7H-Pyridocarbazole, a Probe to Study the Role of Diphosphatidylglycerol in the Functioning of the Cytochrome Oxidase Complex of Mitochondria

Ditercalinium, the generic name for 7H-pyridocarbazole dimer,is a planar aromatic cation which exhibits strong inhibitoryeffects on the respiratory chain of plant mitochondria. By enzymaticassays and spectroscopic studies, the inhibition is shown tooccur at the level of the cytochrome c oxidase complex. It issuggested that the mechanism of inhibition could be due to thecomplexation of the aromatic cation with the diphosphatidylglycerolenvironment essential for cytochrome c oxidase activity ratherthan to a drug-enzyme direct interaction. 7H-pyridocar-bazolecan be used as an appropriate tool to study the role that negativelycharged acidic phospholipids, as diphosphatidylglycerol, playin the functioning of cytochrome c oxidase. (Received July 10, 1989; Accepted February 19, 1990)     

Concentrations and characteristics of procyanidins and other phenolics in apples during fruit growth

Apples (Malus domestica Borkh.) of two table and two cider cultivars were collected during fruit growth and maturation from the end of cell proliferation. Concentrations of flavonoids (flavan-3-ols, dihydrochalcones and flavonols) in the fruit flesh decreased sharply between circa 35 and circa 100 days after flowering. For hydroxycinnamic acids, the decrease appeared slower. In a second experiments apples of the cider cultivars Kermerrien and Avrolles were sampled every 2 weeks from 40 days after flowering to overripeness for a detailed characterisation of polyphenol accumulation kinetics in the fruit flesh. Most polyphenol synthesis had occurred at 40 days after full bloom, though it persisted at a low (Kermerrien) to very low (Avrolles) level during all the fruit growth. All qualitative characteristics of the polyphenols were remarkably stable. The degree of polymerisation of the procyanidins increased slightly in Avrolles and decreased in Kermerrien. This was accompanied by a relative increase in procyanidin B2, while size-exclusion chromatography of Kermerrien polyphenol extracts showed the disappearance of a highly polymerised fraction.     

Adaptation of acidogenic sludge to increasing glycerol concentrations for biohydrogen production

Hydrogen is a promising alternative as an energetic carrier and its production by dark fermentation from wastewater has been recently proposed, with special attention to crude glycerol as potential substrate. In this study, two different feeding strategies were evaluated for replacing the glucose substrate by glycerol substrate: a one-step strategy (glucose was replaced abruptly by glycerol) and a step-by-step strategy (progressive decrease of glucose concentration and increase of glycerol concentration from 0 to 5 g L^?1), in a continuous stirred tank reactor (12 h of hydraulic retention time (HRT), pH 5.5, 35 °C). While the one-step strategy led to biomass washout and unsuccessful H₂ production, the step-by-step strategy was efficient for biomass adaptation, reaching acceptable hydrogen yields (0.4?±?0.1 mol_H2?mol^?1 _{glycerol consumed}) around 33 % of the theoretical yield independently of the glycerol concentration. Microbial community structure was investigated by single-strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) fingerprinting techniques, targeting either the total community (16S ribosomal RNA (rRNA) gene) or the functional Clostridium population involved in H₂ production (hydA gene), as well as by 454 pyrosequencing of the total community. Multivariate analysis of fingerprinting and pyrosequencing results revealed the influence of the feeding strategy on the bacterial community structure and suggested the progressive structural adaptation of the community to increasing glycerol concentrations, through the emergence and selection of specific species, highly correlated to environmental parameters. Particularly, this work highlighted an interesting shift of dominant community members (putatively responsible of hydrogen production in the continuous stirred tank reactor (CSTR)) according to the gradient of glycerol proportion in the feed, from the family Veillonellaceae to the genera Prevotella and Clostridium sp., putatively responsible of hydrogen production in the CSTR.     

Kinetic modelling of steam gasification of various woody biomass chars: influence of inorganic elements

A study was performed on the influence of wood variability on char steam gasification kinetics. Isothermal experiments were carried out in a thermobalance in chemical regime on various wood chars produced under the same conditions. The samples exhibited large differences of average reaction rate. These differences were linked neither with the biomass species nor age and may be related to the biomass inorganic elements. A modelling approach was developed to give a quantitative insight to these observations. The grain model was used on one biomass of reference for temperatures between 750 and 900 °C and steam partial pressures between 0 and 0.27 bar. The model was applied to the other samples through the addition of an integral parameter specific to each sample. A satisfactory correlation was found between this parameter and the ratio potassium/silicium. This result highlighted the catalytic effect of potassium and inhibitor effect of silicium on the reaction.     

Isolation and characterization of novel bacterial strains exhibiting ligninolytic potential

Background

The number of biotransformations that use nicotinamide recycling systems is exponentially growing. For this reason one of the current challenges in biocatalysis is to develop and optimize more simple and efficient cofactor recycling systems. One promising approach to regenerate NAD⁺ pools is the use of NADH-oxidases that reduce oxygen to hydrogen peroxide while oxidizing NADH to NAD⁺. This class of enzymes may be applied to asymmetric reduction of prochiral substrates in order to obtain enantiopure compounds.

Results

The NADH-oxidase (NOX) presented here is a flavoenzyme which needs exogenous FAD or FMN to reach its maximum velocity. Interestingly, this enzyme is 6-fold hyperactivated by incubation at high temperatures (80°C) under limiting concentrations of flavin cofactor, a change that remains stable even at low temperatures (37°C). The hyperactivated form presented a high specific activity (37.5 U/mg) at low temperatures despite isolation from a thermophile source. Immobilization of NOX onto agarose activated with glyoxyl groups yielded the most stable enzyme preparation (6-fold more stable than the hyperactivated soluble enzyme). The immobilized derivative was able to be reactivated under physiological conditions after inactivation by high solvent concentrations. The inactivation/reactivation cycle could be repeated at least three times, recovering full NOX activity in all cases after the reactivation step. This immobilized catalyst is presented as a recycling partner for a thermophile alcohol dehydrogenase in order to perform the kinetic resolution secondary alcohols.

Conclusion

We have designed, developed and characterized a heterogeneous and robust biocatalyst which has been used as recycling partner in the kinetic resolution of rac-1-phenylethanol. The high stability along with its capability to be reactivated makes this biocatalyst highly re-useable for cofactor recycling in redox biotransformations.