cinnamon on CYP2D. This finding suggests that it should be taken into consideration the possible metabolism-related pharmacokinetic drug–cinnamon interactions.5. Additionally, type 2 diabetes condition reduced the enzyme activity and metformin consumption reversed this reduction; however, the significance of the latest is not clear.

Effects of obesity on liver cytochromes P450 in various animal models.

The prevalence of obesity and other obesity-related diseases is increasing worldwide. Obesity is a disease characterized by increased body weight, or a condition resulting from excessive accumulation of body fat. Due to increased body fat deposits, obesity has also been associated with increased mortality resulting from higher incidence rates of hypertension, diabetes, or various types of cancer, such as breast, colorectal, cervical and prostate cancer. Physiological changes associated with obesity are likely to result in altered drug biotransformation. The main enzymes enabling the oxidative biotransformation of most drugs are cytochromes P450 (CYPs). The review summarizes how pathophysiological factors, especially obesity, affect properties (e.g. enzyme activity, protein expression, gene expression) of CYP enzymes in various experimental models of human obesity. Results reported by various authors suggest that obesity is associated with a decrease of CYP activities (except for the CYP2C and CYP2E1 enzymes). The only exception is mouse obesity induced by monosodium glutamate (administered to newborn mice) as it usually leads to increased CYP expression. Selecting an animal model that is as close as possible to the properties of human obesity is of paramount importance.

Oriented Coimmobilization of Oxidase and Catalase on Tailor-Made Ordered Mesoporous Silica.

Mesoporous silica materials are promising carriers for enzyme immobilization in heterogeneous biocatalysis applications. By tailoring their pore structural framework, these materials are designable for appropriate enzyme binding capacity and internal diffusivity. To supply O2 efficiently to solid-supported immobilized enzymes represents a core problem of heterogeneously catalyzed oxidative biotransformations. In this study, therefore, we synthesized and compared three internally well-ordered and two amorphous silica materials as enzyme carriers, each of those with pore sizes of ≥10 nm, to enable the coimmobilization of d-amino-acid oxidase (79 kDa) and catalase (217 kDa). Both enzymes were fused to the silica-binding module Zbasic2 to facilitate their selective and oriented immobilization directly from crude protein mixtures on native silica materials. Analyzing the effects of varied pore architecture and internal surface area on the performance of the immobilized bienzymatic system, we showed that a uniform pore structural framework was beneficial for enzyme loading (≥70 mg protein/g carrier), immobilization yield (≥90%), surface and pore volume filling without hindered adsorption, and catalytic effectiveness (≥60%) of the coimmobilizate. Using the best carrier LP-SBA-15, we obtained a solid oxidase-catalase preparation with an activity of 2000 μmol/(min g_material) that was recyclable and stable during oxidation of d-methionine. These results affirm a strategy of optimizing immobilized O2-dependent enzymes via tunable internal structuring of the silica material used as carrier. They thus make a significant advance toward the molecular design of heterogeneous oxidation biocatalysts on mesoporous silica supports.

Hepatic microsomal metabolism of BDE-47 and BDE-99 by lesser snow geese and Japanese quail

In the present study, we investigated the oxidative biotransformation of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) and 2,2′,4,4′,5-pentabromodiphenyl ether (BDE-99) by liver microsomes from wild lesser snow geese (Chen caerulescens caerulescens) and domesticated Japanese quail (Coturnix japonica). Formation of hydroxy-metabolites was analyzed using an ultra-high performance liquid chromatography-tandem mass spectrometry-based method. Incubation of BDE-47 with avian liver microsomes produced sixteen hydroxy-metabolites, eight of which were identified using authentic standards. The major metabolites formed by liver microsomes from individual lesser snow geese were 4-hydroxy-2,2′,3,4′-tetrabromodiphenyl ether (4-OH-BDE-42), 3-hydroxy-2,2′,4,4′-tetrabromodiphenyl ether (3-OH-BDE-47), and 4′-hydroxy-2,2′,4,5′-tetrabromodiphenyl ether (4′-OH-BDE-49). By comparison, 4-OH-BDE-42 and 4′-OH-BDE-49, but not 3-OH-BDE-47, were major metabolites of Japanese quail liver microsomes. Unidentified metabolites included monohydroxy- and dihydroxy-tetrabromodiphenyl ethers. Incubation of BDE-99 with avian liver microsomes produced seventeen hydroxy-metabolites, twelve of which were identified using authentic standards. The major metabolites formed by lesser snow goose liver microsomes were 2,4,5-tribromophenol, 3-OH-BDE-47, 4′-OH-BDE-49, 4-hydroxy-2,2′,3,4′,5-pentabromodiphenyl ether (4-OH-BDE-90), and 5′-hydroxy-2,2′,4,4′,5-pentabromodiphenyl ether (5′-OH-BDE-99). By comparison, the major metabolites produced by liver microsomes from Japanese quail included 6-hydroxy-2,2′,4,4′-tetrabromodiphenyl ether (6-OH-BDE-47) and 2-hydroxy-2′,3,4,4′,5-pentabromodiphenyl ether (2-OH-BDE-123), but not 3-OH-BDE-47. Unidentified metabolites consisted of monohydroxy-pentabromodiphenyl ethers, monohydroxy-tetrabromodiphenyl ethers and dihydroxy-tetrabromodiphenyl ethers. Another difference between the two species was that formation rates of BDE-47 and BDE-99 metabolites were greater with liver microsomes from male than female Japanese quail, but a sex difference was not observed with lesser snow geese.

Metabolic engineering of Gluconobacter oxydans 621H for increased biomass yield.

The obligatory aerobic acetic acid bacterium Gluconobacter oxydans incompletely oxidizes carbon sources regio- and stereoselectively in the periplasm and therefore is used industrially for oxidative biotransformations, e. g., in vitamin C production. However, it has a very low biomass yield as the oxidized products largely remain in the medium and cannot be used for anabolism. Cytoplasmic carbon metabolism occurs via the pentose phosphate pathway and the Entner-Doudoroff pathway, whereas glycolysis and the tricarboxylic acid cycle are incomplete. Acetate is formed as an end product via pyruvate decarboxylase and acetaldehyde dehydrogenase. In order to increase the biomass yield from glucose, we sequentially replaced (i) gdhS encoding the cytoplasmic NADP-dependent glucose dehydrogenase by the Acetobacter pasteurianus sdhCDABE genes for succinate dehydrogenase and the flavinylation factor SdhE (strain IK001), (ii) pdc encoding pyruvate decarboxylase by a second ndh gene encoding a type II NADH dehydrogenase (strain IK002.1), and (iii) gdhM encoding the membrane-bound PQQ-dependent glucose dehydrogenase by sucCD from Gluconacetobacter diazotrophicus encoding succinyl-CoA synthetase (strain IK003.1). Analysis of the strains under controlled cultivation conditions in bioreactors revealed for IK003.1 that neither gluconate nor 2-ketogluconate was formed, but some 5-ketogluconate. Acetate formation was eliminated, and comparable amounts of pyruvate were formed instead. CO2 formation by IK003.1 was more than doubled compared to the reference strain. Growth of IK003.1 was retarded, but the biomass yield of this strain was raised by 60%. IK003.1 serves as suitable host for oxidative biotransformations and for further metabolic engineering.

Integral biochemical index of the state of aquatic organisms under polluted conditions

The state of marine and estuary organisms living in the polluted coastal zone of Peter the Great Bay in the Sea of Japan has been estimated using molecular biomarkers of oxidative stress and biotransformation that characterize changes in animal metabolism under the effect of pollutants. The integral biochemical indices were calculated and graphically represented for mussels Crenomytilus grayanus collected at marine stations with different degrees of pollution and for the Japanese mitten crab Eriocheir japonica from the estuaries of rivers flowing into Peter the Great Bay, based on statistical processing of the results. It has been noted that the values of the indices correspond to the degree of pollution of animal sampling areas.

Electrochemistry coupled to (LC-)MS for the simulation of oxidative biotransformation reactions of PAHs

Electrochemistry coupled to liquid chromatography and mass spectrometry was used for simulating the biological and environmental fate of polycyclic aromatic hydrocarbons (PAHs) as well as for studying the PAH degradation behavior during electrochemical remediation. Pyrene and benzo[a]pyrene were selected as model compounds and oxidized within an electrochemical thin-layer cell equipped with boron-doped diamond electrode. At potentials of 1.2 and 1.6 V vs. Pd/H2, quinones were found to be the major oxidation products for both investigated PAHs. These quinones belong to a large group of PAH derivatives referred to as oxygenated PAHs, which have gained increasing attention in recent years due to their high abundance in the environment and their significant toxicity. Separation of oxidation products allowed the identification of two pyrene quinone and three benzo[a]pyrene quinone isomers, all of which are known to be formed via photooxidation and during mammalian metabolism. The good correlation between electrochemically generated PAH quinones and those formed in natural processes was also confirmed by UV irradiation experiments and microsomal incubations. At potentials higher than 2.0 V, further degradation of the initial oxidation products was observed which highlights the capability of electrochemistry to be used as remediation technique.